Journal of Experimental Botany

Traynor, Mary;Roberts, Jerry

doi: 10.1093/jxb/erm041pmid: N/A

It is nearly three years since the Journal of Experimental Botany (JXB) launched its Open Access (OA) experiment and during this time approximately 30% of primary research papers have been made freely available online from the time of publication. Early indications show that OA publication increases impact as full text downloads are, on average, 17% higher and citations 14% higher than for those papers kept under subscription control. The JXB now wishes to thank all those who have supported this initiative and extend the experiment to enable more authors to take advantage of OA publication. Substantial costs are incurred in journal publication; the full cost of publishing a paper in the JXB is around £1500 ($2800) and considerably more in many similar journals. In the first phase of our OA experiment, costs were met in part by OA fees from authors (£250 per paper) and by two grants from the UK Joint Information Systems Committee, which is a government organization concerned with ‘innovative use of Information and Communications Technology to support education and research’. However, income from the sale of subscriptions has continued to fund the major part of our publication costs. The JXB is wholly committed to broadening access to its publications and maximizing impact; but at the same time the JXB must remain financially viable. Currently, the only way we are able to continue to publish without imposing large publication fees or page charges is through the maintenance of our subscriber base. It is with this in mind that we have designed the next phase in our OA experiment. All papers accepted after the beginning of April whose corresponding author's institution subscribes to JXB will be published as OA. Our expectation is that this will represent around 70% of our primary papers. Authors not from a subscribing institution who wish to take advantage of OA publication will have to pay the full costs (£1500/$2800/€2250). Over the last few years, we have been careful to develop the JXB, ensuring that our subscribers receive value in addition to our primary papers. In 2006, the JXB published five Special Issues and five Focus Sections (see below) plus reviews and opinion papers, all these remain under subscription control. An institutional subscription to the JXB therefore provides exclusive access to: special issues, reviews and focus sections; the 30% of primary papers that remain under subscription control; plus OA publication for members of the institution. We hope that you can see we are working hard at building a viable business model that can provide a sustainable service to the plant science community. The cost of a full online subscription to the JXB in 2007 is £816 ($1469). On a cost per page basis this is a very competitive price; it is cheaper than any other comparable plant science journal without page charges or subsidy. We are interested in your views and opinions; please send all responses to [email protected] Special Issues 2006 Phenotypic Plasticity and the Changing Environment Plants and Salinity Plant Proteomics Oxygen Metabolism, ROS and Redox Signalling in Plants Major Themes in Flowering Research Focus Sections 2006 The Visible Plant Cell: Biosensors and Bioreporters Nitric Oxide Signalling: Plant Growth and Development Phloem–Insect Interaction Leaf Metabolism and Development Surviving in a Hostile Environment: Barrier Properties of Cuticles and Periderms Published by Oxford University Press [2007] on behalf of the Society for Experimental Biology

Sablowski, Robert

doi: 10.1093/jxb/erm002pmid: 17293602

Abstract Meristems provide new cells to produce organs throughout the life of a plant, and their continuous activity depends on regulatory genes that balance the proliferation of meristem cells with their recruitment to organogenesis. During flower development, this balance is shifted towards organogenesis, causing the meristem to terminate after producing a genetically determined number of organs. In Arabidopsis, WUSCHEL (WUS) specifies the self-renewing cells at the core of the shoot meristems and is a key target in the control of meristem stability. The development of a determinate floral meristem is initiated by APETALA1/CAULIFLOWER (AP1/CAL) and LEAFY (LFY). The latter activates AGAMOUS (AG), partly in co-operation with WUS. AG then directs the development of the innermost floral organs and at the same time antagonizes WUS to terminate the meristem, although the mechanism of WUS repression remains unknown. All these genes participate in a series of regulatory feedback loops that maintain stable expression patterns or promote sharp developmental transitions. Although the regulators of meristem maintenance and determinacy in Arabidopsis are widely conserved, their interactions may vary in other species. AGAMOUS, determinacy, flower development, shoot meristem, WUSCHEL Introduction The apical meristems function as the main sources of new cells to sustain plant growth. The regular recruitment of meristem cells to form new organs and tissues is balanced by cell proliferation within the meristem to maintain its size relatively stable. This steady-state can persist throughout the life of the plant, but in many cases the meristem is genetically programmed to stop producing new cells at a specific developmental stage. In these cases, the meristem is said to be determinate. A determinate meristem produces a part of the plant body with a predictable size and form, such as the flower, whereas indeterminate meristems produce parts of the plant whose size and shape depend on the local environment, such as branches and roots that grow to variable lengths. The positioning of determinate and indeterminate meristems varies between species and is a major determinant of plant architecture. Here, the genetic control of meristem determinacy in Arabidopsis and the applicability of this knowledge to other species is reviewed. In the Arabidopsis shoot, determinacy is a property of the floral meristems, whereas the vegetative and inflorescence meristems are indeterminate. The indeterminate growth of the vegetative and inflorescence meristems requires a specific set of regulatory genes, whose activity is antagonized by flower-specific regulators. For this reason, it is useful to start with a description of how the activity of indeterminate meristems is maintained. Maintenance of indeterminate meristems The indeterminate growth of the vegetative and inflorescence meristems is sustained by small groups of self-renewing cells that are functionally similar to stem cells in animals (Sablowski, 2004). These cells are located in the central zone (CZ) of the meristem, while some of their descendants are displaced to the peripheral zone (PZ), where they are recruited to form new organ primordia (Fig. 1). Below the CZ, the rib meristem (RM) sustains stem growth. The CZ and PZ functions have been well studied in Arabidopsis, although the RM has received much less attention. Superimposed on the CZ/PZ organization, the typical tunica/corpus structure found in angiosperms can also be distinguished, with two external layers (L1 and L2) in which most cell divisions are oriented tangentially to the meristem surface, while the inner layer does not have clearly oriented divisions (Carles and Fletcher, 2003). Although the functional significance of the L1–L3 layering is not well understood, the existence of these clonally distinct layers has been essential to reveal the role of intercellular communication in meristem function. Fig. 1. Open in new tabDownload slide Structure of the shoot apical meristem. The concentric L1, L2, and L3 cell layers are indicated by different shades of colour. The central zone (CZ) is marked in blue, the peripheral zone (PZ) in green, and the position of the rib meristem (RM) is indicated below the meristem dome. The region that expresses WUS is circled in red; the white arrow represents the intercellular signal produced by the WUS-expressing cells to maintain cell identity and proliferation in overlying CZ. P indicates organ primordia produced on the flanks of the meristem. SHOOT MERISTEMLESS (STM) and WUSCHEL (WUS) are two regulatory genes with central roles in shoot meristem development. STM and WUS genes function synergistically during meristem development and are required not only for the establishment of the shoot meristem during embryogenesis, but also for subsequent maintenance of the vegetative, inflorescence and floral meristems (Clark et al., 1996; Laux et al., 1996; Long et al., 1996; Gallois et al., 2002; Lenhard et al., 2002). STM encodes a homeodomain protein expressed throughout the meristem (Long et al., 1996) and has been proposed to delay differentiation to allow enough cells to bulk up before recruitment into organogenesis (Lenhard et al., 2002). WUS also encodes a homeodomain-containing protein and is essential to specify the stem cells present in the CZ: in wus mutants, the defective CZ cannot keep up with organ recruitment in the PZ, and the meristem is quickly consumed (Mayer et al., 1998). Shoot meristem activity is eventually reinitiated in the axils of the leaves, only to terminate again; this intermittent meristem activity can carry on to the reproductive phase, when incomplete flowers are produced because of premature termination of the floral meristem (Fig. 3). Not only is WUS necessary to maintain the CZ cells, but ectopic expression of WUS is also sufficient to convert cells in organ primordia and even root meristems into cells with characteristics of the shoot meristem CZ (Schoof et al., 2000; Gallois et al., 2004). Although WUS is required to maintain stem cells in all layers of the CZ, it is expressed only in a few L3 cells in the centre of the meristem (Mayer et al., 1998) (Fig. 1). Because its effects are seen in cells that do not express WUS, an intercellular signal is believed to mediate these effects. The size and location of the WUS-expressing region of the meristem are maintained in spite of the continuous cell proliferation within the meristem, implying that WUS expression must be adjusted constantly according to the position of cells in the meristem. Although both STM and WUS are essential for meristem maintenance, the evidence so far suggests that WUS has a more prominent role in the developmental control of meristem size and stability. One of the mechanisms that fine-tune WUS expression is the CLAVATA (CLV) signalling pathway, which represses WUS (reviewed by Carles and Fletcher, 2003). The signal in this case is the secreted polypeptide CLV3, whose biologically active form has recently been shown to be a dodecapeptide corresponding to the C-terminal region of the CLV3 product (Ito et al., 2006). CLV3 is produced in the L1 and L2 layers of the CZ and moves into the inner layers, where it is perceived by a receptor containing the CLV1 and CLV2 polypeptides. Mutations in any of the clv genes have a similar effect: WUS expression increases and the CZ gradually enlarges. WUS activates expression of CLV3 in the overlying region of the meristem and therefore limits its own expression, so it is believed that meristem size is stabilized by the WUS/CLV regulatory loop (Fletcher et al., 1999; Brand et al., 2000). Consistent with this idea, increases in CLV3 rapidly repress WUS expression and shut down meristem activity (Reddy and Meyerowitz, 2005), but, surprisingly, it has been found that the meristem eventually adjusts to changes in CLV3 expression and that plants can grow normally with CLV3 levels ranging from 3-fold lower to 3-fold higher than the wild type (Muller et al., 2006). The implication is that additional mechanisms, independent of the CLV loop, stabilize WUS expression and meristem size. It has also been revealed that CLV3 itself has multiple functions, including the control of cell division rates in the meristem and repression of CZ identity in neighbouring PZ cells (Reddy and Meyerowitz, 2005). A number of additional regulatory genes control the position and number of cells expressing WUS and therefore also have a role in controlling meristem stability. One of them is ULTRAPETALA (ULT) (Carles et al., 2004), which antagonizes WUS; accordingly, ult mutants have enlarged inflorescence meristems and supernumerary floral organs. The HD-ZIPIII genes CORONA (CNA), PHABULOSA (PHAB), and PHAVOLUTA (PHAV) (the latter two better known for their role in controlling organ polarity) also restrict the size of the WUS-expressing domain and meristem size (Prigge et al., 2005; Williams et al., 2005). Chromatin remodelling factors participate in preventing WUS expression outside its normal domain (Kaya et al., 2001; Bertrand et al., 2003; Takeda et al., 2004) and in directly activating it in its normal region (Kwon et al., 2005). Another known positive regulator of WUS is STIMPY (STIP), which encodes a protein of the same family as WUS and is required to maintain WUS expression in the meristem; STIP, however, has a more general role in maintaining cell divisions in the meristem, a role that can be bypassed by exogenously added sucrose (Wu et al., 2005). To integrate all these inputs, WUS could be expected to have complex cis-regulatory sequences. Surprisingly, however, much of the WUS expression pattern can be directed by a short (57 bp) sequence, suggesting that the integration of multiple inputs converges at a relatively simple regulatory element in the WUS gene (Baurle and Laux, 2005). From the work reviewed above, WUS emerges as a central regulator of shoot meristem identity and stability. To balance the recruitment of cells away from the meristem with the supply of new meristem cells, a constant and precise pattern of WUS expression must be maintained within a population of cells that proliferates continuously. This is achieved by multiple regulatory inputs, many of which act to repress WUS outside its normal expression domain. From vegetative to floral meristems The dynamic balance between shoot meristem activity and organ initiation is maintained during most of the plant's growth, but it is eventually tipped in favour of organogenesis during floral development. The suppression of indeterminate growth in the floral meristem depends on floral-specific regulatory genes, whose expression is embedded within a programme of gene expression that is initiated at the transition to reproductive development. During this transition, the vegetative meristem is initially converted into the inflorescence meristem, which then produces floral meristems on its flanks. The transition from vegetative to reproductive development is controlled by multiple environmental and endogenous signals that ultimately converge on key regulators of floral identity: APETALA1 (AP1)/CAULIFLOWER (CAL) and LEAFY (LFY) (reviewed by Komeda, 2004; Blazquez et al., 2006) (Fig. 2). Fig. 2. Open in new tabDownload slide Diagram of the interactions between regulators of floral meristem identity, floral organ development, and determinacy. Activation is indicated by an arrow and repression by a blunted line. The repression of WUS by AG is indicated by a dashed line to emphasize that it is likely to be indirect. AP1 and CAL encode closely related MADS-domain transcription factors that are necessary and sufficient for the transition from inflorescence to floral meristem. In the double mutant ap1-1 cal-1, the primordia produced on the flanks of the inflorescence meristem fail to develop as flowers, and instead function as new inflorescence meristems, which go on to produce their own primordia, repeating the process until a large mass of meristems accumulates at the inflorescence apex, which resembles a cauliflower curd (Bowman et al., 1993). Conversely, overexpression of AP1 is sufficient to convert the inflorescence meristem into a terminal flower (Mandel and Yanofsky, 1995). Consistent with their role in specifying floral meristems, AP1 and CAL are expressed as soon as the floral primordium emerges from the inflorescence meristem (Kempin et al., 1995). LFY also encodes a transcriptional regulator that specifies floral identity and consequently promotes determinacy (Weigel et al., 1992). This has been shown both by loss of lfy function, which converts floral meristems to inflorescence shoots (Schultz and Haughn, 1991), and by the effect of ectopic LFY expression, which converts the inflorescence meristem into a terminal floral meristem (Weigel and Nilsson, 1995). LFY promotes the transition from inflorescence to floral meristem largely by activating AP1 (Mandel and Yanofsky, 1995; Wagner et al., 1999), but subsequently has a central and AP1-independent role in controlling floral development. In addition to being activated by LFY, AP1/CAL are redundantly activated by the FT gene (Ruiz-Garcia et al., 1997) (Fig. 2). Recent evidence suggests that FT is expressed in leaves and that its RNA or protein is transported to the apex as part of a mobile flowering signal that is produced in response to long days (Abe et al., 2005; Huang et al., 2005). After reaching the apex, FT is believed to interact with the bZIP protein FD to activate AP1/CAL (Abe et al., 2005; Wigge et al., 2005). LFY itself is also activated by FT (Huang et al., 2005), besides being activated by gibberellin, which also functions as a signal to promote the shift to reproductive development (Blazquez et al., 1998). As mentioned above, to maintain the indeterminate inflorescence meristem, AP1/CAL and LFY must be activated only in the floral primordia. The expression of LFY and AP1/CAL in the inflorescence meristem is prevented by TERMINAL FLOWER (TFL), which encodes a homologue of FT but has the opposite function, i.e. it antagonizes floral development (Bradley et al., 1997; Kardailsky et al., 1999; Kobayashi et al., 1999) (Fig. 2). In the tfl mutant, ectopic expression of LFY and AP1 transforms the whole inflorescence meristem into a floral meristem. TFL is expressed just below the inflorescence meristem, indicating that it functions non-cell-autonomously to prevent LFY and AP1 expression and the consequent termination of the inflorescence meristem. The interactions between FT, AP1/CAL, LFY, and TFL not only delimit where floral meristems develop, but also establish regulatory loops that ensure a sharp and stable transition to floral identity. After the initial activation by FT/FD, LFY and AP1/CAL reinforce each other's expression: LFY activates AP1 directly (Wagner et al., 1999) and AP1 helps to maintain LFY expression in part by antagonizing TFL (Liljegren et al., 1999). Together, LFY and AP1/CAL then activate the floral development programme. Genetically programmed termination of the floral meristem As described above, AP1 and LFY promote floral meristem identity and consequently determinacy. Termination of the meristem, however, is not a direct effect of these regulatory genes, but part of the flower development programme set in motion by AP1 and LFY. After being initiated on the flanks of the inflorescence meristem, the floral meristem produces four whorls of organs, which in wild-type Arabidopsis typically contain four sepals, four petals, six stamens, and two fused carpels (Fig. 3). The identity of each type of organ is specified by a specific combination of MADS-domain proteins that are believed to form multiprotein complexes, each complex able to control the set of target genes required for the development of a particular organ type (reviewed by Krizek and Fletcher, 2005). One of these MADS-domain proteins is AP1 which, after its earlier role in specifying floral identity, participates in the development of the perianth organs (sepals and petals). LFY also remains active after floral initiation and activates genes encoding MADS-domain proteins required for stamen and carpel development: APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) (Fig. 2). Among these, AG is especially relevant here because it has the additional role of controlling meristem determinacy. In strong ag mutants such as ag-1 or ag-3 (Fig. 3), stamens are replaced by petals (reflecting the organ identity function) and carpels are replaced by a reiteration of the sequence sepals–petals–petals, produced by an active meristem at the centre of the flower (revealing both the organ identity and the determinacy functions of AG). Fig. 3. Open in new tabDownload slide Flowers of wild-type Arabidopsis, ag-3 mutant, and wus-1 mutant. Floral organs are indicated: sepals (Se), petals (Pe), stamens (St), and carpels (Ca). Note the indefinite production of sepals and petals in the ag mutant and the premature termination of flower development in the wus mutant. To terminate the meristem, AG would be expected to antagonize the function of meristem maintenance genes such as STM or WUS. Given the prominent role of WUS in the control of meristem size and stability, an attractive idea would be that AG adds a further negative input to shut down WUS and terminate the floral meristem. Several lines of evidence have confirmed that this is the case. WUS is initially expressed in the floral meristem, but its expression decreases at the stage when AG is activated and disappears by the time carpel primordia are initiated (Mayer et al., 1998). In contrast, WUS remains active in the centre of the indeterminate floral meristem of the ag-1 mutant (Lenhard et al., 2001; Lohmann et al., 2001). As mentioned before, WUS is required for the maintenance of all shoot meristems, including the floral meristem; the premature termination of the floral meristem in the wus-1 mutant (Fig. 3) is opposite to the extended meristem activity in ag mutants. WUS is essential for the indeterminacy seen in ag flowers, because the flowers of the double mutant wus-1 ag-1 look indistinguishable from those of wus-1 (Laux et al., 1996). Conversely, forcing an increase in WUS expression in the floral meristem (using LFY, AG, or AP3 promoters to express WUS) promotes indeterminacy in spite of AG activity (Lenhard et al., 2001). The experiments described above also revealed that WUS activated AG: ectopic WUS not only prolonged meristem activity, but also led to ectopic stamen and carpel development in an AG-dependent way (Lenhard et al., 2001; Lohmann et al., 2001). Supporting the suggestion that it can activate AG, the WUS protein bound in vitro to regulatory sequences present in AG and activated transcription of a reporter containing these sequences in yeast cells (Lohmann et al., 2001). In the yeast experiments, however, transcription was only activated when WUS and LFY were combined, and not by either protein alone. The activation of AG by WUS and LFY combined would explain why AG is activated by WUS only during floral development. It must be noted, however, that WUS must be a redundant activator of AG, which still functions in wus mutant flowers to direct the development of stamens (Fig. 3). Moreover, the AG expression domain is wider than that of WUS, so direct activation by WUS can only occur in a subset of the AG-expressing cells (so far, there is no evidence that the WUS protein moves between cells). Nevertheless, the overall conclusion is that AG functions in a negative feedback loop that terminates WUS expression and meristem activity in the floral bud. While the activation of AG by WUS (at least in part of the floral meristem) appears to result from direct binding of WUS to the AG gene, the repression of WUS by AG is unlikely to be direct. Experiments using mosaic expression of AG have shown that determinacy is lost when AG expression is absent from the L2 layer of the floral meristem (Sieburth et al., 1998), where WUS is not expressed (Mayer et al., 1998). This implies that AG must function across cell boundaries to antagonize WUS, and that coincident expression of AG and WUS in the L3 layer is not sufficient to terminate the flower. Another reason why the repression of WUS by AG is probably indirect is that there is a delay between the activation of AG in the floral bud (stages 2–3) and down-regulation of WUS (stage 6, which occurs ∼12 h later; Smyth et al., 1990). Such a delay would be unexpected if AG functioned in a simple transcriptional cascade to down-regulate WUS. What could be the signal that mediates the repression of WUS by AG? Non-cell-autonomous repression of WUS brings to mind the CLV pathway, so AG might stimulate the negative feedback loop involving CLV3. However, clv mutants have a much milder effect on floral determinacy than ag mutations; in other words, AG is still largely able to terminate the meristem in the absence of CLV function. In addition, the ag-2 clv1-1 double mutant has a stronger increase in floral meristem activity than ag-2 or clv1-1 alone (Clark et al., 1993), indicating that CLV and AG functions converge to limit meristem activity. Therefore, the CLV pathway appears unlikely to play a major role in mediating the determinacy effect of AG. Other genes are known to promote floral determinacy, but are also unlikely to mediate AG functions. One of them is SUPERMAN (SUP), which limits stamen number and is believed to function non-cell-autonomously to control cell proliferation in the centre of the floral meristem (Schultz et al., 1991; Bowman et al., 1992; Sakai et al., 1995). WUS is required for the decreased determinacy seen in the sup mutant, because the meristem termination in wus-1 flowers is epistatic over the increase in organ number seen in the sup-6 mutant (Laux et al., 1996). The interaction between sup-1 and ag-1 mutations, however, is synergistic, indicating that they control meristem activity through parallel pathways (Bowman et al., 1992). The ULT gene, which as described above antagonizes WUS, also limits the number of floral organs. In this case, strong ag mutations are epistatic over ult (Carles et al., 2004), suggesting that the role of ULT in floral determinacy is contained within the functions activated by AG. However, as in the case of clv mutants, the loss of determinacy in ult mutants is much weaker than in ag mutants, showing that ult is largely dispensable for the determinacy function of AG. One way to reveal the downstream effectors that mediate the determinacy role of AG is to use expression arrays to screen for genes regulated by AG. Gomez-Mena et al. (2005) used inducible AG in an ap1-1 cal mutant background to screen for AG targets during the early stages of stamen and carpel development. Among the genes activated by AG in this study was GA4, whose product catalyses the final step in the biosynthesis of bioactive gibberellin, suggesting that one of the early functions of AG is to activate gibberellin biosynthesis. Because gibberellin is believed to antagonize meristem activity (reviewed by Shani et al., 2006), a localized increase in gibberellin levels might mediate the meristem-antagonizing function of AG. To test this idea, it would be necessary to see whether floral meristems become indeterminate in the absence of gibberellin, but this has not been possible so far because even severe gibberellin-deficient mutants such as ga1-3 are believed still to produce low levels of gibberellin (Hedden and Phillips, 2000). Activation of GA4 in the early stages of floral development was confirmed by Wellmer et al. (2006), who used inducible AP1 in the ap1-1 cal-1 background to produce a time-course of changes in gene expression during early floral development. This study, however, also showed activation of genes that encode GA2-oxidases, which inactivate gibberellin. In the vegetative meristem, GA2-oxidases are expressed at the base of the meristem and organ primordia, and have been proposed to prevent diffusion of gibberellin from developing organs into the meristem (Jasinski et al., 2005). Thus it is possible that GA2-oxidases are required to protect the floral meristem from gibberellin produced by the organ primordia before meristem termination is due. The exact location and timing of GA4 and GA2-oxidase expression during early flower development, however, remain unknown. Gibberellin appears unlikely to be the only phytohormone whose levels are relevant to meristem termination. It has been proposed that meristem activity requires at the same time low gibberellin levels and cytokinin biosynthesis, both of which are promoted by STM (Jasinski et al., 2005; Yanai et al., 2005). The positive role of cytokinin in meristem maintenance is also consistent with the finding that one of the functions of WUS is to repress genes that antagonize cytokinin responses (Leibfried et al., 2005). Therefore, it might be expected that termination of the floral meristem would be associated not only with an increase in gibberellin activity, but also with a decrease in the levels or sensitivity to cytokinin. So far, however, no connection has been noted between AG and genes involved in cytokinin production or responses. In summary, during floral development, the repressive input that restricts the location and level of WUS expression is increased by genes such as AG and SUP to promote meristem determinacy. Precisely how these regulators antagonize WUS is still unknown, although phytohormones are plausible candidates to mediate the non-cell-autonomous effect of AG on meristem termination. Relevance to other species Of the regulators of meristem maintenance and determinacy described above, AG has been the most intensively studied from the evolutionary point of view (Theissen et al., 2000; Irish, 2003). One of the interesting twists of AG function in other species is that the organ identity and determinacy functions are not always carried out by a single gene: in maize, these functions are performed by different AG paralogues (Mena et al., 1996). The organ identity and determinacy functions of AG are also separable in Arabidopsis: loss of determinacy, but not of organ identity, occurs in plants with partial loss of AG function caused by antisense RNA or by a weak allele (ag-4, which produces a mutant AG protein with an internal deletion) (Mizukami and Ma, 1995; Sieburth et al., 1995). The fact that the two functions of AG are genetically separable suggests that the organ identity function does not overlap significantly with the meristem termination function. This in turn is compatible with the idea that these functions of AG were acquired at different times during evolution (and were subsequently separated again in maize), although it is not clear what the ancestral function was. The association between AG homologues and reproductive shoot development in gymnosperms has been used to suggest that the role of AG in reproductive organ identity is ancient, but at the same time it has been noted that these reproductive shoots are determinate (Irish, 2003). The large increase in the number of perianth organs seen in ag mutants is reminiscent of double flowers, such as roses and carnations, that have been selected in many species by horticulturalists. The resemblance raises the question of whether similar genes are involved, and in some examples this appears to be the case. In Japanese morning glory, a double mutant flower phenotype first described in the 18th century is caused by mutation of an AG homologue (Nitasaka, 2003). In rose, two AG homologues have been identified, one of which has the same type of internal deletion as the ag-4 allele, which as described above causes loss of determinacy (Kitahara and Matsumoto, 2000). Whether this allele has played a role in the selection of modern double roses, however, remains to be seen. It must be noted that the much increased number of petals and stamens and eventual termination of the floral meristem in roses could also be caused by a localized enlargement of the floral meristem, as seen in the sup or ult mutants. Other key players in meristem identity and determinacy, such as WUS, LFY, and TFL, are also clearly conserved and carry out comparable functions in other species (Stuurman et al., 2002; Schwarz-Sommer et al., 2003; Angenent et al., 2005; Kieffer et al., 2006). Some of the regulatory connections between these genes, however, are variable. In tomato, for example, the TFL orthologue SELF-PRUNING maintains indeterminacy in the inflorescence meristem, but does not do so by antagonizing expression of the LFY orthologue, FALSIFLORA (Pnueli et al., 1998; Molinero-Rosales et al., 1999). The floral-specific repression of WUS may also differ in plants in which the central region of the floral meristem gives rise to the placenta instead of carpel primordia. This is the case in Impatiens, where AG does not appear to be sufficient to terminate the floral meristem (Ordidge et al., 2005; Chiurugwi, 2007). Similarly, it has been noted in petunia that repression of WUS does not coincide with the activation of AG orthologues, but occurs later, when other MADS proteins are expressed in the centre of the flower to specify ovule identity. When expressed during the vegetative phase, these ovule identity genes terminate the meristem, suggesting that they could mediate WUS repression (Ferrario et al., 2006). Other aspects of meristem determinacy are even more clearly divergent, particularly when determinacy is controlled during developmental steps that have no obvious equivalent in Arabidopsis. In maize, the inflorescence meristem does not give rise to floral meristems directly, but instead gives rise to two intermediate types of meristems, the spikelet pair and the spikelet meristem (see review by Bortiri and Hake, 2007). The regulatory genes ramosa1 (Vollbrecht et al., 2005), ramosa2 (Bortiri et al., 2006), and branched silkless1 (Chuck et al., 2002) control the determinacy of the spikelet pair and spikelet meristems, and do not appear to have counterparts that control meristem determinacy in dicotyledonous plants. Another way in which the control of determinacy differs across plants is in its reversibility. In an annual plant such as Arabidopsis, it is clear why commitment to flowering and floral development should be irreversible and followed by death of the plant. In perennial plants, reversion to vegetative growth after the flowering season occurs from meristems that have not been converted to reproductive development (i.e. there is no reversion), but in some cases true reversion occurs, exemplified by plants showing pseudovivipary and by Impatiens shifted to long days after flowering (reviewed by Tooke et al., 2005). Stable developmental transitions are often caused by autoregulatory loops that translate a transient stimulus into a stable regulatory change (Davidson et al., 2002). In Arabidopsis, such autoregulatory loops occur in multiple stages in the control of flowering and determinacy, including autoactivation by FT (Huang et al., 2005), the reciprocal activation of LFY and AP1/CAL mentioned above, and positive autoregulation of AG (Gomez-Mena et al., 2005). In Impatiens, reversion to vegetative development in long days correlates with the interrupted production of a leaf-derived flowering signal (Tooke et al., 2005) and could be due to a failure to establish autoregulatory loops, such as the FT autoactivation loop. In conclusion, to understand evolutionary variation in meristem determinacy and in plant development in general, a future challenge will be to reveal not only the conserved and divergent regulators of meristem activity, but also how diversity is created by changes in the regulatory connections between those genes. Work in my laboratory is funded by the Biotechnology and Biological Sciences Research Council and by the European Union. References Abe M , Kobayashi Y , Yamamoto S , Daimon Y , Yamaguchi A , Ikeda Y , Ichinoki H , Notaguchi M , Goto K , Araki T . 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Bortiri, Esteban;Hake, Sarah

doi: 10.1093/jxb/erm015pmid: 17337752

Abstract All plant organs are produced by meristems, groups of stem cells located in the tips of roots and shoots. Indeterminate meristems make an indefinite number of organs, whereas determinate meristems are consumed after making a specific number of organs. Maize is an ideal system to study the genetic control of meristem fate because of the contribution from determinate and indeterminate meristems to the overall inflorescence. Here, the latest work on meristem maintenance and organ specification in maize is reviewed. Genetic networks, such as the CLAVATA components of meristem maintenance and the ABC programme of flower development, are conserved between grasses and eudicots. Maize and rice appear to have conserved mechanisms of meristem maintenance and organ identity. Other pathways, such as sex determination, are likely to be found only in maize with its separate male and female flowers. A rich genetic history has resulted in a large collection of maize mutants. The advent of genomic tools and synteny across the grasses now permits the isolation of the genes behind inflorescence architecture and the ability to compare function across the Angiosperms. Determinacy, flowers, inflorescence, maize, meristem, spikelets Introduction A remarkable aspect of plants is their ability to produce new organs throughout their life. This capacity is achieved by the action of meristems, groups of self-renewing stem cells located at the tips of shoots and roots (Steeves and Sussex, 1989). Divisions in the meristem give rise to cells with different fates. Cells in the centre of the meristem, the central zone, continue to replenish the meristem, maintaining a defined size. Cells in the periphery of the meristem are in the morphogenetic zone from which organs eventually arise. The balance of these two processes, organogenesis and self-perpetuation, guarantees prolonged activity and such a meristem is said to be indeterminate. In contrast, determinate meristems, such as those that produce flowers, are consumed after making a certain number of organs. The maize inflorescence provides an excellent model to study the developmental control of meristems because it is shaped by both indeterminate and determinate meristems. In addition, maize has a rich genetic history and several mutations affecting discrete stages of inflorescence development have been described (Neuffer et al., 1997). These mutants may have abnormal meristem size or mis-specification of organ identity, or both. The genetics of inflorescence and flower development in maize and other grasses has been recently reviewed by other authors (McSteen et al., 2000; Bommert et al., 2005a). Current knowledge of grass inflorescence development is briefly summarized here and the latest work is reviewed. The focus is on maize, but some discoveries in rice are also included. The basic unit of grass inflorescence architecture is the spikelet, a compact axillary branch that consists of two bracts subtending one to several reduced flowers (Clifford, 1987). Maize is a monoecious plant that produces male flowers on a terminal tassel (Fig. 1A) and female flowers on lateral ears (Fig. 1B), which arise in the axils of vegetative leaves. The tassel initiates several long, indeterminate branches at the base while the ear consists of a single spike with no long branches. The tassel's main spike and branches, and the entire ear, produce short branches (spikelet pairs) that bear two spikelets (Figs. 1C, D, 2A–D). The branches and spikelet pairs arise in the axils of small, undeveloped leaves referred to as bracts. In maize, spikelet and spikelet pair meristems are considered determinate because they produce a defined number of organs (Vollbrecht et al., 2005). Fig. 1. View largeDownload slide Wild-type maize inflorescences. (A) Tassel bearing spikelet pairs (inset) on branches and main spike. (B) Ear showing rows of kernels. (C) Scanning electron microscopy of a tassel primordium initiating spikelet pair meristems. (D) Scanning electron microscopy of an ear. Spm: spikelet pair meristem. Sm: spikelet meristem. Fig. 1. View largeDownload slide Wild-type maize inflorescences. (A) Tassel bearing spikelet pairs (inset) on branches and main spike. (B) Ear showing rows of kernels. (C) Scanning electron microscopy of a tassel primordium initiating spikelet pair meristems. (D) Scanning electron microscopy of an ear. Spm: spikelet pair meristem. Sm: spikelet meristem. Inflorescence meristem size and maintenance Unlike its ancestor teosinte, which is induced to flower by short days, maize undergoes transition from the vegetative to reproductive phase after producing a fixed number of leaves (Irish and Nelson, 1988). The most important known regulator of the transition to reproductive stage in maize is indeterminate1 (id1). The id1 gene encodes a zinc-finger protein that is produced in young leaves. ID1 functions non-autonomously to signal to the shoot apical meristem (SAM) for the transition to a reproductive stage. Mutants of id1 form many more leaves than wild-type maize and show vegetative reversion in the tassel with plantlets arising in male spikelets, often complete with roots (Colasanti et al., 1998). In the eudicots Antirrhinum and Arabidopsis, the genes FLORICAULA (FLO) and LEAFY (LFY) are necessary for the production of flowers (Coen et al., 1990; Weigel et al., 1992). flo mutants produce inflorescence branches in the position of flowers while lfy mutants produce flowers with features of the inflorescence. Maize has duplicate orthologues of FLO/LFY, zea flo/lfy1 (zfl1) and zfl2. Phenotypic analyses of zfl1 and zfl2 mutants shows that their function is somewhat conserved in maize. While single zfl1 and zfl2 mutants have mild phenotypes, the double mutants do not undergo normal transition to reproductive development and have abnormal terminal inflorescences. These are described as ‘tassel ears’ because they are branched inflorescences with female branches enveloped in husk leaves that terminate in a spike of male flowers. Development of spikelets, paleas, and lemmas of zfl1/zfl2 mutants appear to be normal, but flowers show various defects associated with a lack of determinacy and organ identity: normal carpels fail to form and, instead, the female floral meristem produces several ‘carpelloid’ organs with silks and vegetative outgrowths. Similarly, in the tassel, paleas and lemmas are normal but stamens do not develop and are sometimes replaced by lemma or palea-like organs (Bomblies et al., 2003). One of the key processes in meristem maintenance is the CLAVATA (CLV) pathway, originally described in Arabidopsis and named after three genes, CLV1, CLV2, and CLV3. Mutations in these genes cause an enlargement of the shoot and flower meristems, resulting in flowers with extra floral parts (Clark et al., 1993,Clark et al., 1995, Kayes and Clark, 1998). CLV1 and CLV2 belong to a large gene family and both contain a transmembrane domain. CLV1 is a receptor-like kinase (RLK) with a leucine-rich repeat region (LRR), and CLV2 is a receptor-like protein (RLP) with an LRR but, unlike CLV1, it lacks a cytoplasmic tail and has no kinase function (Jeong et al., 1999). CLV3 encodes an extracellular protein that putatively interacts with CLV1 and CLV2 to form a complex that triggers a signalling pathway in the central zone of the SAM resulting in the restriction of stem cell accumulation through the transcription factor WUSCHEL (Carles and Fletcher, 2003). In recent years it has become evident that much of the CLV pathway is conserved between Arabidopsis and grasses. In maize, the gene thick tassel dwarf1 encodes an LRR-RLK and is the putative orthologue of CLV1 (Bommert et al., 2005b). Another maize gene, fasciated ear2 encodes an LRR-RLP, similar to CLV2 (Taguchi-Shiobara et al., 2001). The inflorescence phenotypes of td1 and fea2 mutants are similar. Mutations result in fasciated ears, an increase in spikelet density in the tassel due to a thicker rachis, and an increase in stamen number in male florets. Ears have increased seed row number and are shorter and fatter than wild type. These phenotypes are consistent with the observed increase in size of all inflorescence meristems of td1 and fea2 mutants. A dominant maize mutant that causes fasciation of the ear, Fasciated ear1 (Fas1) (Fig. 3F), has recently been identified and its role in the CLV pathway is being investigated (China Lunde, unpublished results). Mutations in rice orthologues of CLV1 and CLV3 show similar phenotypes to maize td1 and fea2 mutants, especially the enlargement of shoot apical and floral meristems. The FLORAL ORGAN NUMBER1 (FON1) gene, which encodes an LRR-RLK similar to CLV1 and td1, is expressed in vegetative and reproductive meristems and in all floral organ primordia (Suzaki et al., 2004). Despite its broad expression pattern, mutations in FON1 only affect the floral meristems by increasing the numbers of palea, lemma, stamens, and pistils. Mutations in FON4, a gene with sequence similarity to CLV3, cause enlargement of shoot apical, inflorescence, and floral meristems (Chu et al., 2006). Consequently, FON4 mutants have thicker stems, additional inflorescence branches, and extrafloral organs. FON4 is expressed in the central zone of vegetative, inflorescence, and floral meristems, similar to CLV3. Another rice CLV3-like gene, FON2, has a broad expression pattern, but its loss of function only affects the specification of flower organ number, while inflorescences are normal (Suzaki et al., 2004). This suggests that in vegetative and inflorescence meristems, FON4 acts redundantly with FON2 to limit meristem size, but both are needed in floral meristems for proper flower formation. An opposite group of maize mutants fail to produce branches and spikelets. barren inflorescence2 (bif2) mutants have few if any branches in both ears and tassels. Bracts are not affected, as demonstrated by a double mutant with tasselsheath, which makes enlarged bracts (McSteen and Hake, 2001). bif2 encodes a serine/threonine protein kinase co-orthologous to PID of Arabidopsis (P McSteen and S Hake, unpublished data). PID is responsible for the polar localization of the auxin efflux carrier PIN proteins (Friml et al., 2004), and both PIN and PID loss-of-function mutants have inflorescences that lack leaves and axillary branches and flowers (Galweiler et al., 1998; Christensen et al., 2000). kn1 loss-of-function mutants show a mild barren phenotype. Ears often fail to form and if they do, have few kernels and extra silks. Tassels have fewer branches and fewer spikelet pairs (Kerstetter et al., 1997). Mutants of the rice gene LAX PANICLE (LAX) and its maize orthologue barren stalk1 (ba1) also show a decrease in production of axillary branches. Although the inflorescence defects are similar, the effect on vegetative branching differs. ba1 mutant plants fail to produce any tillers while tillering in lax mutants is only mildly affected (Komatsu et al., 2003a; Gallavotti et al., 2004). This difference is probably due to redundancy of LAX and SMALL PANICLE (SPA) since the lax/spa double mutants have a significantly lower numbers of tillers (Komatsu et al., 2003a). The pattern of expression of ra2, a marker of the very early stages of axillary branching (Bortiri et al., 2006), can be used to examine the stage at which a mutant departs from normal development. Expression of ra2 in bif2 tassels show that this mutant initiates fewer than normal meristems, but they are of normal size. Consequently, bif2 inflorescences form some ‘escape’ axillary branches and spikelet pairs (McSteen and Hake, 2001). On the other hand, ba1 tassels have a normal distribution of axillary meristems as determined by the domains of ra2 expression but the size of the meristem anlagen is smaller, indicating that meristems of ba1 mutants fail to grow because they do not reach a critical size (E Bortiri et al., unpublished results). Axillary meristem initiation and determinacy Spikelet pairs are a derived feature, present only in the Andropogoneae tribe, a monophyletic group that includes species such as maize, sorghum, and sugar cane (LeRoux and Kellogg, 1999; Group, 2001). Both spikelet pairs and indeterminate branches originate from axillary meristems; however, unlike other axillary meristems of racemose inflorescences, spikelet pair meristems terminate after the production of two spikelets. For this reason they are considered determinate. The phylogenetic placement of spikelet pairs suggests that a novel genetic programme arose in the Andropogoneae to specify the fate of determinate axillary meristems. The ramosa mutants (ra1, ra2, ra3) provide the starting material to study the molecular basis of the spikelet pair developmental programme. All ra mutants have axillary meristems with increased indeterminacy. Ears of strong ra mutant alleles make branches and, in tassels, spikelet pairs are replaced by indeterminate branches. The long branches at the base of the tassel show increased degrees of branching (Fig. 3A–C). ra1 encodes a Cys2-His2 zinc-finger of the plant-specific EPF subclass and it is expressed at the base of the spikelet pair meristem (Vollbrecht et al., 2005). ra2 encodes a plant-specific LOB domain transcription factor containing a cysteine-rich region and a leucine-zipper-like region. RA2 of maize, sorghum, barley, and rice has a conserved C-terminus domain in addition to the LOB domain (Bortiri et al., 2006). ra2 is expressed in the anlagen of indeterminate long branches, spikelet pair meristems, and spikelet meristems. ra3 encodes a trehalose-6-phosphate phosphatase (TPP) and is expressed at the base of spikelet pair meristems (Satoh et al., 2006). Levels of ra1 transcript are very low in both ra2 and ra3 mutant inflorescences and ra1/ra2, and ra1/ra3 double mutants show an additive phenotype (Vollbrecht et al., 2005; Satoh et al., 2006). In addition, ra2 expression is normal in ra1 and ra3 mutants (Bortiri et al., 2006), and ra3 transcript levels are not changed in ra2 and ra1 mutants (Satoh et al., 2006). These data suggest that ra2 and ra3 are upstream of ra1, but in different pathways, and both are necessary for normal transcription of ra1. This finding would explain why ra1 is not expressed in branch meristems, which have ra2 but lack ra3 expression. Orthologues of ra2 and ra3 have been found in other grasses, including rice, and they have a similar expression pattern in those grass species (Bortiri et al., 2006; Satoh et al., 2006). However, a ra1 orthologue has not been found in rice, or other grasses outside the Andropogoneae (E Vollbrecht, EA Kellogg, and S Malcomber, unpublished results). Judging from the conservation of their sequence and expression patterns, it appears that RA2 and RA3 have been recruited to regulate ra1, a gene whose function arose in the Andropogoneae, and the three act together to impose determinate fate to the spikelet pair meristems. The function of ra2 and ra3 in other grasses is not yet known, but it is speculated that they modulate the extent of branching. Other mutants with loss of determinacy in both tassels and ears are branched silkless1 (bd1), indeterminate spikelet1 (ids1), tassel seed4 (ts4), fuzzy tassel, and the dominant mutant Tassel seed6 (Ts6). bd1 and ids1 encode proteins containing one or two AP2 domains, respectively, and both affect the spikelet meristem (Chuck et al., 1998, 2002), however, the fates of these meristems differ. bd1 mutants show a very striking phenotype in the ear in which the spikelet is replaced by a sterile, indeterminate branch. Each branch produces spikelet pair meristems, similar to the branches of the tassel. In the tassel, the spikelet meristems are also indeterminate, but produce spikelets in a distichous pattern, eventually producing fertile flowers (Chuck et al., 2002). The rice mutant, frizzy panicle, is an orthologue of bd1 with a similar mutant phenotype (Komatsu et al., 2003b). ids1 mutants also have a loss of determinacy in the SM, however, the SM retains its identity, and makes extra flowers and glumes. The ear and tassel are similar, although the ear is not fertile (Chuck et al., 1998). The fuzzy tassel mutant also makes extra flowers per spikelet and multiple sterile flower parts but, unlike ids1, it lacks normal glumes (Fig. 3F). bd1 is expressed only in ears and tassels in a very narrow domain at the flank of spikelet meristems (Chuck et al., 2002). The expression of bd1 is seen as the spikelet pair meristem divides to produce a spikelet meristem. The expression appears first at the base of one spikelet meristem, then at the base of the other. The expression marks a position between glume and spikelet meristem. ids1 is expressed more broadly, although it has a narrow domain of action. It is expressed in SPM and SM and in palea, lemma, and stamen initials. Expression is not seen in the carpel and glumes (Chuck et al., 1998). Ts6 and ts4 have similar phenotypes (Fig. 3D, E). They both have feminized tassels (see discussion below of sex determination), but can be male fertile depending on inbred background (E Bortiri and S Hake, personal observations). Meristem determinacy is affected at different stages of inflorescence development in these two mutants. Analysis by Erin Irish shows that in ts4, spikelet pair meristems are transformed into indeterminate branches bearing additional spikelet pairs, while in Ts6 the pedicellate spikelet meristems makes more flowers (Irish, 1997). SEM analysis suggests that the branching patterns in ts4 are not as regular as seen in ids1 or bd1 mutants (Irish, 1997; G Chuck and S Hake, unpublished results). Sex determination in maize flowers All maize flowers initiate a palea, lemma, two lodicules, three stamens, and three carpels, which fuse to make a single pistil. After initiation, pistil primordia in tassel flowers abort, and stamen primordia in the ear show cell-cycle arrest (Dellaporta and Calderon-Urrea, 1994). In addition, the lower floret of the ear also arrests. As a result, tassel spikelets bear two functional staminate flowers, but in the ear only one pistillate flower develops to maturity. Several mutants have been found that alter sex determination of either male or female flowers. A special class of dwarf plants is andromonoecious, with male flowers in the tassel and perfect flowers in the ear. Most andromonoecious dwarfs are defective in GA biosynthesis (Phinney, 1956), although the dominant mutant, D8 is defective in GA response (Harberd and Freeling, 1989). D8 is homologous to GAI of Arabidopsis and contains an N-terminal deletion of the DELLA domain (Peng et al., 1999). An understanding of how GA regulates sex determination is not known, but the finding that GA levels are 100-fold lower in developing tassel spikelets suggests that low GA levels are required for staminate flower development and higher levels trigger stamen abortion (Rood and Pharis, 1980). An additional effect of GA-deficient mutants in maize is a reduction in tassel branch number. This is most obvious in anther ear1, which encodes an ent-kaurene synthase, an enzyme that catalyses an early step in the GA biosynthesis (Bensen et al., 1995). Thus GA may regulate not only stamen abortion in the ear, but also tassel branching. tassel seed1 (ts1) and ts2 have completely feminized tassels independent of background. In addition, the lower floret of the ear fails to abort. ts2 encodes a short-chain alcohol dehydrogenase that is presumed to lead to pistil abortion (DeLong et al., 1993). Expression of ts2 is not seen in ts1 mutants, thus ts1 is thought to operate upstream of ts2 (Calderon-Urrea and Dellaporta, 1999). ts2 is expressed in all pistil primordia including those of the ear, thus Calderon-Urrea and Dellaporta propose that the upper floret of the ear is protected from the TS2 imposed pistil cell death post-transcriptionally (Calderon-Urrea and Dellaporta, 1999). silkless1 (sk) has an opposite phenotype to ts1 and ts2; the tassel is normal, but the ears are without pistils and are thus sterile. Double mutants with ts2 show that ts2 is epistatic to sk (Jones, 1932; Veit et al., 1991; Irish et al., 1994; Calderon-Urrea and Dellaporta, 1999). These results suggest that SK negatively regulates TS2 in the upper floret of the ear, thus only this floret is protected from the cell death mediated by TS2. Double mutants between ts2 and D8-Mp1 show an additive phenotype with perfect flowers (stamens and pistils) in both ear and tassel (Veit et al., 1991; Irish et al., 1994). This results shows that the TS2 and GA pathways operate independently and the pistil development in the tassel is not dependent on the loss of stamens. Maize floral organ specification The ABC model of flower development was originally described for the eudicots Arabidopsis and Antirrhinum. This model holds that A-class genes specify sepal fate in the first flower whorl, A plus B genes specify petals in the second whorl, B plus C genes give rise to stamens, and C genes alone are needed for carpel development in the fourth whorl (Coen and Meyerowitz, 1991). This model has been expanded recently to incorporate D class genes, responsible for the development of ovules, and E-class genes, which are necessary for normal expression of all the above-mentioned genes. With the exception of the A-class gene APETALA2, all of those genes are members of the MADS-box family of transcription factors and they act by forming dimers and complexes of higher order (de Folter et al., 2005). silky1 (si1) is a MADS-box gene related to Arabidopsis APETALA3 and Antirrhinum DEFICIENS (B-class). Mutations in si1 transform lodicules into bract-like organs reminiscent of paleas or lemmas (Ambrose et al., 2000), and stamens into pistils. This phenotype is similar to a loss of function of B-class genes in eudicots. si1 is expressed in the centre of the floral meristem at the time that the lemma and palea are produced. Later, expression is restricted to the region of the floral meristem that will give rise to lodicules and stamens. The finding that SILKY1 has biochemical properties of B-class proteins and can rescue an Arabidopsis ap3 mutant shows that B-class function is conserved between grasses and Arabidopsis (Whipple et al., 2004). AGAMOUS and PLENA, the Arabidopsis and Antirrhinum C-class genes, specify stamen and carpel identity in the third and fourth whorls and also confer floral meristem determinacy. In ag and ple mutants, flowers produce sepals and petals in a reiterative fashion (Yanofsky et al., 1990; Bradley et al., 1993). Maize and rice have duplicate AG-like genes and they appear to have evolved partial subfunctionalization. Mutations in zag1 cause indeterminate growth of pistil primordia giving rise to more than one silk and undifferentiated masses of tissue inside the ovary (Mena et al., 1996). In the tassel some silks occasionally develop, indicating that pistil abortion is not complete, but the stamens are normal. The lack of effect on stamen identity has been explained by the presence of zmm2, another MADS box gene highly similar to AG and PLE. The expression patterns of zag1 and zmm2 are largely non-overlapping because zag1 transcript levels are higher in pistils while zmm2 is expressed in stamens, suggesting a sex organ specialization that explains the lack of phenotype in tassel flowers of zag1 (Mena et al., 1996). A similar finding has been described in rice. Both OsMADS3, the orthologue of zmm2, and OSMADS58, the orthologue of zag1, are expressed at the site of stamen and pistil initiation. Mutations in OSMADS3 and OSMADS58 have consequences for organ specification, i.e. transformation of stamens into lodicules, and increased number of carpels. However, OSMADS58 appears to have a role in floral meristem determinacy because mutants consistently had indeterminate organ development. In addition, the effects of OSMADS3 mutations on stamen identities are more severe than those of OSMADS58 (Yamaguchi et al., 2006). Although mutations in zmm2 have not yet been isolated, there is now evidence indicating that while zmm2/OSMADS3 and zag1/OSMADS58 contribute to stamen and carpel specification, their contributions are unequal, with the former being more important for stamen identity and the latter for proper carpel development and floral meristem determinacy. Double mutant analysis of si1 and zag1 show the expected phenotype for a BC double mutant, i.e. loss of lodicules and stamen identity and, instead, formation of several whorls of lemma/palea-like organs, indicating the loss of floral meristem determinacy as well as organ identity defects (Ambrose et al., 2000). The origin of the sterile floral parts of grasses has been a mystery for many years. In the last few years it has become evident that the ABC model of flower development applies, with some modifications, to maize and rice. For example, the phenotypes of mutations in maize B- and C-class genes, and their orthologues in rice (Nagasawa et al., 2003), indicates that lodicules and petals share a common ancestor and develop in equivalent whorls of grass and eudicot flowers, respectively (Irish, 2000). The interpretation of lemma and palea, however, is more difficult because homeotic transformations in maize B- and C-class mutants generate leaf-like organs that have characteristics of both (Ambrose et al., 2000), although, in rice, mutations in SUPERWOMAN1 (SPW1) transform lodicules into palea-like organs (Nagasawa et al., 2003). One hypothesis suggests that the lemma arose from reductions and fusions of bracts that formed outside the flower in the common ancestor of grasses and sister lineages (Whipple and Schmidt, 2006). Quantitative trait loci controlling inflorescence development Much of the natural variation in inflorescence shape observed in maize and other grass species are actually due to the cumulative effect of several loci. Therefore, and because of the economical importance of maize and grasses in general, the study of quantitative trait loci (QTL) is an important field of cereal genetics aimed at yield improvement. Quantitative studies have been energized recently by the advancement of genomic tools such as the sequencing of the rice genome and the rapid development of very dense genetic maps in several grass species. As a consequence, QTL mapping with greatly improved resolution power is now a powerful tool to uncover genes that control important traits. Recently, two reports have characterized the contribution of QTL to inflorescence architecture in grasses. Using two sorghum inbred lines with different inflorescences, Brown et al. (2006) mapped QTL for number of branches of first to third order, branch length, and rachis diameter, among others traits. Their findings suggest that branches of different orders are under the control of different loci. Two genes, Dwr3 (br2 in maize), and Sbra2 (the orthologue of maize ra2) mapped to two regions with QTL. Dw3, which encodes a P-glyocoprotein responsible for auxin transport (Multani et al., 2003), mapped to QTL for plant height, and rachis and branch length. The Sbra2 gene closely co-localized to one of two QTL detected for primary branch number. Using maize tassels, Upadyayula and colleagues identified two QTL for higher branch number, five for spikelet pair density on the central spike, and two for spikelet pair density on the branches (Upadyayula et al., 2006). It is interesting that the latter two sets of QTL (spikelet pairs on the central spike versus that on primary branches) are non-overlapping, again indicating that different loci have prominent roles at different stages of development. In ears, QTL were found that control kernel number per row, kernel density, row number per ear, ear diameter, and ear weight. Some genes identified by mutant phenotypes co-localize to QTL. These include ra1, which maps closely to a QTL for branch number, td1, which is in the same region as QTL that control ear weight, tassel branch angle, and spikelet pair density on primary branches, ra2 which maps to the region with a QTL for kernel number per row, and fea2, which localizes to a region with a QTL for branch number. Some QTL were found in regions with no known genes, indicating that QTL mapping can help to identify novel genes involved in inflorescence development (Upadyayula et al., 2006). Conclusions Grasses are the most important crop worldwide, but research to understand the mechanisms of organogenesis has been limited. The development of new techniques in combination with the power of maize genetics has unleashed a new era in grass biology research. Rice has a fully sequenced genome and maize and sorghum are being sequenced. Transformation is now routine in rice and a new organism, Brachypodium distachyon, with a very short life-cycle and small genome, will be fully sequenced shortly (Draper et al., 2001; Vogel et al., 2005). Genomic synteny in the family has been used successfully to clone genes in maize and wheat. Most of the genetic networks of meristem determinacy and organ specification are conserved between maize and rice, with the notable exception of ra1 (Vollbrecht et al., 2005). Future experiments will take advantage of the synteny in the grasses to unravel the mechanisms behind the variation in inflorescence architecture. Fig. 2. View largeDownload slide Maize is a monoecious plant with staminate flowers borne in a tassel and pistillate flowers in ears. (A) A pair of staminate flowers each showing a lemma, a palea, and three stamens (the two lodicules are not seen here). (B) A row of pistillate flowers showing one silk (style) each. (C, D) A schematic representation showing the arrangement of flowering organs in a pair of staminate (C) and pistillate (D) spikelets. Fig. 2. View largeDownload slide Maize is a monoecious plant with staminate flowers borne in a tassel and pistillate flowers in ears. (A) A pair of staminate flowers each showing a lemma, a palea, and three stamens (the two lodicules are not seen here). (B) A row of pistillate flowers showing one silk (style) each. (C, D) A schematic representation showing the arrangement of flowering organs in a pair of staminate (C) and pistillate (D) spikelets. Fig. 3. View largeDownload slide Maize inflorescence and flowering mutants. (A–C) The ramosa mutants have increased indeterminacy of lateral organs, transforming determinate meristems (spikelet pairs) into branches with varying degrees of indeterminacy. (A) ra1. (B) ra2. (C) ra3. (D) tasselseed4 (ts4) is a mutant that fails to abort pistils in tassel spikelets and also shows increased branching. Notice the proliferation of silks due to pistil formation in the tassel (picture courtesy of George Chuck). (E) Ts6 also has feminization of tassels. In this picture silks have been removed to reveal pistil formation. (F) Fascicled ear1 (Fas1), a mutant with fasciated ear and increased kernel row number (picture courtesy of China Lunde). (G) The fuzzy tassel mutant makes extra flowers per spikelet, multiple sterile flowers parts, and lacks normal glumes. Shown is a segment of the mutant tassel rachis on the left compared with the wild type on the right. Fig. 3. View largeDownload slide Maize inflorescence and flowering mutants. (A–C) The ramosa mutants have increased indeterminacy of lateral organs, transforming determinate meristems (spikelet pairs) into branches with varying degrees of indeterminacy. (A) ra1. (B) ra2. (C) ra3. (D) tasselseed4 (ts4) is a mutant that fails to abort pistils in tassel spikelets and also shows increased branching. Notice the proliferation of silks due to pistil formation in the tassel (picture courtesy of George Chuck). (E) Ts6 also has feminization of tassels. In this picture silks have been removed to reveal pistil formation. (F) Fascicled ear1 (Fas1), a mutant with fasciated ear and increased kernel row number (picture courtesy of China Lunde). (G) The fuzzy tassel mutant makes extra flowers per spikelet, multiple sterile flowers parts, and lacks normal glumes. Shown is a segment of the mutant tassel rachis on the left compared with the wild type on the right. The work was supported by an NSF postdoctoral fellowship to EB and NSF grant 0110189 to SH. 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Génard, M;Bertin, N;Borel, C;Bussières, P;Gautier, H;Habib, R;Léchaudel, M;Lecomte, A;Lescourret, F;Lobit, P;Quilot, B

doi: 10.1093/jxb/erl287pmid: 17283376

Abstract The fruit is a hierarchically organized organ composed of cells from different tissues. Its quality, defined by traits such as fruit size and composition, is the result of a complex chain of biological processes. These processes involve exchanges (transpiration, respiration, photosynthesis, phloem and xylem fluxes, and ethylene emission) between the fruit and its environment (atmosphere or plant), tissue differentiation, and cell functioning (division, endoreduplication, expansion, metabolic transformations, and vacuolar storage). In order to progress in our understanding of quality development, it is necessary to analyse the fruit as a system, in which processes interact. In this case, a process-based modelling approach is particularly powerful. Such a modelling approach is proposed to develop a future ‘virtual fruit’ model. The value of a virtual fruit for agronomists and geneticists is also discussed. Complexity, fruit, hierarchy, integrative biology, model, quality, system Introduction Fruit quality involves a set of traits such as fruit size, overall composition and taste, and proportion of edible tissue. The links between environmental control and quality traits have been extensively investigated (Ho, 1998; Bertin et al., 2000; Wu et al., 2002) and progress in the understanding of the genetic control of quality has been boosted by the emergence of molecular markers and mapping technology (Causse et al., 2002; Lecomte et al., 2004). However, these studies have limited explanatory power since they focus on the links between control variables and quality traits, without explicitly considering the underlying mechanisms. In order to progress in our understanding of quality build-up, it is necessary to analyse the fruit as a system characterized by interacting biological processes. For this purpose, the modelling features of a virtual fruit are presented here. Even though every process involved in fruit physiology cannot be integrated in a virtual fruit, a real degree of complexity is needed. Indeed, fruit has the characteristics of complex systems as defined by Wu and Marceau (2002). In particular, it exchanges energy and/or mass with its environment (atmosphere, plant) and it is composed of a large number of diverse components (different sugars and acids, tissues, etc) which interact with each other non-linearly. This complexity has been investigated recently through the development of several models which describe the main processes involved in the build-up of fruit quality (Génard and Lescourret, 2004) and constitute a strong knowledge base for a virtual fruit project. The goal of this article is to propose a general system of organization for a virtual fruit, including the processes to be considered at different scales, and their control. The value of a virtual fruit for agronomists and geneticists is pointed out. How should the fruit system be represented? In order to unravel the complexity of the fruit system, it is proposed to analyse it following a hierarchical approach (Wu and David, 2002). The fruit is composed of one or several seeds, surrounded by a pericarp, itself composed of three tissues: the endocarp which may be lignified as in the case of stone fruits; the mesocarp which is composed of large cells constituting the main part of the fruit; and the epicarp (skin). This structure can be defined at three main levels. At the higher level, the fruit can be considered as an entity exchanging resources (minerals, sugars, and water) with the plant through the phloem and xylem vessels, and gases (water vapour, CO2, O2, and ethylene) with the atmosphere through the skin. At an intermediate level, the tissues can be treated as separate compartments in interaction and/or competition. Each of these tissues has specific characteristics in terms of ontogenesis, growth features, metabolism abilities, and construction costs (Valantin et al., 1999). At the lower level, the fruit is composed of cells subject to different processes such as cell division, cell differentiation, cell endoreduplication, cell expansion, metabolic transformations, and vacuolar storage. A schematic representation of the virtual fruit organization is presented in Fig. 1. Fig. 1. View largeDownload slide Schematic representation of the virtual fruit organization in levels and objects (on the right). For each level, constraints on the lower level are given in the lower part of the box and initiating conditions to the upper level are given in the upper part of the box. The plant, genetic, and environmental controls are also indicated. Fig. 1. View largeDownload slide Schematic representation of the virtual fruit organization in levels and objects (on the right). For each level, constraints on the lower level are given in the lower part of the box and initiating conditions to the upper level are given in the upper part of the box. The plant, genetic, and environmental controls are also indicated. Which level-to-level and process-to-process connections? In a hierarchical approach, the relationship between two levels is asymmetric: a higher level exerts constraints on the level below it, whereas the lower level provides initiating conditions to the higher level (Fig. 1). At the cellular level, cell differentiation governs the tissue's appearance, and the intensity of cell division and endoreduplication, which are important components of sink strength (Ho, 1996; Sugimoto-Shirasu and Roberts, 2003). The biophysical characteristics of cells govern tissue growth, while cell metabolism and vacuolar storage govern the tissue composition in sugars, acids, and secondary compounds. At the tissue level, the biophysical characteristics of a tissue, its carbon and water status, its growth and maintenance respiration costs, and its size in terms of cell number drive the growth and metabolism at the cell level. The area and biophysical characteristics of skin and the gas concentrations in tissues control the gas exchanges of the fruit with the atmosphere. The phloem and xylem conductances and the osmotic and turgor pressures of tissues determine the water, minerals, and carbon exchanges with the plant. The upper level is the fruit, where the different fluxes (phloemic and xylemic fluxes, transpiration, respiration, etc.) drive the water and carbon status as well as gas concentrations in the fruit tissues. It is also at this level that the relative growth of tissues is co-ordinated. In such a hierarchical system, information passes between the different levels. For instance, from the cell to the fruit level, the cell ploidy governs the cell sink strength and the cell division governs the cell number in the tissues. The cell number and cell sink strength govern the tissue sink strength which is an important factor of the phloemic flux into the fruit. Now from the fruit to the cell level, the phloemic flux drives the assimilate supply to the tissues. This supply is shared among the cells according to their individual sink strength, and the cells can divide, grow, and accumulate sugars and acids. A complete consideration of feedback and interaction mechanisms also requires an accurate representation of the links among physiological processes at a given level. The first process to consider in the fruit life time is cell division. After a first phase of division, cell DNA endoreduplication boosts the cell sink strength. Cell expansion then depends on the flux of water and on metabolic transformations. The metabolic transformations, vacuolar storage, and ethylene metabolism, which determine the biochemical composition and maturation, depend on resources, pH, and other processes such as respiration. A schematic representation of the relationships among processes at the cell level is presented in Fig. 2. At the tissue level, the intensities of cellular processes and of their interrelationships vary according to the type of tissue. Tissue-specific properties such as mechanical properties depend on the interaction between tissue differentiation and cell density in the tissue. At the fruit level, it is important to preserve the respective proportions of the tissues throughout the season by applying laws of assimilate distribution among them. Strong relationships between phloem and xylem fluxes and transpiration exist (Huguet et al., 1998; Li et al., 2001; Kawabata et al., 2005), and a virtual fruit should be able to simulate these relationships. Fig. 2. View largeDownload slide Schematic representation of the relationships between processes at the cell level as considered in the virtual fruit, and links with cell number, size, composition, and maturation (italics). Division was assumed to be related to assimilate supply, water uptake, and other processes through its link with cell growth. The cell growth and expansion were dependent on water uptake, assimilate supply, DNA endoreduplication, and metabolism. The sugar and acid metabolism which determines fruit acidity and sweetness interacts with a number of processes such as assimilate supply and respiration. The ethylene metabolism which was related to fruit maturation and accumulation of secondary compounds was assumed to be controlled by respiration. In numerous cases, feedback loops are involved in the link between processes. Models are available for all the processes except for the secondary metabolism. Fig. 2. View largeDownload slide Schematic representation of the relationships between processes at the cell level as considered in the virtual fruit, and links with cell number, size, composition, and maturation (italics). Division was assumed to be related to assimilate supply, water uptake, and other processes through its link with cell growth. The cell growth and expansion were dependent on water uptake, assimilate supply, DNA endoreduplication, and metabolism. The sugar and acid metabolism which determines fruit acidity and sweetness interacts with a number of processes such as assimilate supply and respiration. The ethylene metabolism which was related to fruit maturation and accumulation of secondary compounds was assumed to be controlled by respiration. In numerous cases, feedback loops are involved in the link between processes. Models are available for all the processes except for the secondary metabolism. Which processes to consider and how? Each process to be included in a single complex model must be described in a simple fashion. Following Tardieu (2003), one can assume that the network of gene regulation is co-ordinated in response to internal or external factors, giving way to meta-mechanisms; this allows a simplified mechanistic representation of the processes. Hereafter, existing process-based models which could be included in a virtual fruit will be reviewed. Cell scale Cell division, differentiation, size increase, metabolic transformations, and vacuolar storage are the essential processes to be considered at the cell scale. They determine important quality traits such as fruit size, sweetness, acidity, and nutritional quality. Cell division: Several comprehensive models of mitotic control have been proposed recently (Novac and Tyson, 1993; Gardner et al., 1998; Ciliberto and Tyson, 2000), but their complexity is too great to be integrated in a virtual fruit. A phenomenological population dynamic model was suggested for the analysis of cell multiplication in growing fruits (Bertin et al., 2003). This model involves a phase of exponential cell proliferative activity, followed by a progressive decrease of proliferative activity as cell division proceeds. The model is simply defined by the rate of decrease of proliferative activity and the cell cycle duration. It is simple enough to be included in a virtual fruit. Future modelling should focus on the effect of environmental factors such as temperature, and plant factors such as carbon supply, on cell division intensity. A possible way would be to consider that cells divide when they double their initial size (Morgan et al., 2004; Yang et al., 2006) and thus to relate the intensity of proliferative activity to cell growth that is itself sensitive to temperature and carbon supply. Cell differentiation: Although the control of cellular differentiation has been investigated from a theoretical viewpoint (Cinquin and Demongeot, 2005), realistic and mechanistic models useful in the framework of a virtual fruit seem out of reach. There is clearly a need for models able to describe the phenomenology of cell differentiation as there is for cell division. Cell size: Cell size increase is driven by two distinct processes (Sugimoto-Shirasu and Roberts, 2003): cell growth, which involves the increase in total cytoplasmic macromolecular mass; and cell expansion, which involves the increase of cell volume through vacuolization. It is stated that the amount of cytoplasm a cell can make and sustain is proportional to the amount of DNA in the nucleus (Sugimoto-Shirasu and Roberts, 2003). Amplifying genome size by endoreduplication contributes to increasing cell size (Kondorosi et al., 2000; Cheniclet et al., 2005). Although accumulating data revealed that endoreduplication is developmentally regulated (Joubès and Chevalier, 2000), it is still too poorly understood in plants to be formalized into a mechanistic model. That is why a phenomenological model of cell endoreduplication was developed in fruits (Bertin et al., 2006). The cells, which stopped mitosis, are assumed to be still able to perform an incomplete cycle, including DNA reduplication, but not cell division. The model considers two types of non-proliferating cells: those which become completely inactive and those which participate in incomplete cycles. The latter cells are distributed over several DNA levels according to the number of incomplete cycles they performed: the cells which participated in k incomplete cycles, increased their DNA level 2k times. Such a model is able to simulate the evolution of endoreduplication during fruit development. It has been shown that endoreduplication varies with environmental factors such as light, temperature, and nutrition (Barow, 2006). Further modelling work could consider such environmental effects. Cell expansion consists essentially of an irreversible increase in cell volume (V) which involves the uptake of water and the plastic deformation of the wall. The following equation deduced from Lockhart (1965) was extensively used to study the change in cell and tissue expansion (Tyree and Jarvis, 1982; Dale and Sutcliffe, 1986):  in which ϕ describes the extensibility of the cell walls, Y is the yield threshold pressure, and P is the turgor pressure. Assuming that the fruit mesocarp behaves as a single large cell, the Lockhart equation has been used by Fishman and Génard (1998) to model its volume increase. There is recent evidence that fruit growth is not always regulated by pressure changes in the fruit (Mingo et al., 2003). Lechaudel et al. (2007) proposed that ϕ and Y varied during fruit development, and Mingo et al. (2003) described a non-hydraulic regulation in cell wall extensibility. More research needs to be undertaken on the variation and control of these parameters. Although plastic variations of cell volume are the main determinants of cell expansion, the elastic variations of cell volume due to turgor pressure changes may also be important; they explain the diurnal tissue shrinkage, which has strong effects on the mineral accumulation in the fruit (Lang and Volz, 1998) and plays an important role in the control of physiological disorders. Therefore, a virtual fruit should combine plastic and elastic deformations. A first attempt has recently been made by Léchaudel et al. (2007) who showed by simulation the strong effect of the elastic modulus on mango fruit shrinkage. More knowledge is now needed on the mechanical properties of the cells and on their variation. An important question emerging from the above considerations is how to link cell growth due to endoreduplication and expansion. It may be assumed that the rate of carbohydrate uptake by cells is proportional to their level of endoreduplication, but more quantitative research is needed to test such an hypothesis. Metabolic transformations and vacuolar storage: Taste mainly results from the accumulation of sugars and acids in fruit cells (Stevens et al., 1979). This accumulation can be controlled through the intensity of metabolic transformations and/or through the vacuolar storage capacity. These processes are well known and have been extensively described in the literature (Ho, 1988; Wink, 1993). On this basis, Génard and Souty (1996) and Génard et al. (2003) designed a mechanistic model called SUGAR to predict the changes in sugar composition during peach fruit development. In this model, the unloaded sugars are either directly stored in the cells, transformed into other sugars, or used to synthesize other compounds (structural carbohydrates, etc). Lobit et al. (2003, 2006) designed two models predicting fruit acidity. The first one described citric acid production and degradation during fruit development by representing the fluxes through the citrate cycle. In the second one, malic acid content was assumed to depend mainly on the cell's capacity to store this acid in its vacuole, i.e. the thermodynamic conditions of the transport from the cytosol to the vacuole. Both citric and malic models were combined with a model of pH calculation (Lobit et al., 2002) into a global model able to predict the titratable acidity of the fruit (Habib, 2000). Compounds from secondary metabolism such as vitamins and carotenoids are essential for the nutritional quality of fruits. Their biosynthetic pathways are often known (Carrari and Fernie, 2006), but the lack of knowledge on their regulation strongly limits modelling capacity in the framework of a virtual fruit. It is known that environmental factors and agricultural techniques affect secondary compounds (Dumas et al., 2003; Gautier et al., 2005a), but more quantitative studies are needed before undertaking modelling. There is a real challenge for the future here. As the biosynthesis of these compounds is partly regulated by ethylene (Marty et al., 2005) and because this hormone is strongly related to maturation and growth duration in climacteric fruits, ethylene production has to be considered in a virtual fruit. Génard and Gouble (2005) have recently proposed a model in which the biosynthetic pathway of ethylene is supplied by ATP and regulated by 1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase. The respiration, which is an essential variable of this model, has been modelled according to the concept of growth and maintenance respiration (Thornley, 1970), but a more physiologically based representation of respiration could be used in the future (Cannel and Thornley, 2000; Dewar, 2000). Although the theories underlying these models are largely different from one another, the same general laws have been used for modelling. In particular, the enzymatic reactions have been described according to the ‘rate law’ of chemical kinetics (Chang, 2000), which states that the rate of a reaction is proportional to the reactant. This conceptual homogeneity should favour the integration of the different models into a virtual fruit. Most of the processes involved at the cell scale have been described through published models and can be assembled as shown in Fig. 2. Tissue scale In a virtual fruit focusing on fruit quality, each of the fruit tissues would be intrinsically characterized by its quality criteria (e.g. sweetness, acidity, and nutritional quality would be considered only in the edible tissues such as pericarp; size would be considered for every tissue) and the related important processes. The process intensity should be tissue specific. For example, growth respiration is known to be higher for lignified tissues than for other tissues (Williams et al., 1987) so the parameters of growth respiration models presented at the cell scale need to be tissue specific. In tomato fruit which has been intensively studied, it is now well accepted that the metabolism in the fruit pericarp is different from that in the placenta, and this is even different from that in the columella (Carrari and Fernie, 2006). It has been also shown that the composition of secondary compounds varied in skin, flesh, and seeds of tomatoes (Toor and Savage, 2005). Similarly, biophysical characteristics such as elasticity, plasticity, and conductance to water are tissue specific. In a mechanistic approach, Bussières (1994) proposed a model of water import in tomato fruit in which conductances of the mesocarp and placenta vessels may be different. In the case of the skin, these biophysical characteristics are especially important because they have strong effects on fruit growth (Thompson et al., 1998; Andrews et al., 2000) and on skin cracking (Gibert et al., 2005). More basic research is needed on the variations of composition and biophysical characteristics of the tissues. Another important point is that the tissue cannot be considered as being a simple juxtaposition of independent cells. Indeed, Bertin (2005) showed that there was a clear linear decrease of cell size with the increase of cell number in tomato pericarp, which indicates a density dependence effect. She also found that pericarp mass increases with increasing cell number in spite of competition, which indicates an undercompensating density dependence. It is clear that competition between cells has to be taken into account in a virtual fruit. One important question for the future is to define how to represent this competition. Lescourret and Génard (2003) proposed a multilevel theory of competition between fruit units which could be adapted to the case of competition among cells. Another important question is the impact of cell competition on cell metabolism and thus on tissue composition. Cell competition also potentially affects the biophysical characteristics of the tissue (e.g. the cell size has an effect on tissue texture; Sams, 1999). There is clearly a need for research on these different points and on how to model the effect of cell competition on cell growth, composition, and biophysics. Fruit scale Important quality traits are manifested at the fruit scale. This is especially true for fruit size, dry matter content, and percentage of edible tissues. Fruit size and dry matter content result from the exchange of resources with the plant and the atmosphere. The carbohydrate supply has often been modelled according to the source/sink concepts (Baumgaertner et al., 1984; Gutierrez et al., 1985; Gary et al., 1998, Léchaudel et al., 2005). An important variable of these models is the fruit demand for carbohydrates, which is positively correlated to the seed number (Lescourret et al., 1998). In some models, a more mechanistic approach has considered the processes involved in sugar unloading from the phloem to the fruit tissues. For example, Fishman and Génard (1998) and Bruchou and Génard (1999) modelled sugar phloem unloading through mass flow, diffusion, and active transport. This approach should be adopted in the virtual fruit to relate water and carbohydrate fluxes mechanistically. The phloem unloading pathway (symplasmic or apoplasmic) is an important aspect of the regulation of sugar unloading, with a possible shift from a symplasmic to apoplasmic pathway during fruit development, as shown for grape berry (Zhang et al., 2006). More modelling effort is needed on this shift. The fluxes of water in xylem and phloem tissue have been extensively modelled at the scale of the vessel (Tyree et al., 1974) or the plant (Boersma et al., 1991; Doussan et al., 1998), but only a few fruit models describe them (Bussières, 1994, 1995; Fishman and Génard, 1998). The differences in water potential, osmotic potential, and turgor pressure between the stem and the fruit have been considered as the driving forces of the water import rate, and the role of pedicel resistance and variation in xylem functionality has been emphasized recently (Bussières, 2002; Drazeta et al., 2004). The water balance of the fruit has been calculated considering water uptake and transpiration per unit of fruit area as constant (Lee, 1990) or variable (Bussières, 1993; Génard and Huguet, 1996; Fishman and Génard, 1998; Léchaudel et al., 2004). These works constitute a basis for modelling the water balance of a virtual fruit. The xylem ‘backflow’ from the fruit to the plant is an important aspect of fruit water balance which is now well known (Lang and Thorpe, 1989; Keller et al., 2006), although it still requires a modelling effort. In a postharvest context, the gas (O2, CO2, ethylene, and water vapour) fluxes through the skin have been related to concentration gradients, according to physical laws of gas diffusion (Ben-Yehoshua and Cameron, 1989). This physical approach could be adequate in the framework of a virtual fruit. A model of fruit surface conductance to water vapour has been proposed recently (Gibert et al., 2005) which could improve the prediction of fruit transpiration as a function of fruit growth. More generally, fruit surface conductance to gas needs to be modelled in the future because it has a strong implication for fruit quality through its effect on fruit physiology and ripening (Paul and Srivastava, 2006). Combining the model of carbon and water balance proposed by Fishman and Génard (1998), the model of Bussières (2002) on pedicel resistance, and that of Gibert et al. (2005) on fruit surface conductance to water vapour would provide a sound basis to model the water and carbon exchanges with the plant and the atmosphere. Biomass allocation to the fruit tissues (which determines the percentage of edible tissues) also needs to be considered at the fruit scale. However, to our knowledge, there is no mechanistic model of biomass allocation into fruit tissues. Establishing empirical laws relating the size of any given tissue to that of another one or to that of the whole fruit may be a solution. Such laws are common in biology, mainly in the framework of allometric growth (Causton and Venus, 1981; West et al., 1997). For example, the dry masses of each fruit tissue and the dry mass of the fruit have been linked by allometric relationships (Léchaudel et al., 2002; Lescourret and Génard, 2005), which seem to be independent of assimilate supply, but vary greatly according to the genotype (Quilot et al., 2004). Derivation of allometric equations could be used in a virtual fruit to distribute the carbon unloaded to the fruit between its different tissues as done by Lescourret and Génard (2005). Which controls for a virtual fruit? Fruit development is controlled by three classes of factors: environmental, genetic, and plant (Fig. 1). The first class includes temperature, light, and air humidity. Such factors influence important processes such as cell cycle duration (Francis and Barlow, 1988), photosynthesis and respiration (Pavel and Dejong, 1993), transpiration (Leonardi et al., 1999), phloemic transport (Guichard et al., 2005), and metabolism (Yamada, 1994). The influence of the main environmental factors on plant physiology has classically been included in most ecophysiological models. Except for temperature effects (Bussières, 1995, 2002), this has been done more rarely for fruit, though its microclimate strongly affects its quality (Gautier et al., 2005b). Recent advances on the climate perceived by individual plant organs (Chelle, 2005) suggest that fruit temperature models will soon be available for coupling with a virtual fruit. A large body of literature shows the importance of genetic factors in the control of fruit quality (Dirlewanger et al., 1999; Causse et al., 2002). Genetic control has recently been integrated in some ecophysiological models (Yin et al., 2004) including fruit models (Quilot et al., 2004). Considering that only a limited number of genes present in the fruit are involved, or that groups of genes have co-ordinated actions (Tardieu, 2003), the genetic control could be defined by a limited set of genotype-specific parameters of the main modelled processes (a few tens to hundreds, compared with the thousands of genes controlling fruit development). A first attempt at genotypic specification for a fruit model has been made by looking for quantitative trait loci (QTLs) of parameters (Quilot et al., 2005b). With the rapid progress of functional genomics, a further step would be, according to Struik et al. (2005), to combine fruit models with genes and gene regulatory networks underlying these QTLs for key subprocesses controlling fruit quality traits. The plant controls fruit quality through resource and hormonal controls. Source–sink relationships for carbon in plants and their effect on fruit growth (Starck et al., 1990; Ho, 1992, 1996) and fruit metabolism (Archbold, 1999; Souty et al., 1999) are well documented. Water relations in plants and their effects on fruit quality have also been intensively studied (Ho et al., 1987; Mitchell et al., 1991). Carbon and water allocation within a plant depends on complex rules linking source organs (leaves for carbon and roots for water) and sink organs such as fruits. The complexity essentially comes from regulations due to feedback mechanisms and interactions among different functions (e.g. between sink growth and leaf photosynthesis). Plant models of carbon and water allocation able to simulate this complexity have been developed during the last 30 years (Le Roux et al., 2001; Van Ittersum et al., 2003), with a recent focus on 3D virtual plants (Dauzat et al., 2001; King, 2005). These models are powerful tools to analyse how the plant affects the carbon and water accumulation in fruit. The phloem sap sugar concentration and water potential in the plant would be the main plant variables controlling a virtual fruit. The virtual fruit will need to be connected to a plant model of carbon and water balance able to predict these two variables. Plant hormonal control of fruit quality is very difficult to take into account even if it has been extensively investigated. More quantitative research is needed in this field. The virtual fruit as a tool for agronomists and geneticists Using a virtual fruit in conjunction with models of plant functioning would make it possible to simulate the effects of agronomical factors, such as water availability, on fruit quality. This has been illustrated in the first version of the virtual fruit by Lescourret and Génard (2005). This first virtual fruit was developed by adapting and integrating into one complex system three existing process-based models describing fruit dry mass growth (Lescourret et al., 1998), sugar accumulation in the flesh (Génard et al., 2003), and fruit fresh mass growth (Fishman and Génard, 1998) by supply–demand, compartmental, and biophysical approaches, respectively. A general scheme of this first virtual fruit is given in Fig. 3. Fig. 3. View largeDownload slide General scheme of the Lescourret and Génard (2005) virtual fruit. Underlined variables are external variables, arrows are data flows, and dashed lines represent feedback information. (This figure has been redrawn from Lescourret F, Génard M. 2005. A virtual peach fruit model simulating changes in fruit quality during the final stage of fruit growth. Tree Physiology25, 1303–1315, and reproduced by kind permission of Heron Publishing.) Fig. 3. View largeDownload slide General scheme of the Lescourret and Génard (2005) virtual fruit. Underlined variables are external variables, arrows are data flows, and dashed lines represent feedback information. (This figure has been redrawn from Lescourret F, Génard M. 2005. A virtual peach fruit model simulating changes in fruit quality during the final stage of fruit growth. Tree Physiology25, 1303–1315, and reproduced by kind permission of Heron Publishing.) The model of Lescourret et al. (1998) describes the carbon balance of a fruit-bearing stem. The daily available pool of carbon assimilates consists of leaf assimilation plus, eventually, the carbon mobilized from reserves. The leaf photosynthesis may be affected by a feedback inhibition through the size of leaf reserves. Carbon is allocated according to organ demands and priority rules. Maintenance respiration costs have first priority, whereas vegetative and reproductive growth are given second and third priorities, respectively. The carbon demand for fruit growth emphasizes the role of fruit history, in terms of both sink size and sink activity. It also emphasizes the role of developmental time. The incoming carbon flow is shared between the flesh and the endocarp+seed according to an equation derived from an empirical relationship between endocarp+seed dry mass and total fruit dry mass. Using the model of Génard et al. (2003), the fruit flesh carbon is then partitioned into several compounds: four sugars (sucrose, sorbitol, glucose, and fructose), other fruit compounds globally considered (starch and structural carbohydrates), and respired CO2. The rates of change of the amounts of carbon in the four sugar compounds are described through a set of differential equations. According to the Fishman and Génard (1998) model, the flow of water to the fruit is driven by differences in hydrostatic and osmotic pressures between xylem or phloem and fruit. The fruit osmotic pressure induced by sugars is calculated by means of the ‘sugar accumulation’ model. The hydrostatic pressure is calculated assuming that the growth predicted by the Lockhart equation is equal to the water inflows minus fruit transpiration. Fruit transpiration is calculated from skin conductance to water vapour and the vapour pressure difference between the air and the fruit. The inputs of this first virtual fruit are weather data (i.e. global radiation, temperature, and air relative humidity), stem water potential, and leafy shoot and fruit number on the stem (Fig. 3). Although each of the three initial submodels has already been validated, the virtual fruit was evaluated as a whole. It was successfully done for several quality traits (fruit fresh mass, proportion of flesh in the total mass, dry matter content of flesh, sucrose, sorbitol, glucose, and fructose concentrations in the flesh, and sweetness index) on peach fruit using experiments from three different years. The relative root mean squared error of prediction calculated using a cross-validation approach (Wallach et al., 2001) was smaller than 20% in most cases (Lescourret and Génard, 2005). Moreover, a simplified version (for sugars only) was tested on 87 different peach genotypes, and the error of prediction was also smaller than 20% in most cases (Quilot et al., 2005a). Virtual experiments using this model showed that applying a water stress to peach trees after a period of normal water supply resulted in an intense slowing down of growth. During the same period, the fruits grown under continuous water stress experienced continuous growth. This suggests that continuously stressed fruit may adapt to drought. In the virtual fruit, this adaptation was based on a ‘sugar signal’, i.e. the increase of sugar concentration during the stress period being able to promote the growth. Thus it appears that a virtual fruit can develop complex adaptations. Combining a virtual fruit with a virtual plant could give rise to new tools for agronomists in charge of technical innovations to be able to improve fruit quality. Virtual experiments in which several agronomic factors could be combined would make it possible to find the best technical combinations in terms of fruit quality. Geneticists have made much progress in the last decade thanks to molecular marker technologies, which enable dissection of the variation of traits into effects of QTLs. However, they still encounter two major difficulties. First, the quality traits that are breeding targets result from many overlapping processes and are thus controlled by many genes. The second difficulty results from the fact that these characters are under the influence of the environment. This often results in strong genotype×environment interactions which make the genetic analysis and its application to breeding difficult. Studying a quantitative trait via a virtual fruit makes it possible to deal with both difficulties simultaneously. Instead of directly looking for QTLs controlling this trait, it is more efficient to look for QTLs controlling model parameters because they are assumed to be independent of the environment. This approach was applied by Quilot et al. (2005a, b) to peach fruit. Through the study of the co-locations between QTLs of parameters and traits, physiological meanings of QTLs of traits have been proposed. For instance, on the first linkage group (LG1), QTLs for fresh fruit mass are located in the same region as QTLs for parameters involved in sugar metabolism and early fruit growth. On LG2, LG4, and LG7, they are co-located with QTLs for parameters involved in water fluxes in the fruit (hydraulic conductance and permeation coefficient of the fruit surface to water vapour). Quilot et al. (2005a, b) also observed pleiotropic effects. For instance, QTLs for parameters involved in sugar metabolism were located in the same region as a QTL for total sugar concentration on LG1 and in the same region as a QTL for dry matter content of flesh on LG3. Moreover, when parameters are well predicted by QTLs, it is then possible to predict, through the virtual fruit, the quality traits (i.e. fruit mass) of any given genotype in a given environment. Assuming for instance that one QTL could be manipulated to increase the intensity of sucrose uptake by the fruit, the Lescourrret and Génard (2005) ‘Virtual Fruit’ model predicts an increase of both carbon and water stored in the fruit and thus an increase of fruit size (Fig. 4). It also predicts an increase in fruit sugar concentration and leaf photosynthesis, and a decrease in the leaf carbohydrates reserve pool (Fig. 3). Indeed, the change in assimilate uptake by the fruit alters not only the fruit function but also the source–sink relationship within the plant. This example shows that a virtual fruit can be a powerful tool for analysing the response of genotypes, in terms of fruit quality and plant functioning, to contrasting environmental conditions and to look for the genotype adaptation. This opens up the way to new breeding approaches based on simulation results. Fig. 4. View largeDownload slide The Lescourret and Génard (2005) virtual fruit model was used to analyse the effect of a genetic change in mean sugar uptake by the peach fruit on the dynamics of its quality [fruit mass and sugar concentration (g 100 g−1 fresh mass), carbohydrate reserve concentrations in the leaves (g 100 g−1 dry mass), and leaf photosynthesis] from 81 d to 139 d after full bloom. That model is a combination of a ‘carbon submodel’, describing the management of carbon in the fruit-bearing shoot and the growth in dry mass of the vegetative and fruit parts, a ‘sugar submodel’ that partitions the carbon into the four sugars present in the peach fruit flesh, and a ‘water submodel’ describing the management of water in the fruit. Fig. 4. View largeDownload slide The Lescourret and Génard (2005) virtual fruit model was used to analyse the effect of a genetic change in mean sugar uptake by the peach fruit on the dynamics of its quality [fruit mass and sugar concentration (g 100 g−1 fresh mass), carbohydrate reserve concentrations in the leaves (g 100 g−1 dry mass), and leaf photosynthesis] from 81 d to 139 d after full bloom. That model is a combination of a ‘carbon submodel’, describing the management of carbon in the fruit-bearing shoot and the growth in dry mass of the vegetative and fruit parts, a ‘sugar submodel’ that partitions the carbon into the four sugars present in the peach fruit flesh, and a ‘water submodel’ describing the management of water in the fruit. Conclusion Fruit quality traits are quantitative, complex, and controlled by environment and genes. The variation of these traits with environment and genotype has been traditionally studied following a quantitative genetics approach such as correlation and marker–trait association analysis. The combined physiological and genetic approach of our virtual fruit research is more able to take into consideration the internal regulations and their genetic and environmental controls. The application of the first version of a virtual fruit to agronomic and genetic questions is encouraging and is an important step towards quality trait modelling of fruits using different biological scales for explanation (Struik et al., 2005). Gutiérrez et al. (2005) proposed a systems approach to understand plant biology from the molecular to the ecological scale. This systems biology is based on the integration of existing knowledge about biological components and building of the system as a whole. In accordance with this viewpoint, Yin et al. (2004) proposed that a new generation of models should enable us to narrow the gap between genes and complex phenotypes. Concerning fruit quality, this new generation is really needed to accompany the advances in fruit genomics (Baxter et al., 2005). We believe that a virtual fruit able to generate complex phenotypes thanks to its emerging properties belongs to this new generation. Abbreviation Abbreviation QTL quantitative trait locus We thank G Wagman and A Hall for revising the manuscript. 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Aarnes, H; Eriksen, AB; Petersen, D; Rise, F

doi: 10.1093/jxb/erl247pmid: 17210989

Abstract 14N-NMR and 31P-NMR have been used to monitor the in vivo pH in roots, stems, and needles from seedlings of Norway spruce, a typical ammonium-tolerant plant. The vacuolar and cytoplasmic pH measured by 31P-NMR was found to be c. pH 4.8 and 7.0, respectively, with no significant difference between plants growing with ammonium or nitrate as the N-source. The 1H-coupled 14 NH4+ resonance is pH-sensitive: at alkaline pH it is a narrow singlet line and below pH 4 it is an increasing multiplet line with five signals. The pH values in ammonium-containing compartments measured by 14N-NMR ranged from 3.7 to 3.9, notably lower than the estimated pH values of the Pi pools. This suggests that, in seedlings of Norway spruce, ammonium is stored in vacuoles with low pH possibly to protect the seedlings against the toxic effects of ammonium ( NH4+ ) or ammonia (NH3). It was also found that concentrations of malate were 3–6 times higher in stems than in roots and needles, with nitrate-grown plants containing more malate than plants grown with ammonium. Ammonium, cytoplasmic pH, in vivo 14N-NMR, in vivo 31P-NMR, malate, organic acids, Picea abies (L.) Karst., vacuolar pH Introduction Conifers and ericaceous plants adapted to acid soils with low or no nitrification have a preference for NH4+ as the main nitrogen source. These plants can tolerate high concentrations of NH4+ and have been found to possess a reduced capacity to use NO3− (Kronzucker et al., 1995, 1997; Forde and Clarkson, 1999; Britto et al., 2001a; Britto and Kronzucker, 2002; Schjoerring et al., 2002), although in Picea abies and Vaccinium myrtillus short- and long-term field studies have shown the same capacity for NH4+ and NO3− utilization (Persson et al., 2003). Sources of NH4+ in soils are the ammonification of organic nitrogen and/or atmospheric wet- and dry-deposition of NH4+ . High concentrations of NH4+ can be harmful to non-adapted plants and toxicity can be avoided by rapidly converting NH4+ to amino acids in the roots or by sequestering NH4+ in a vacuolar reservoir. NH4+-toxicity syndromes are mostly found in NO3−-adapted plants and are still not well understood (Mehrer and Mohr, 1989; Britto et al., 2001a, b; Britto and Kronzucker, 2002). NH4+ uptake by the roots is accompanied by the uptake of monovalent anions or the release of H+-ions resulting in acidification of the soil (Marschner et al., 1991; Forde and Clarkson, 1999; Schjoerring et al., 2002) which can be one of the causes of ammonium toxicity (Britto and Kronzucker, 2002). Most plants are susceptible to NH4+ toxicity when NH4+ is the only nitrogen source, an exception is, for instance, wetland rice that can tolerate very high concentrations of NH4+ when growing on flooded anoxic soil with low nitrification (Britto et al., 2001b). Primary assimilation of NH4+ into amino acids is catalysed by the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT). Interestingly, although glutamine synthetase has a Km for NH4+ at μmolar concentration, NH4+-adapted plants have been found to contain millimolar concentrations of NH4+ (Lee and Ratcliffe, 1991; Aarnes et al., 1995; Britto et al., 2001a). We have earlier reported high concentrations of NH4+ in all parts of spruce seedlings quantified by 14N-NMR and 15N-NMR and have found indications that NH4+ can be stored in acidic compartments (Aarnes et al., 1995). In vivo31P-NMR (Ratcliffe, 1994) can be used to estimate intracellular vacuolar and cytoplasmic pH (Kime et al., 1982; Martin et al., 1982; Gerendás et al., 1990; Katsuhara et al., 1997; Espen et al., 2004; Pfeffer et al., 2004). In vivo14N-NMR and 31P-NMR were used to compare the pH in vacuoles and cytoplasm of spruce seedlings growing with NH4+ or NO3− as the nitrogen source. In addition, the amount of malate and citrate was monitored in different parts of the plants since these organic acids can play a role in cellular pH homeostasis (Britto and Kronzucker, 2005). Materials and methods Plant material Seeds of Norway spruce [Picea abies (L.) Karst.] provenance B2 were grown for 3 weeks in vermiculite/perlite at an air temperature of 22 °C and a relative humidity of 60% in a climate-conditioned growth room in continuous light (250 μmol m−2 s−1, using metal halide lamps, Kolorarc 400 W from General Electric Co., USA) as described by Aarnes et al. (1995). Groups of 20 plants were removed from the growth medium, rinsed in distilled water, and transferred to drams glass containing 10 ml nutrient solution with different concentrations of nitrogen (5–25 mM N), supplied as (NH4)2SO4 or KNO3. The plants were grown for 2 d in the different nutrient solutions in the same light and temperature regime as described above. The pH of the nutrient solutions was determined with a glass electrode (Radiometer, Denmark). NMR One hundred seedlings were divided into roots, stems, and needles that were wrapped in Teflon tape and put into a 10 mm NMR tube with circulating air-bubbled buffer (25 mM MES pH 6.0, 0.01 mM CaSO4) at 20 °C (Lee and Ratcliffe,1983). 31P-NMR and 14N-NMR spectra were obtained by using a Bruker DPX300 spectrometer with a BBO 10 mm probe head. 31P-NMR spectra were recorded at 121.51 MHz, with an acquisition time of 0.85 s, a relaxation delay of 1 s, and a pulse angle of 90°. Chemical shifts were compared to 17 mM methylene diphosphonic acid in a capillary tube as external standard resonating at 16.9 ppm compared with H3PO3. The standard calibration curve that related chemical shifts of intracellular 31P resonances to different pH values was made according to Martin et al. (1982) and Roberts et al. (1981). The intracellular pH of the Pi pools was determined by using the chemical shifts of the 31P-NMR spectra. 14N-NMR spectra were recorded at 21.68 MHz, a 70° pulse angle, a recycle time of 4 s, and 1024 scans. The pH value of the ammonium-containing compartments was determined by using the pH-dependent 1H-coupled line shape of 14 NH4+-1H-coupled spectra. Solutions with 25 mM ammonium-acetate at different pH were used as pH standards. NO3− was used as a standard (0 ppm). Extraction and determination of malate and citrate Groups of 20 plants were washed in distilled water, divided into roots, stems, and needles and homogenized by grinding in a mortar with 6 ml ice-cold distilled water. The homogenates were centrifuged for 15 min at 10 000 g (4 °C). The supernatant was used to measure the pH and the concentrations of organic acids by a coupled enzymatic UV-assay (340 nm) for food analysis using test combinations for L-malic acid, citric acid, and oxalic acid from Boehringer Mannheim/Roche. Results A typical in vivo31P-spectrum of roots of Norway spruce seedlings with peaks for vacuolar and cytoplasmic Pi, sugar phosphates, and nucleoside phosphate is shown in Fig. 1. Similar spectra were obtained from stems and needles. The spectra give information about the pH in vacuoles and in the cytoplasm by comparing 31P-NMR Pi signals with a calibration curve showing the pH dependence of the Pi chemical shift (ppm). A pH of c. 4.8 in vacuoles and c. 7.0 in the cytoplasm were found, with no significant difference in vacuolar and cytoplasmic pH for plants growing with NO3− or NH4+ (Table 1). Fig. 1. Open in new tabDownload slide In vivo31P-NMR spectrum of roots of 3-week-old Norway spruce seedlings grown with 10 mM (NH4)2SO4 as nitrogen source. (1) Methylene diphosphonic acid (standard). (2) Sugar phosphates. (3) Vacuolar Pi. (4) Cytoplasmic Pi. Table 1. Cytoplasmic and vacuolar pH estimated from chemical shifts of 31P-NMR spectra of roots, stems and needles of 3-week-old Norway spruce seedlings growing on either (NH4)2SO4 or KNO3 as N-source (10 mM N) N-source pH Roots Stems Needles (NH4)2SO4 Vacuolar Pi 4.85±0.57 4.93±0.60 4.90±0.85 (NH4)2SO4 Cytoplasmic Pi 6.98±0.15 7.10±0.15 6.95 KNO3 Vacuolar Pi 4.68±0.08 4.91±0.53 4.70±0.28 KNO3 Cytoplasmic Pi 7.03±0.15 7.13±0.15 7.00±0.11 N-source pH Roots Stems Needles (NH4)2SO4 Vacuolar Pi 4.85±0.57 4.93±0.60 4.90±0.85 (NH4)2SO4 Cytoplasmic Pi 6.98±0.15 7.10±0.15 6.95 KNO3 Vacuolar Pi 4.68±0.08 4.91±0.53 4.70±0.28 KNO3 Cytoplasmic Pi 7.03±0.15 7.13±0.15 7.00±0.11 Values are means ±SD, n=1–3. Open in new tab Table 1. Cytoplasmic and vacuolar pH estimated from chemical shifts of 31P-NMR spectra of roots, stems and needles of 3-week-old Norway spruce seedlings growing on either (NH4)2SO4 or KNO3 as N-source (10 mM N) N-source pH Roots Stems Needles (NH4)2SO4 Vacuolar Pi 4.85±0.57 4.93±0.60 4.90±0.85 (NH4)2SO4 Cytoplasmic Pi 6.98±0.15 7.10±0.15 6.95 KNO3 Vacuolar Pi 4.68±0.08 4.91±0.53 4.70±0.28 KNO3 Cytoplasmic Pi 7.03±0.15 7.13±0.15 7.00±0.11 N-source pH Roots Stems Needles (NH4)2SO4 Vacuolar Pi 4.85±0.57 4.93±0.60 4.90±0.85 (NH4)2SO4 Cytoplasmic Pi 6.98±0.15 7.10±0.15 6.95 KNO3 Vacuolar Pi 4.68±0.08 4.91±0.53 4.70±0.28 KNO3 Cytoplasmic Pi 7.03±0.15 7.13±0.15 7.00±0.11 Values are means ±SD, n=1–3. Open in new tab Figure 2 shows in vivo14N-NMR spectra of NH4+ in roots, stems, and needles compared with spectra of different NH4+-pH standards. At increasing acidity intensity the five peak signal at around 355 ppm in the 14 NH4+-NMR spectra becomes increasingly pronounced. Above pH 4 the 1H-coupled 14 NH4+-NMR signal consists of only one peak. Using the right peak of the quintet signal as a reference, the pH in roots, stems, and needles was estimated to be 3.9, 3.9, and 3.7, respectively. The 14N-NMR spectra also showed a peak from the α-amino nitrogen in amino acids at c. −335 ppm. The amino acid nitrogen peak was more pronounced in stems and needles than in roots. Fig. 2. Open in new tabDownload slide 1H-coupled in vivo14N-NMR spectra of roots, stems, and needles of 3-week-old Norway spruce seedlings growing with 10 mM (NH4)2SO4 as nitrogen source. 14N-NMR spectra pH standards (25 mM ammonium acetate) at pH 3.04, 3.35, 3.59, and 3.82. The amounts of malate and citrate in water extracts of roots, stems and needles are shown in Table 2. Concentrations of malate were 3–6 times higher in stems than in roots and needles. Malate concentrations were significantly lower in roots and stems of NH4+-fed plants compared to plants grown with NO3− or without nitrogen. Except for stems of NO3−-fed plants, there were no significant differences between plants growing on NO3− or water (Table 2). Concentrations of oxalate were relatively high with 29.6±2.4; 19.8±0.9, and 40±4.8 μmol g−1 FW in needles, stems and roots, respectively. Oxalate concentrations did not appear to be affected by the nitrogen source. Based on a dry weight of 15% the concentrations of NH4+ in spruce plants were recalculated from the 14 NH4+ NMR spectra to be 5–54 mM for plants growing in nutrient solutions with 0.5–50 mM NH4+ ⁠. The highest content of ammonium was found in roots and needles of plants growing in nutrient solution containing 2.5 mM (NH4)2SO4. An increase in the nitrogen level of the nutrient solution up to 50 mM NH4+ did not further affect the NH4+ content of the plants. Similar results were obtained from 15N NMR spectra from plants growing on 15 NH4+ as shown previously (Aarnes et al., 1995). Recalculation of these data gave slightly lower amounts of 15 NH4+ in roots ranging from 9–23 mM, and up to 13 mM in the stems. However, very low concentrations of 15 NH4+ were found in needles. In this case the highest amount of NH4+ was found in roots of plants growing in a nutrient solution with 2.5 mM 15NH4NO3. Table 2. Concentrations of malate and citrate in roots, stems, and needles of 3-week-old Norway spruce seedlings growing on either (NH4)2SO4 or KNO3 as N-source (10 mM N) N-source Acid Roots (μmol g−1 FW) Stems (μmol g−1 FW) Needles (μmol g−1 FW) ( NH4+ )2SO4 Malate 1.40±0.22 8.00±0.70 1.60±0.25 ( NH4+ )2SO4 Citrate 0.49±0.18 0.60±0.16 0.35±0.05 KNO3 Malate 3.19±0.60 11.24±1.20 1.72±0.42 KNO3 Citrate 0.77±0.32 0.84±0.11 0.56±0.31 None (control) Malate 2.92±0.65 10.37±0.91 1.75±0.11 None (control) Citrate 0.82±0.38 0.76±0.17 0.51±0.12 N-source Acid Roots (μmol g−1 FW) Stems (μmol g−1 FW) Needles (μmol g−1 FW) ( NH4+ )2SO4 Malate 1.40±0.22 8.00±0.70 1.60±0.25 ( NH4+ )2SO4 Citrate 0.49±0.18 0.60±0.16 0.35±0.05 KNO3 Malate 3.19±0.60 11.24±1.20 1.72±0.42 KNO3 Citrate 0.77±0.32 0.84±0.11 0.56±0.31 None (control) Malate 2.92±0.65 10.37±0.91 1.75±0.11 None (control) Citrate 0.82±0.38 0.76±0.17 0.51±0.12 Nitrogen-free nutrient solution was used as a control. Values are means ±SD, n=4–5. The pH in the external root medium was pH 3.62±0.12, pH 6.40±0.09, and pH 6.38±0.11 for plants growing with (NH4)2SO4, KNO3, and nitrogen-free nutrient solution, respectively. Open in new tab Table 2. Concentrations of malate and citrate in roots, stems, and needles of 3-week-old Norway spruce seedlings growing on either (NH4)2SO4 or KNO3 as N-source (10 mM N) N-source Acid Roots (μmol g−1 FW) Stems (μmol g−1 FW) Needles (μmol g−1 FW) ( NH4+ )2SO4 Malate 1.40±0.22 8.00±0.70 1.60±0.25 ( NH4+ )2SO4 Citrate 0.49±0.18 0.60±0.16 0.35±0.05 KNO3 Malate 3.19±0.60 11.24±1.20 1.72±0.42 KNO3 Citrate 0.77±0.32 0.84±0.11 0.56±0.31 None (control) Malate 2.92±0.65 10.37±0.91 1.75±0.11 None (control) Citrate 0.82±0.38 0.76±0.17 0.51±0.12 N-source Acid Roots (μmol g−1 FW) Stems (μmol g−1 FW) Needles (μmol g−1 FW) ( NH4+ )2SO4 Malate 1.40±0.22 8.00±0.70 1.60±0.25 ( NH4+ )2SO4 Citrate 0.49±0.18 0.60±0.16 0.35±0.05 KNO3 Malate 3.19±0.60 11.24±1.20 1.72±0.42 KNO3 Citrate 0.77±0.32 0.84±0.11 0.56±0.31 None (control) Malate 2.92±0.65 10.37±0.91 1.75±0.11 None (control) Citrate 0.82±0.38 0.76±0.17 0.51±0.12 Nitrogen-free nutrient solution was used as a control. Values are means ±SD, n=4–5. The pH in the external root medium was pH 3.62±0.12, pH 6.40±0.09, and pH 6.38±0.11 for plants growing with (NH4)2SO4, KNO3, and nitrogen-free nutrient solution, respectively. Open in new tab Discussion In the present study the pH in different parts of cells in roots, stems, and needles of Norway spruce seedlings has been measured by in vivo NMR methods. Cytoplasmic and vacuolar pH in roots, stems, and needles was found to be 7.0 and 4.8, respectively, while 14N-NMR spectra of NH4+ showed a pH of about 3.8. The estimated cytoplasmic pH (around 7.0) is lower than reported pH values of 7.7 for roots of Pinus sylvestris and 7.5 in leaves of Pisum sativum (Gerlitz and Werk, 1994; Bligny et al., 1997). This may be due to mild hypoxia in the samples (Felle, 2005), most probably because of slow buffer circulation and aeration instead of oxygenation. Hydroponics were used for growth of the seedlings, a process that differs from the natural situation involving ectomycorrhiza. The position of the Pi signal is insensitive to a pH below 5, allowing only approximate estimates of pH in this range by 31P-NMR. To circumvent these limitations the 13C-NMR signal of malate (Chang and Roberts, 1989) and 19F-NMR (Pfeffer et al., 2004) have been used previously. In contrast to the 31P-NMR signal, the line-shape of the ammonium signal is clearly sensitive to pH changes in the acidic region, allowing a more accurate estimation of the pH in the ammonium-containing compartment. At low pH the nitrogen atoms will always carry four hydrogen atoms splitting the shape of the coupled- 14 NH4+ resonance into a five-peak signal. The amount of NH3 will increase with increasing pH, but the three hydrogen atoms will not stay long enough at the nitrogen atom for the NMR response to be quartet. Because hydrogen jumps on and off, the nitrogen nucleus has no time to see the effect of hydrogen, and at a pH above pH 4 the signal becomes a single peak, as for a decoupled signal, eventually changing to a narrow single peak between pH 4 and 6. The 31P-NMR method used here to determine the pH of cytoplasm and vacuole only estimates the pH in phosphate compartments which, according to the 14N-NMR results, seem to be less acidic than the NH4+ compartments. As NH4+ and NH3 (ammonia) are in a pH-dependent equilibrium an acidic NH4+ compartment will keep the pH considerably lower than the pKa value ensuring an NH4+/NH3 equilibrium displaced towards NH4+ ⁠. Acidic NH4+ compartments separated from the main phosphate storage pools could explain the pH values found here. However, it is also possible that Pi is stored in the NH4+-storing vacuoles because the position of the Pi signal in 31P-NMR is relatively insensitive to pH changes below 5 (Pfeffer et al., 2004). Belton et al. (1985) have also proposed NH4+ compartments in their study of inorganic nitrogen metabolism in NH4+-fed barley roots with the 14N-NMR method, but pH values as low as those reported here have only been described for vacuoles of some hyperacidifying plants (Felle, 2005). Neither the intra-thylakoidal space of chloroplasts under full illumination, nor vesicles of the endosomal Golgi-related complex, have been shown to have such low pH. Roberts and Pang (1992) has used both the 31P-NMR and 13C-NMR method to examine the NH4+ distribution between cytoplasm and vacuoles in maize root tips. They found rapid movement and accumulation of NH4+ in the vacuole, but no significant effects of NH4+ treatments on cytoplasmic pH with the 31P-NMR method. The more sensitive 13C-method showed an increase in short-term NH4+-induced vacuolar pH (Roberts and Pang, 1992). Gerendás et al. (1990) and Gerendás and Ratcliffe (2000) have used in vivo31P-NMR spectroscopy and have shown ammonium-induced changes in both cytoplasmic and vacuolar pH values in root tissues from maize seedlings grown in alkaline or acidic nutrient solution with NH4+ ⁠. In spruce, which is a gymnosperm, the situation might be different from maize where Espen et al. (2004) have found that nitrate uptake is an acidifying process. In this study, no effect of different nitrogen sources on the pH in cytoplasm and vacuoles could be measured by 31P-NMR. This could be due to NH4+ compartmentation and might represent an effective system for homeostasis of cellular pH. However, in our studies, the K+-levels in the nutrient solution are allowed to vary widely between treatment since KNO3 was used as nitrate source, and both the K and N levels are high compared with nutrient levels actually present in the natural environment. Separate acid compartments could allow high concentrations of NH4+ in cells without harmful effects for the plant. We have earlier reported high levels of NH4+ (4–46 μmol NH4+ g−1 FW in roots, stems, and needles of young Norway spruce seedlings growing in nutrient solutions with NH4+-N ranging from 5–50 mM as measured by 14N- and 15N-NMR (Aarnes et al., 1995). In these analyses the 15N-estimates of NH4+ levels were slightly lower than the 14N-estimates. In vivo14N-NMR methods allow direct determination of ammonium concentrations and its subcellular distribution in cells (Mesnard and Ratcliffe, 2005). Comparing decoupled and coupled in vivo14N-NMR spectra, Lee and Ratcliffe (1991) estimated vacuolar and cytoplasmic concentrations of NH4+ in maize roots and found up to 15 mM NH4+ vacuoles. Britto et al. (2001b) used the short-lived radiotracer 13N for the estimation of cytosolic concentrations of NH4+ in root cells of barley and rice. Notably high levels of NH4+ were found, 358 mM and 232 mM for barley and rice, respectively. Similarly, high NH4+ levels were reported in the cytosol of root cells of aspen (134 mM), Douglas-fir (77 mM), and white spruce (33 mM) (Kronzucker et al., 2003), all in line with the high NH4+ levels we have found in roots of Norway spruce (Aarnes et al., 1995). Millimolar concentrations of NH4+ were also found in the xylem sap and leaf apoplast of oilseed rape and tomato (Schjoerring et al., 2002), and in the cytosol of both NH4+-sensitive and -tolerant plants grown in root medium with high NH4+-concentrations (Kronzucker et al., 2003). Interestingly, the highest cytosolic NH4+ concentrations were found in NH4+-sensitive plant species, indicating an uncontrolled accumulation of NH4+ in these plants. One explanation for NH4+ toxicity could be an increased energetic burden for the sensitive plants (Britto et al., 2001b; Kronzucker et al., 2003). In addition to fluxes, assimilation of NH4+ is important for the regulation at the cytosolic level. It was concluded earlier that, by inhibiting GS by methionine sulphoximine, spruce plants use the GS/GOGAT NH4+-assimilation pathway (Aarnes et al., 1995). By the use of 15N-NMR spectroscopy, high concentrations of alanine, glutamine, and arginine have been found, the latter two being efficient NH4+-storage and NH4+-transport forms. Spruce plants can also use urea as a nitrogen source, and the arginine and ornithine cycle are important in the further assimilation of NH4+ (Thorpe et al., 1989; Aarnes et al., 1995). When K15NO3 was used in the nutrient solution, a small signal from NH4+ was obtained in the roots, in stems a very small signal, and in needles no signal was found at all. The lack of 15N signals in needles, when plants were grown in 5 mM K15NO3, can be a dilution effect where it was not possible to label NH4+100% (Aarnes et al., 1995) However, the low amount of NH4+ found in the NO3−-fed plants is the reason why only data from NH4+-grown plants are presented in this study. The import of NH4+ is coupled to the export of protons, explaining the low pH in the root medium of plants growing on NH4+ ⁠. H+-ATPase can generate pH gradients that mediate secondary transport and play a role in pH homeostasis. Chang and Roberts (1989) suggested that malate can provide H+ that can be stored in compartments in the cell. Davies (1986) has proposed a biochemical pH-stat based on the formation and removal of carboxyl groups from malate or oxalate in order to maintain pH homeostasis. A revision of the classical malate biochemical pH-stat hypothesis has been suggested (Britto and Kronzucker, 2002, 2005), emphasizing the importance of the membrane proton system and reaffirming the anaplerotic role of PEP carboxylase within the context of N metabolism, assimilation, and storage. High concentrations of malate were found in the stem, suggesting a role of malate in the transport of nitrogen from root to shoots in spruce seedlings. High malate concentrations in the stem might also lead to the accumulation of malate in xylem parenchyma cells, even at low malate concentrations in the xylem sap. High concentrations of water-soluble oxalate, which is probably present in vacuoles or cell walls, was also found. Organic acids were extracted with water, and a denaturing extraction agent to reduce the possible action of enzymes during homogenization of tissues would have been better. 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This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) © 2007 The Author(s).

Lin, Wuling;Peng, Yanhui;Li, Guowei;Arora, Rajeev;Tang, Zhangcheng;Su, Weiai;Cai, Weiming

doi: 10.1093/jxb/erl255pmid: 17237160

Abstract The suppression subtractive hybridization technique was used to identify differentially expressed genes between hormone-autotrophic and hormone-dependent Panax ginseng callus lines. A tonoplast intrinsic protein cDNA (PgTIP1) was found to be highly and specifically expressed in hormone-autotrophic ginseng cells, which was slightly up-regulated by cytokinin while significantly down-regulated when treated with auxin. PgTIP1 encodes a polypeptide of 250 amino acids which shows sequence and structure similarity with tonoplast aquaporins in plants. The water channel activity of PgTIP1 was demonstrated by its expression in Xenopus laevis oocytes. When over-expressed in Arabidopsis thaliana, PgTIP1 substantially altered the plant's vegetative and reproductive growth and development. Arabidopsis plants over-expressing PgTIP1 showed significantly enhanced seed size and seed mass plus greatly increased growth rate compared with those of the wild type. Moreover, the seeds from PgTIP1 over-expressing Arabidopsis had 1.85-fold higher fatty acid content than the wild-type control. These results demonstrate a significant function of PgTIP1 in the growth and development of plant cells. Arabidopsis, ginseng, habituation PgTIP1, tonoplast intrinsic protein Introduction The growth and development of plant cells depend on tight regulation of cellular water movement and homeostasis. Aquaporins (AQPs), which facilitate and regulate passive exchange of water across membranes (Agre, 1992; Chrispeels and Agre, 1994; Schäffner, 1998), belong to a highly conserved membrane protein family MIP (major intrinsic protein). In higher plants, AQPs are divided into four subfamilies: PIPs, TIPs, NIPs, and SIPs, mainly based on the membrane location and function (Chaumont et al., 2001; Johanson et al., 2001; Baiges et al., 2002; Quigley et al., 2002). Aquaporin expression can be regulated at both the RNA and protein levels. Over-expression or antisense/knockout reduction of certain AQPs highlights their important roles in numerous physiological processes in plants (Kaldenhoff et al., 1995, 1998; Gerbeau et al., 2002; Javot et al., 2003; Aharon et al., 2003; Uehlein et al., 2003; Hanba et al., 2004; Hachez et al., 2006). Although the discovery of AQPs has resulted in a paradigm shift in the understanding of plant water relations, a comprehensive picture of their physiological role(s) in plant growth and development remains elusive. It is well established that in vitro plant cell/tissue cultures require exogenous supply of plant hormones (auxins and cytokinins) for their sustained growth (Collin and Edwards, 1998). However, certain cell-lines, although originally grown in hormone-based culture medium, may lose this dependency on one or more externally supplied plant hormones for growth and become hormone-autotrophic or habituated/autonomous (Meins, 1989; Gaspar et al., 2002). Habituation, a stable heritable competency of plant cells to proliferate without hormonal supply, is a distinct response from the one associated with tumour development: tumours are mediated by pathogens or result from genetic transformation (Gaspar, 1998). Despite the reports on habituation in many cell types (Jäger et al., 1997), what controls this phenomenon is not well understood. Although increasing the levels of auxins and cytokinins would result in cell expansion, the physiological factor that directly drives plant cell expansion is turgor pressure, which is mainly generated by a rapid influx of water into the cells. The uptake of water by expanding plant cells may well involve AQPs. Evidence showed that some AQPs were up-regulated by exogenous auxin during cell growth (Werner et al., 2001; Ozga et al., 2002). We have on hand habitutated callus line from Panax ginseng that exhibits autotrophy for both auxin and cytokinin. No significant difference between these two callus lines was found with respect to the level of active free auxin and cytokinin concentration by ELISA measurement (data not shown). In order to explore the molecular differences between habituated and non-habituated ginseng calli, the differentially expressed genes were screened using the suppression subtractive hybridization (SSH) method. Besides the down-regulation of some cDNAs in habituated cells, for example, early auxin responsive gene GH3, periodic tryptophan protein gene PWP, aconitase gene ACO, retrotransposon-like gene, and many other genes with so far unknown functions, an aquaporin gene (PgTIP1) was detected, which belongs to plant TIPs subfamily, and was specifically and highly expressed in hormone-autotrophic ginseng cells. The results from our study, which demonstrated that PgTIP1 significantly altered the growth and developmental attributes of plants when over-expressed in Arabidopsis thaliana, are reported here. Materials and methods Plant cell cultures Hormone-autotrophic (H; habituated) and hormone-dependent (NH; non-habituated) Panax ginseng calli were cultured on hormone-free 67V medium and 67V medium, respectively (Veliky and Martin, 1970); the latter supplemented with 1.5 mg·l−1 dichlorophenoxyacetic acid (2,4-D), 1 mg l−1 indole-3-acetic acid (IAA), 0.1 mg l−1 naphthalene acetic acid (NAA), and 0.25 mg l−1 kinetin (KT), pH 5.8. Both callus lines were maintained at 24 °C in the dark. Suspension cultures of the ginseng calli were maintained at 24 °C in 250 ml flasks with 50 ml medium; cultures were aerated by shaking at 0.5 g on a rotary shaker in the dark, and were subcultured every 28 d. Suppression subtractive PCR and northern blot analysis PolyA+ RNA were isolated from hormone-autotrophic and hormone-dependent ginseng cells using mRNA purification kit (Qiagen). The polyA+ samples were used to construct driver- and tester-cDNAs following the protocol of the PCR-Select cDNA subtraction kit provided by the manufacturer (Clontech). Selectively amplified products were inserted into pMD18-T vector using a T/A cloning kit (Takara). Northern blot analysis was performed using the digoxigenin-dUTP system (Roche) and the cDNA fragment screened from SSH library was labelled as a probe for hybridization. Full-length cDNA cloning of PgTIP1 and bioinformatic analysis A cDNA library of hormone-autotrophic ginseng cells was constructed and screened according to the instructions in ZAP Express Predigested Vector Kit and ZAP Express Predigested Gigapack Cloning Kit (Stratagene). Nucleotide and deduced amino acid sequences were analysed with Bioedit software. Homology searches were made in all major databases. Alignments of amino acid sequences were generated and edited with DNAStar software. Predicted stereo structure of PgTIP1 and AQP2 and ar/R region simulation were performed at http://swissmodel.expasy.org (Schwede et al., 2003). Hormone treatments and analysis of PgTIP1 expression The phytohormones KT and 2, 4-D or both were added to ginseng suspension cultures at the final concentration of 0.25 mg l−1 and 1.5 mg·l−1, respectively. To determine the transcript levels in different hormone-treated samples, the real-time quantification of RNA target was performed in the Rotor-Gene 3000 real-time thermal cycling system using SYBR Green RT-PCR kit (Toyobo). The PCR primers for Act were 5′-GTGTTGCCCCAGAAGAGC-3′ in sense and 5′-CAGAATCCAGCACAATACCT-3′ in antisense orientation, and those for PgTIP1 were 5′-CTCAGGCTTGGCATTTAG-3′ and 5′-CCCAGTTCTCCCTTCTTT-3′, respectively. The reaction mixture (25 μl) contained 200 ng of total RNA, 0.5 μM of each primer, and appropriate amounts of enzymes and fluorescent dyes as recommended by the manufacturer. The Rotor-Gene 3000 cycler was programmed as follows: 2 min at 95 °C for pre-denature; 40 cycles of 15 s at 94 °C, 15 s at 55 °C, 20 s at 72 °C for Act and 30 s at 94 °C, 30 s at 55 °C, 30 s at 72 °C for PgTIP1. The data were collected during the extension step. No detectable fluorescence signal was detected in control samples where H2O was added to the reaction mixture instead of RNAs. A possible contamination by genomic DNA of the RNA sample was carefully monitored and avoided. Xenopus oocyte expression The coding region of PgTIP1 and AQP2 (positive control) were cloned into pXBG-ev1 vector (Li et al., 2000) using a Bgl II restriction site. After digestion and linearization of the plasmid, the complementary RNAs (cRNA) were synthesized in vitro using the mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit (Ambion). Oocyte preparation, injection, and expression were performed as described by Daniels et al. (1996). Osmotic water permeability of oocytes was determined essentially as described by Weig et al. (1997). Generation of PgTIP1-overexpressing Arabidopsis plants and their phenotypic analysis The ORF of PgTIP1 was cloned into pHB vector (Mao et al., 2005) using a HindIII and a XbaI restriction site to generate double 35S:PgTIP1 transgene. Arabidopsis plants (ecotype Columbia-0) were transformed with this transgene using the floral-dipping method (Clough and Bent, 1998). Independent hygromycin-resistant lines (T0) were isolated and amplified. Experiments were conducted with homozygote T2 plants. The root length measurements were made on 1-week-old plants cultured with MS medium on a vertical plate. Leaf histological analysis (fifth-leaf samples, 2-week-old) was performed according to Hu et al. (2003). Average mass of the seeds produced by PgTIP1 over-expressing Arabidopsis plants was determined by weighing mature dry seeds in batches of 1000; at least three sample batches (of 1000 each) were weighed for a given data point and average value was from three independent transgenic lines. Size distributions of WT and transgenic seed populations were analysed by a scanning electron microscope equipped with ‘smileview’ software (JEDL JSM-6360LV, Japan). Fatty acid extraction was performed as described by Fiehn et al. (2000) using 0.3 g dry seeds. Nonadecanoic acid methyl ester stock solution (2 mg ml−1 CHCL3) and ribitol stock solution (0.2 mg ml−1 H2O) were used as internal standards for the lipid phase and the polar phase, respectively. Selected subsamples were injected in a GC-mass spectrometer (6890N GC System/5973 MS Selective Detector) and resultant electron ionization mass spectra were used to identify and quantify individual fatty acid species. The quantity of each fatty acid was determined by comparison with the internal standard and average value of total fatty acid content was from three independent transgenic lines as example. Seeds were collected from Arabidopsis thaliana plants that were grown at 22 °C with a 16/8 h day/night cycle in a greenhouse. Leaf net photosynthetic rates, stomatal conductance, intercellular CO2 concentration, and transpiration rates were measured by a portable gas analysis system, Li-Cor 6400 with a light-emitting diode light source (Li-Cor Inc. Lincoln, Nebraska, USA). Results PgTIP1 belongs to the MIP super gene family SSH employing the mRNAs from autonomous and hormone-dependent ginseng cells allowed the isolation of a cDNA for tonoplast intrinsic protein that is referred to here as PgTIP1. The full-length cDNA of PgTIP1 includes a 750 bp open reading frame (GenBank accession number DQ237285), and encodes a protein of 250 amino acids (Fig. 1). BLASTX and ClustalX analyses (Thompson et al., 1997) of this gene indicated high similarities to putative plant aquaporins: PgTIP1 deduced protein is most similar to NtAQP1 (80.8% identity), a tonoplast aquaporin, and has high homology to AtTIP1;1, OsTIP1;1, and ZmTIP1;2 at 78.8%, 77.2%, and 68.4%, respectively. The hydrophobicity profiles of PgTIP1, as determined by using BioEdit and TMHMM (http://www.cbs.dtu.dk/services/TMHMM), indicated six highly hydrophobic regions (A–F) corresponding to membrane-spanning putative α-helices that are characteristic of AQPs. It also has two NPA domains, which form the water pore within the membrane lipid bilayer. Secondary and stereo structural analyses (see Fig. 1 in the supplementary data at JXB online) revealed the similarity of three-dimensional structure of PgTIP1 to that of aquaporin 2 (AQP2). The conserved narrow selectivity filter region (the aromatic/Arg [ar/R] filter) of PgTIP1 is formed by H-65(H2), I-186(H5), A-195(LE1), and V-201(LE2) (see Fig. 2 in the supplementary data at JXB online). This ar/R tetrad was identical to that of the Group I TIP in Arabidopsis (AtTIP1;1) (Wallace and Roberts, 2004), which has already been shown to have high water channel activity. Fig. 1. View largeDownload slide The sequence alignment of PgTIP1 and other plant TIPs. Fig. 1. View largeDownload slide The sequence alignment of PgTIP1 and other plant TIPs. PgTIP1 is a novel gene that is specifically expressed in habituated ginseng cells and inhibited by auxin PgTIP1 fragment was strongly expressed in autonomous ginseng cells but was not detected in hormone-dependent cells as indicated by northern blot analysis (Fig. 2A). This is a first report on an aquaporin expression response in context with the phenomenon of autonomous growth. Using the real-time reverse transcriptase (RT)-PCR method, the expression of PgTIP1 under different hormone treatments was studied in suspension-cultured hormone-autotrophic ginseng cells. Data indicated that the PgTIP1 was up-regulated by kinetin (KT, 0.25 mg l−1) treatment while significantly down-regulated by 1.5 mg l−1 2, 4-dichlorophenoxyacetic acid (2, 4-D); its expression was also down-regulated when the cells were exposed to both the hormones together (Fig. 2C). Fig. 2. View largeDownload slide PgTIP1 expression in habituated and non-habituated ginseng cells: effect of hormones. (A) Northern blot analysis of PgTIP1 in habituated (H) and non-habituated (NH) ginseng cells. (B) Habituated ginseng calli (H) and non-habituated ginseng calli (NH). (C) PgTIP1 transcription in hormone-autotrophic suspension ginseng cells when treated with 0.25 mg l−1 KT and/or 1.5 mg l−1 2,4-D for 6, 12, 24, 48, 96 h, respectively; the data are given as the mean ±SE (n=3). Fig. 2. View largeDownload slide PgTIP1 expression in habituated and non-habituated ginseng cells: effect of hormones. (A) Northern blot analysis of PgTIP1 in habituated (H) and non-habituated (NH) ginseng cells. (B) Habituated ginseng calli (H) and non-habituated ginseng calli (NH). (C) PgTIP1 transcription in hormone-autotrophic suspension ginseng cells when treated with 0.25 mg l−1 KT and/or 1.5 mg l−1 2,4-D for 6, 12, 24, 48, 96 h, respectively; the data are given as the mean ±SE (n=3). Water-channel activity of PgTIP1 To find out whether PgTIP1 is a functional aquaporin, water-channel activity of PgTIP1 was assayed in the Xenopus oocytes system; AQP2 was included as a positive control in these assays. Three days after cRNA or water injection, the rate of cell volume change (Fig. 3A) and the osmotic Pf values (Fig. 3B) were calculated in the presence of an osmotic gradient. The Pf of PgTIP1-expressing, AQP2-expressing, and water-injected oocytes was 3.19 × 10−2 cm s−1, 2.36×10−2 cm s−1, and 0.14×10−2 cm s−1, respectively. Oocytes expressing PgTIP1 yielded 23-fold and 1.35-fold higher Pf than that of the water-injected oocytes and the positive control, respectively, suggesting that PgTIP1 is, indeed, a functional aquaporin with high water-channel activity. Fig. 3. View largeDownload slide Water-channel activity PgTIP1. (A) Initial swelling rates of Xenopus laevis oocytes injected with cRNA encoding PgTIP1, mammalian AQP2 (as positive control) or water (as negative control). The rate of oocyte swelling upon immersion in hypo-osmotic medium is plotted as V/V0 versus time, where V is the volume at a given time point and V0 is the initial volume. (B) Osmotic water permeability coefficient (Pf) of oocytes injected with cRNA encoding PgTIP1, AQP2, or water. The Pf values were calculated from the initial rate of oocyte swelling. Data are given as the mean ±SE (n=30). Fig. 3. View largeDownload slide Water-channel activity PgTIP1. (A) Initial swelling rates of Xenopus laevis oocytes injected with cRNA encoding PgTIP1, mammalian AQP2 (as positive control) or water (as negative control). The rate of oocyte swelling upon immersion in hypo-osmotic medium is plotted as V/V0 versus time, where V is the volume at a given time point and V0 is the initial volume. (B) Osmotic water permeability coefficient (Pf) of oocytes injected with cRNA encoding PgTIP1, AQP2, or water. The Pf values were calculated from the initial rate of oocyte swelling. Data are given as the mean ±SE (n=30). Increased growth rate and enhanced seed size, mass, and fatty acid content in Arabidopsis plants over-expressing PgTIP1 Specific expression of PgTIP1 in hormone-autotrophic ginseng cells suggested its potential involvement in cell division and/or growth. A PgTIP1 over-expression construct was generated in Arabidopsis to explore its physiological function, if any. The transcription of PgTIP1 was observed in roots, stems, leaves, flowers, and siliques of Arabidopsis transformants by real-time RT-PCR analysis (data not shown). Among 24 independent transformed lines, 22 (approximately 92%) PgTIP1 over-expressers exhibited faster growth rate than the wild-type (WT) control as evidenced by the root elongation, leaf expansion, and weight increase of the aerial parts (Fig. 4). Root length of the one-week-old transgenic Arabidopsis seedlings was significantly greater than that of the WT control (Fig. 4A, B). The overall leaf-size was bigger in transgenic plants as indicated by the length and width of the leaf-blade and the petiole length of 3-week-old; fifth leaves (Fig. 4C, E, F). Histological observations using 2-week-old seedlings also indicated the size of mesophyll cells in transgenic plants to be bigger than that of the WT leaves (Fig. 4D). The increase in the weight of the aerial parts over time (days after germination) for the WT and PgTIP1 over-expressors demonstrated faster and stronger growth of transgenic plants (Fig. 4G). PgTIP1 over-expression also resulted in precocious flowering in Arabidopsis plants; transgenic plants flowered at least 3 d earlier than WT (Fig. 4H). The most intriguing phenotype of Arabidopsis over-expressing PgTIP1 was that of the size and mass of mature seeds in that both attributes were significantly higher in transgenic plants compared to the WT (Fig. 4I, J; Table 1). Fig. 4. View largeDownload slide Phenotypic and molecular characterization of PgTIP1 over-expressing Arabidopsis plants. (A) One-week-old seedlings of WT and transgenic Arabidopsis cultured with MS medium on a vertical glass plate. (B) Root length of 1-week-old seedlings from WT and transgenic Arabidopsis. Data are given as the mean ±SE (n=50). (C) Three-week-old seedlings of WT and PgTIP1 over-expressing Arabidopsis plants. (D) Transverse sections of the fifth leaves (2-week-old) of WT control and PgTIP1 over-expressing plants. The palisade (p) and spongy mesophyll (s) cells are indicated. Bars=100 μm. (E) Morphology of 3-week-old fifth leaves. (F) Dimensions of 3-week-old fifth leaves as shown in (E). Data are given as the mean ±SE (n=15). (G) The aerial parts weight over time (days after germination) for the WT and PgTIP1 over-expressing Arabidopsis plants. (a) Fresh weight increases during one month of growth after germination. (b) Dry weight increases during one month of growth after germination. Data are given as the mean ±SE (n=15). (H) Four-week-old WT and PgTIP1 over-expressing Arabidopsis plants, indicating precocious flowering in transgenic plants. (I) Mature dried seeds from WT and PgTIP1 over-expressing Arabidopsis plants. (J) Scanning electron micrographs of mature Arabidopsis seeds from WT control and PgTIP1 over-expressing Arabidopsis plants. Bars=100 μm. Fig. 4. View largeDownload slide Phenotypic and molecular characterization of PgTIP1 over-expressing Arabidopsis plants. (A) One-week-old seedlings of WT and transgenic Arabidopsis cultured with MS medium on a vertical glass plate. (B) Root length of 1-week-old seedlings from WT and transgenic Arabidopsis. Data are given as the mean ±SE (n=50). (C) Three-week-old seedlings of WT and PgTIP1 over-expressing Arabidopsis plants. (D) Transverse sections of the fifth leaves (2-week-old) of WT control and PgTIP1 over-expressing plants. The palisade (p) and spongy mesophyll (s) cells are indicated. Bars=100 μm. (E) Morphology of 3-week-old fifth leaves. (F) Dimensions of 3-week-old fifth leaves as shown in (E). Data are given as the mean ±SE (n=15). (G) The aerial parts weight over time (days after germination) for the WT and PgTIP1 over-expressing Arabidopsis plants. (a) Fresh weight increases during one month of growth after germination. (b) Dry weight increases during one month of growth after germination. Data are given as the mean ±SE (n=15). (H) Four-week-old WT and PgTIP1 over-expressing Arabidopsis plants, indicating precocious flowering in transgenic plants. (I) Mature dried seeds from WT and PgTIP1 over-expressing Arabidopsis plants. (J) Scanning electron micrographs of mature Arabidopsis seeds from WT control and PgTIP1 over-expressing Arabidopsis plants. Bars=100 μm. Table 1. Morphometric measurements of dimensions and mass of seeds from the WT and PgTIP1 over-expressing Arabidopsis plants: data are given as means ±SE   n  WT  PgTIP1  Seed length (μm)  150  470±40  577±42  Seed width (μm)  150  275±21  320±23  Thousand seeds weight (mg)  3  17±1  34±1    n  WT  PgTIP1  Seed length (μm)  150  470±40  577±42  Seed width (μm)  150  275±21  320±23  Thousand seeds weight (mg)  3  17±1  34±1  View Large Differential expression of aquaporin genes is essential to plant growth and stress tolerance but their effect on seed characteristics has never been reported. In order to explore this intriguing transgenic effect further, the protein, sugar and fatty acid content of the seeds from transgenic and WT plants was analysed. Total protein and sugar contents of the seeds showed no significant difference between the transgenic and WT plants (data not shown), while the total fatty acid content per unit weight of the seeds from transgenic plants was ∼1.85-fold of that from the WT. Furthermore, compositional analysis of fatty acids showed some differences between the two seed types; with the relative proportion of 18:1 increased and 18:3 decreased in the PgTIP1 over-expressors (Table 2). Table 2. Fatty acid content and composition of seeds from the WT and PgTIP1 over-expressing Arabidopsis plants: the data are given as the mean ±SE (n=3) Genotype  Fatty acid content (mg g−1 seed)  Fatty acid composition (mol %)      16:0  18:0  18:1  18:2  18:3  20:1  Othersa  WT  89.6±4.8  8.6±0.6  6.2±0.4  27.4±1.8  28.0±1.2  5.0±0.3  19.4±0.9  5.4±0.4  PgTIP1  166.4±6.8  8.5±0.6  4.5±0.3  35.8±2.2  25.6±1.6  0.7±0.1  20.1±1.1  4.8±0.3  Genotype  Fatty acid content (mg g−1 seed)  Fatty acid composition (mol %)      16:0  18:0  18:1  18:2  18:3  20:1  Othersa  WT  89.6±4.8  8.6±0.6  6.2±0.4  27.4±1.8  28.0±1.2  5.0±0.3  19.4±0.9  5.4±0.4  PgTIP1  166.4±6.8  8.5±0.6  4.5±0.3  35.8±2.2  25.6±1.6  0.7±0.1  20.1±1.1  4.8±0.3  a ‘Others’ are primarily 14:1, 20:0, 20:2, 22:0, and 22:1 fatty acids, the relative composition of which did not change significantly between WT and PgTIP1 over-expressing Arabidopsis seeds. View Large Transpiration and photosynthetic behaviour of Arabidopsis plants over-expressing PgTIP1 Leaf transpiration rate (Tr), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and leaf net photosynthetic rates (Pn) in WT and PgTIP1 over-expressing plants were measured when leaves were fully expanded. Results indicated that Arabidopsis plants over-expressing PgTIP1 had higher Gs and Tr than the WT plants (Fig. 5A, C), indicating a stronger water absorption and transpiration ability. A higher Ci concentration was also detected in transgenic Arabidopsis (Fig. 5B), but the level of Pn in PgTIP1 over-expressing Arabidopsis had no significant change compared to the WT plants (Fig. 5D); higher Ci in PgTIP1 over-expressing Arabidopsis may be a consequence of greater Gs (potentially resulting in greater CO2 influx) but similar Pn rates of WT and transgenic plants. The explanation for no apparent difference in the Pn rates for the two genotypes, despite the difference in their Ci levels, could not be determined in this study. It is noteworthy, however, that stomatal conductance (Gs) is just one of the factors that would control the CO2 acquiring capacity and Pn level in transgenic Arabidopsis. We still postulate that the transgenic Arabidopsis plants may accumulate more assimilation product due to their larger leaf size. Fig. 5. View largeDownload slide Leaf stomatal conductance (Gs) (A), intercellular CO2 concentration (Ci) (B), transpiration rate (Tr) (C), and leaf net photosynthesis rates (Pn) (D) in WT control and PgTIP1 over-expressing Arabidopsis. Values indicate a mean of three measurements with standard deviations, each with a sample size of eight leaves. One of the triplicate trials is shown. Regression analysis confirmed that the Gs, Ci, and Tr values of PgTIP1 over-expressing plants differ significantly from those of the WT (asterisk, P≤0.01). Fig. 5. View largeDownload slide Leaf stomatal conductance (Gs) (A), intercellular CO2 concentration (Ci) (B), transpiration rate (Tr) (C), and leaf net photosynthesis rates (Pn) (D) in WT control and PgTIP1 over-expressing Arabidopsis. Values indicate a mean of three measurements with standard deviations, each with a sample size of eight leaves. One of the triplicate trials is shown. Regression analysis confirmed that the Gs, Ci, and Tr values of PgTIP1 over-expressing plants differ significantly from those of the WT (asterisk, P≤0.01). Discussion Habituation refers to a naturally occurring phenomenon whereby callus cultures, upon continued subculture, lose their requirement for auxin, cytokinin, or both and it is considered to be an in vitro epigenetic switch to autotrophy (Meins, 1983; Syono and Fujita, 1994). The physiological basis of habituation is still unknown. General opinion is that habituation results from an enhanced accumulation by cells of the hormone for which they are habituated. But there are reports that auxin and cytokinin are present in both cell types at roughly the same concentration (Meins, 1989). It is noteworthy that no significant difference with respect to the endogenous levels of auxin or cytokinin was observed between the habituated and non-habituated ginseng cells in this study. Based on the latest research on a cytokinin-habituated callus line in Arabidopsis (Pischke et al., 2006), it also seems less likely that habituation is caused by an over-production of endogenous hormones, and more likely that it may be caused by altered expression of one or more other genes, for example, cytokinin-signalling genes. Cytokinin sensitivity may be modulated through regulation of cytokinin-receptor production. And epigenetic changes, instead of increases in hormone concentration, contribute to the acquisition of auxin/cytokinin-habituation. All these indicate that the endogenous hormone level might not be the key factor in the habituation course and habituation may arise from processes downstream from perception of hormone stimuli, which control cell division and expansion, as in animal cancer cells where activation and expression of genes bypass the requirement for specific growth factors (Hagège et al., 1994). Furthermore, over-expression of a specific gene has been shown artificially to confer habituation in callus tissues (Kakimoto, 1996; Hwang and Sheen, 2001; Sakai et al., 2001; Osakabe et al., 2002). It is speculated that altered hormone signalling routes and hormone sensitivity might lead to this complicated phenomenon. In fact, the proliferation of habituated ginseng callus tissues was inhibited by exogenously applied auxin (the same concentration as in the media for non-habituated cell lines) in our study, which suggested that the sensitivity to auxin in a habituated ginseng cell line might be enhanced during the habituation course. The down-regulation of PgTIP1 by exogenous auxin might be a side-off effect rather than the result of a directly negative regulation. High level and specific expression of PgTIP1 in the habituated cell line, as observed in the present study, should result from an acclimation to the environment (exogenous hormones subtracted from the media). Plant vacuole is a multifunctional organelle with important roles in space filling, osmotic adjustment, storage, and digestion. Vacuole biogenesis and enlargement require transport of osmotically active substances across the tonoplast, followed by a rapid influx of water into the vacuole. This influx generates the turgor pressure that drives cell expansion and maintains the cell shape. Rapid cell expansion may require a high hydraulic permeability of the tonoplast to support water entry into the vacuole. Although vacuole volume increase can never be triggered by the water channel function of aquaporins for the passive process of water transport via them; the uptake of water by expanding vacuoles may well involve tonoplast aquaporins (Chaumont et al., 1998; Javot and Maurel, 2002). Reisen et al. (2003) reported that the heterologous expression of a cauliflower tonoplast aquaporin (BobTIP26;1, orthologous to AtTIP1;1) in tobacco suspension cells had no effect on the growth rate, but the cells were larger than in the wild-type. In present study, it is shown that PgTIP1, which is highly and specifically expressed in hormone-autotrophic ginseng cells, has a high water-channel activity. In the roots and leaves of two detected transgenic lines, PgTIP1 has a ∼20-fold higher transcription than endogenous AtTIP1;1 and AtTIP1;2 (data not shown), both known to be highly expressed in Arabidopsis plants. Transgenic Arabidopsis plants over-expressing PgTIP1 had faster growth rate, longer roots, and bigger leaves and leaf cells; these results support the interpretation that TIPs might be involved in cell enlargement by modulating the permeability of the tonoplast. The volume increase of leaf cells might be triggered by enlargement of the vacuolar compartment facilitated by accumulating osmotically active substances, and following water influx into the vacuole, which was fine regulated by TIPs. Whereas, the principal function of vacuoles is the maintenance of cell turgor, they can also accumulate macromolecules and secondary metabolites (Marty, 1999). Plant cells have different types of vacuoles that can coexist in the same cell and the different TIP isoforms may have entirely different or similar functions. While α-TIP alone is a marker for autophagic vacuole, it, coupled with δ- or γ-TIP, is involved in the protein storage function. By contrast, γ-TIP alone marked the lytic vacuoles and it, combined with δ-TIP, has a role in the storage of vegetative storage proteins and pigments (Jauh et al., 1999). Takahashi et al. (2004) isolated three novel γ-TIP cDNAs in rice, OsTIP1, OsTIP2, and OsTIP3, and demonstrated their specialized function. By contrast with OsTIP2 and OsTIP3, which are expressed specifically in roots and seeds, respectively, OsTIP1 was expressed in mature seed embryos and during early seed germination. These observations indicate that different TIP isoforms, alone or in combination with each other, play complicated physiological functions in plants. Storage vacuole is one of the most important vacuole types in plants, especially in seed cells. A greatly enhanced seed size and mass, and the significantly increased seed fatty acid content of Arabidopsis plants over-expressing PgTIP1 indicate that PgTIP1 might have a role in substance storage and metabolism, in addition to involving in cell expansion. From sequence homology, protein structure to water channel activity, PgTIP1 shares characteristics with Arabidopsis tonoplast TIP1;1, which has been classified as a γ-TIP. The physiological role of AtTIP1;1 was investigated in plants using RNA interference (Ma et al., 2004). Data indicated that a strong down-regulation of AtTIP1;1 led to plant death and suggested an essential physiological role of this otherwise highly expressed isoform. Transcript, metabolite profiling, and the cellular localization data suggested a role for AtTIP1;1 in carbon distribution, possibly by regulation of vesicle trafficking towards the central vacuole. We postulate that, similar to AtTIP1;1, PgTIP1 might be involved in substance storage and metabolism in the seeds of Arabidopsis plants over-expressing this gene. As mentioned earlier, AQPs might have physiological functions other than facilitating water movement across cellular membranes. AQP over-expression highlights those physiological processes in which AQPs act as bottlenecks. Transgenic tobacco over-expression of AtPIP1;2 significantly increased plant growth and transpiration rate, stomatal density, and photosynthetic activity (Aharon et al., 2003). Similarly, transgenic rice over-expression of HvPIP2;1 also resulted in faster growth rate and higher internal CO2 (by 40%), stomatal conductance (by 27%), and CO2 assimilation (by 14%) than the wild-type plants (Hanba et al., 2004). These studies indicated PIPs may play an important role in regulating plant vigour. However, the mechanism of the involvement of PgTIP1 in this process might be different from that of PIPs, since the photosynthetic activity of transgenic Arabidopsis plants over-expressing PgTIP1 was found to be similar to that of wild-type plants. However, it has been shown here that PgTIP1 might be involved in cell enlargement via transport of water across tonoplast, which often represents a limiting factor that regulates plant vigour. Further understanding of the physiological roles of PgTIP1 in plant growth and development should be helpful in understanding AQP functions in plants more comprehensively. We thank Dr HongQuan Yang for providing the pHB vector. We are grateful to Dr DaQuan Xu for his technical suggestions and assistance. This work was supported by the Chinese Academy of Sciences (Grant No.KSCX2-SW-329), Institute of Plant Physiology and Ecology and National Natural Science Foundation of China (Grant No. 30570157), and by Hatch Act and State of Iowa funds. 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Jr, Charles Stewart,;Mazourek, Michael;Stellari, Giulia M.;O'Connell, Mary;Jahn, Molly

doi: 10.1093/jxb/erl243pmid: 17339653

Abstract Capsaicin, the pungent principle in hot peppers, acts to deter mammals from consuming pungent pepper pods. Capsaicinoid biosynthesis is restricted to the genus Capsicum and results from the acylation of the aromatic compound, vanillylamine, with a branched-chain fatty acid. The presence of capsaicinoids is controlled by the Pun1 locus, which encodes a putative acyltransferase. In its homozygous recessive state, pun1/pun1, capsaicinoids are not produced by the pepper plant. HPLC analysis confirmed that capsaicinoids are only found in the interlocular septa of pungent pepper fruits. Immunolocalization studies showed that capsaicinoid biosynthesis is uniformly distributed across the epidermal cells of the interlocular septum. Capsaicinoids are secreted from glandular epidermal cells into subcuticular cavities that swell to form blisters along the epidermis. Blister development is positively associated with capsaicinoid accumulation and blisters are not present in non-pungent fruit. A genetic study was used to determine if the absence of blisters in non-pungent fruit acts independently of Pun1 to control pungency. Screening of non-pungent germplasm and genetic complementation tests identified a previously unknown recessive allele of Pun1, named pun12. Sequence analysis of pun12 revealed that a four base pair deletion results in a frameshift mutation and the predicted production of a truncated protein. Genetic analysis revealed that pun12 co-segregated exactly with the absence of blisters, non-pungency, and a reduced transcript accumulation of several genes involved in capsaicinoid biosynthesis. Collectively, these results establish that blister formation requires the Pun1 allele and that pun12 is a recessive allele from C. chinense that results in non-pungency. Blister, capsaicin, capsaicinoids, Capsicum, pepper, pungency, secretion Introduction Capsaicin and its analogues, collectively called capsaicinoids, are the pungent principle of peppers (Thresh, 1876). Peppers (Capsicum spp.) are well known for their ability to cause an intense organoleptic sensation of heat when consumed. Capsaicinoids are produced only within the genus Capsicum and their presence has driven the domestication of several Capsicum species (Walsh and Hoot, 2001). Peppers probably originated in Bolivia, as this area contains many of the 20–27 recognized species of Capsicum (Andrews, 1984; Hunziker, 2001; Walsh and Hoot, 2001). Bell pepper seeds were first traded nearly 500 years ago, and archeological evidence indicates peppers have a history of human use dating back nearly 9000 years, making Capsicum one of the oldest plant genera to be domesticated (Boswell, 1937; Pickersgill, 1966; Heiser Jr 1969; Basu and Krishna De, 2003). Ecological studies suggest that capsaicinoids evolved as a means of directed deterrence (Tewksbury and Nabhan, 2001). Birds, the favoured seed-dispersal agents of wild Capsicum, do not perceive capsaicinoids. After ingestion by avian frugivores, seeds are transported within the birds’ gut, and then excreted, often in favourable habitats under trees that provide appropriate light levels for plant growth. Furthermore, passage through the avian digestive system appears to promote the germination of pepper seeds (Tewksbury and Nabhan, 2001). In contrast to birds, mammals are extremely sensitive to capsaicinoids, perceiving pain when capsaicinoids contact epidermal tissues. Furthermore, mammals possess crushing molars and harsh, acidic, digestive systems that harm pepper seeds (Tewksbury and Nabhan, 2001). The pain response to capsaicinoids is due to the activation of the TRPV1 (VR1) receptor in mammals by capsaicinoids; in birds, this receptor does not respond to capsaicinoids (Caterina et al., 1997, 2000; Jordt and Julius, 2002). In fact, the TRPV1 receptor is involved in several pain-sensing pathways, which has prompted much basic and clinical research on the use of capsaicinoids to treat a variety of human ailments, including arthritis, bladder and digestive problems, and cancer (Deal et al., 1991; Chancellor and De Groat, 1999; Han et al., 2001; Surh, 2002; Cruz, 2004; Srinivasan, 2005). Natural capsaicin analogues, which may function as neurotransmitters, have been found in mammalian brains, and endorphins have been shown to be released in the brain when capsaicin is consumed (Appendino et al., 2002; Huang et al., 2002; Chu et al., 2003). It is generally accepted that capsaicinoids are produced solely in pepper fruits, although the location of the biosynthesis and accumulation of capsaicinoids within the fruits has been debated. A recent report describes the detection of capsaicinoids in vegetative organs, and others have reported small amounts of capsaicinoids in seeds (Ohta, 1962; Balbaa et al., 1968; Estrada et al., 2002). Within the pepper fruit, capsaicinoids chiefly accumulate along the epidermal cells of the interlocular septum which defines the fruit locules and is derived from the tissue connecting the placenta to the pericarp (Judd et al., 1999). The epidermal cells of the interlocular septum have been implicated in capsaicinoid biosynthesis, based on morphological changes during fruit development and the existence of osmiophilic granules in these cells (Furuya and Hashimoto, 1954; Ohta, 1962; Suzuki et al., 1980). In pungent varieties, epidermal protrusions or blisters arise from the lifting of the cuticle layer from the cell wall during the filling of subcuticular cavities with capsaicinoids (Rowland et al., 1983; Zamski et al., 1987). Very little is known about the regulation of capsaicinoid biosynthesis. Capsaicinoids are produced by condensation of a branched-chain fatty acid, derived from either valine or leucine, with vanillylamine, derived from phenylalanine (Fig. 1a) (Bennett and Kirby, 1968; Leete and Louden, 1968; Suzuki et al., 1981; Sukrasno and Yeoman, 1993). In previous research, the identity of Pun1, a putative acyltransferase named AT3, was reported (Stewart et al., 2005). The Pun1 locus, first investigated nearly a century ago, is responsible for non-pungency throughout C. annuum based on inferred breeding pedigrees and sequencing results (Webber, 1911; Stewart et al., 2005). Non-pungent C. annuum genotypes were observed to have a 2.5 kb deletion spanning the putative promoter and first exon of AT3. The pun1 allele defined by this large deletion is the only known mutation to date that has a qualitative effect on the presence/absence of capsaicinoids (Webber, 1911; Blum et al., 2002). Fig. 1. View largeDownload slide Tissue specificity of capsaicinoid accumulation in pepper fruits. (a) Current model of the capsaicinoid biosynthetic pathway. Enzymes are shown adjacent to the reactions they catalyse. For those enzymes underlined, the gene encoding them has been cloned in Capsicum. Kas is the only enzyme functionally characterized in Capsicum; all other enzyme attributions are based on nucleotide similarity and differential gene expression. Pal, phenylalanine ammonia lyase; Ca4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate CoA ligase; HCT, hydroxycinnamoyl transferase; C3H, coumaroyl shikimate/quinate 3-hydroxylase; COMT, caffeic acid O-methyltransferase; pAMT, aminotransferase; BCAT, branched-chain amino acid transferase; Kas, 3-keto-acyl ACP synthase; ACL, acyl carrier protein; Fat, acyl-ACP thioesterase; ACS, acyl-CoA synthetase; CS, capsaicin synthase. (b) C. chinense ‘Habanero’ fruit dissected and seeds removed to show interlocular septum, placenta, and pericarp locations. (c) HPLC analysis of capsaicinoid accumulation in leaf, root, pericarp, interlocular septum, and seed tissues of Habanero. Values represent the mean ±SD of at least three plants. Fig. 1. View largeDownload slide Tissue specificity of capsaicinoid accumulation in pepper fruits. (a) Current model of the capsaicinoid biosynthetic pathway. Enzymes are shown adjacent to the reactions they catalyse. For those enzymes underlined, the gene encoding them has been cloned in Capsicum. Kas is the only enzyme functionally characterized in Capsicum; all other enzyme attributions are based on nucleotide similarity and differential gene expression. Pal, phenylalanine ammonia lyase; Ca4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate CoA ligase; HCT, hydroxycinnamoyl transferase; C3H, coumaroyl shikimate/quinate 3-hydroxylase; COMT, caffeic acid O-methyltransferase; pAMT, aminotransferase; BCAT, branched-chain amino acid transferase; Kas, 3-keto-acyl ACP synthase; ACL, acyl carrier protein; Fat, acyl-ACP thioesterase; ACS, acyl-CoA synthetase; CS, capsaicin synthase. (b) C. chinense ‘Habanero’ fruit dissected and seeds removed to show interlocular septum, placenta, and pericarp locations. (c) HPLC analysis of capsaicinoid accumulation in leaf, root, pericarp, interlocular septum, and seed tissues of Habanero. Values represent the mean ±SD of at least three plants. Studies of this locus have revealed that AT3 transcript is expressed specifically in the placenta and begins to accumulate along with several other capsaicinoid biosynthetic genes at 20 days post-anthesis (dpa). It was noted that several capsaicinoid biosynthetic genes, including AT3, were either not expressed or reduced in expression when non-pungent and pungent genotypes were compared (Stewart et al., 2005). Similarly, Curry et al. (1999) and Aluru et al. (2003) observed that transcript accumulation of several capsaicinoid biosynthetic genes was correlated with the level of pungency. In the book, The Paprika, Somos described the presence of blisters that contained capsaicin and suggested it may be possible to regulate capsaicinoid production by regulating the presence of blisters (Somos, 1984). A recent report termed these blister structures ‘vesicles’ and stated that the absence of these capsaicinoid-accumulating vesicles is inherited as a single recessive gene at a new locus designated ‘loss-of-vesicles’ (lov) (Votava and Bosland, 2002). This locus, lov, was proposed as a second locus, in addition to Pun1, that has a qualitative effect on pungency. The current study was undertaken critically to test the genetic relationship between blisters, capsaicinoid biosynthesis, lov, and Pun1. The localization of capsaicinoid biosynthesis and accumulation to the epidermal cells of the interlocular septa in pungent fruits is reported here. The presence of blisters in pungent fruits represents sites of capsaicinoid secretion and storage and is precisely related to capsaicinoid accumulation. A recessive allele of the Pun1 locus was identified and it is shown that in the allelic state at Pun1, transcript accumulation of capsaicinoid biosynthetic genes and capsaicinoid accumulation are highly correlated. Based on these results, it is concluded that mutations at a single locus are responsible for non-pungency within C. annuum and C. chinense and that this locus is also responsible for the presence/absence of the blistered structures that contain capsaicinoids. Materials and methods Plant material Seeds of C. chinense ‘Habanero’ and C. annuum ‘Maor’ were obtained from commercial sources (Tomato Growers Supply Co., Ft. Myers, FL). C. chinense NMCA 30036, previously described by Votava and Bosland (2002), was obtained from the NMSU Chile Pepper Institute. For genetic studies in the field, plants were sown in the greenhouse and then transplanted to fields located in Varna, NY during the summer of 2004. For gene expression studies, all plant materials were grown and maintained in greenhouse conditions at 27 °C day/night with supplemental lighting unless otherwise noted. Scanning-electron and light microscopy Interlocular septum tissue from fresh, unfixed, immature green Habanero and NMCA 30036 fruits were removed and dissected cross-sectionally by hand into 5 mm2 pieces using a razor blade. The samples were mounted on aluminium stubs and coated twice under vacuum with a film of fine-grain gold using a Denton Vacuum Sputter Coater. The surface of the sections was examined under a Hitachi S-3200N Variable Pressure Scanning Electron Microscope (SEM). For light microscopic analysis, the interlocular septa of ripe Habanero fruits were fixed and embedded into low viscosity resin (Electron Microscopy Sciences, Hatfield, PA) as described in Rowland et al. (1983) with the following modifications. After fixation, samples were washed three times with cold 50 mM PIPES buffer, pH 5.6, dehydrated in a graded ethanol series, 30 min at room temperature for each step, and incubated overnight in 100% ethanol at 4 °C. Ethanol was removed through a graded acetone/ethanol series, 30 min at 4 °C for each step, and soaked in acetone for 2–3 h. Samples were infiltrated with resin as described in the manufacturer's protocol. Sections 1 μm thick were cut using a glass knife and stained using a 1% (w/v) crystal violet solution. An Olympus BX60 compound microscope attached to a digital camera and computer for video-capture was used to view samples. Blisters on the epidermis of the interlocular septum of Habanero and NMCA 30036 were photographed at 10 d intervals through fruit development using a dissecting microscope attached to a digital camera mounted with the ScopeTronix MaxView Plus System (Scopetronix, Cape Coral, FL). Images were taken of multiple fruit from multiple plants. Immunolocalization of capsaicinoids Thick sections, approximately 8–10 mm wide, were dissected from the interlocular septa of immature green Habanero and NMCA 30036 fruits (approximately 30 dpa) using a double-edge razor blade. Samples were immediately placed in fixative consisting of 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2, and incubated overnight at 4 °C. Samples were washed five times with 0.1 M sodium phosphate buffer and dehydrated through a graded ethanol series, 30 min at room temperature for each step. Samples were incubated overnight at 4 °C in 100% ethanol, then moved through a graded Histoclear (National Diagnostics, Atlanta, GA) series, 1.5 h at each step, and held overnight in 100% Histoclear at ambient temperature. Samples were infiltrated with Paraplast (Kendall, Mansfield, MA) as per the manufacturer's instructions. Sections, approximately 8 μm thick, were cut, paraffin removed, and rehydrated in a graded ethanol series. A rabbit capsaicin antibody (Beacon Analytical Systems Inc., Portland, ME) was used for immunolocalization at a 1:250 dilution using the ImmPRESS Anti-Rabbit Ig Peroxidase Kit (Vector Laboratories, Burlingame, CA) following the manufacturer's instructions. According to the manufacturer, the capsaicin antibody has reactivity against capsaicin and dihydrocapsaicin, the dominant capsaicinoids in commercial peppers. Sections were viewed using an Olympus BX50 microscope attached to a digital camera. HPLC and ELISA analysis of pungency Extracts of Habanero, Maor, NMCA 30036, and F2 progeny from the cross of Habanero and NMCA 30036 were prepared and analysed for capsaicinoid accumulation via HPLC as previously described in Stewart et al. (2005). Interlocular septa from ripe Habanero fruits were removed and either directly dried or gently brushed with a small spatula to remove blisters and then dried. Capsaicinoids were extracted and analysed via HPLC as previously described in Stewart et al. (2005). The presence/absence of pungency in fruit produced for the genetic complementation tests was assessed with ELISA due to the greater sensitivity of this method for capsaicinoid detection. Multiple ripe fruits from Maor, NMCA 30036, and the F1 progeny of the complementation cross were analysed for capsaicinoid accumulation using the Capsaicin High Sensitivity Plate Kit (Beacon Analytical Sys. Inc, Portland, ME) that utilizes an antibody that specifically recognizes the capsaicinoid small molecule as the epitope. The manufacturer's protocol was followed with the followng modification. Methanol extracts of the dried pepper samples were not diluted prior to analysis. Inheritance and co-segregation of blisters, pungency, and Pun1 Ripe fruit from F1 and F2 populations (n=495), derived from C. chinense Habanero and C. chinense NMCA 30036 and its reciprocal cross, were analysed for pungency and presence/absence of blisters, and then genotyped to determine which Pun1 allele(s) were present. Plants were scored twice independently as pungent or non-pungent by a panel of three trained individuals. Non-pungent phenotypes were confirmed using HPLC as described above. The presence/absence of blisters along the interlocular septum of the fruits was visually scored and the absence of blisters was independently verified by two observers. A PCR-based, co-dominant marker based on the following primers 5′-ATGTCAACCGGCCAGCAGCAT-3′, 5′-CTGATTCTTCTGCCACCTTCAATCCC-3′, 5′-CCCACAATCAAACTTACTTGAAC-3′ was used to determine the allelic state at Pun1 for all individuals. PCR was performed using Advantage 2 Polymerase (Clontech, Palo Alto, CA) on DNA isolated from the interlocular septa according to the method of Doyle and Doyle (1990) with one modification. Microfuge tubes were treated with polyvinylpolypyrrolidone (Sigma P6755), prior to DNA extraction to minimize interference of phenolic compounds during PCR. RNA and total protein from interlocular septa of mature green F2 plants, confirmed homozygous dominant or recessive at Pun1, were isolated as described below to analyse gene expression. Capsaicinoid accumulation in the same samples was analysed via HPLC as described above. Genetic complementation tests to determine whether the absence of pungency in NMCA 30036 and Maor is due to defects at the same or different loci were conducted by crossing NMCA 30036×Maor. The interspecific F1 progeny were confirmed to be hybrids visually and by genotyping. Reciprocal crosses in the opposite direction did not pollinate. Ripe F1 fruits from each cross were assayed for capsaicinoid accumulation via ELISA as described above. RNA gel blot analysis RNA was extracted from pepper fruits at time points through fruit development for gel blot analysis as described in Stewart et al. (2005). Fruits from multiple plants were pooled together to minimize variation due to environmental effects. Due to small fruit size, whole fruits with seeds removed were used for all 10 dpa time points. Ten μg of RNA was transferred to Hybond N+ membrane (Amersham Bioscience, Piscataway, NJ). Full-length AT3 cDNA was radiolabelled using the Prime-It RmT Random Labeling Kit (Stratagene, La Jolla, CA) and hybridized to the membrane according to standard protocols (Sambrook and Russell, 2001). Pal and Kas, known capsaicinoid biosynthetic genes, were radiolabelled and used as probes as described previously (Curry et al., 1999; Aluru et al., 2003; Blum et al., 2003). Filters were washed twice at 65 °C in 2× SSC/0.1% SDS for 10 min and twice in 1× SSC/0.1% SDS for 10 min. Filters were exposed on a PhosphorImager storage screen (Molecular Dynamics). RNA gel bots were replicated at least twice from independent extractions. Immunoblotting of AT3 Habanero, Maor, and NMCA 30036 fruits were harvested at 10 d intervals. Total protein was extracted and blotted as described in Stewart et al. (2005). For detection of the AT3 protein, a rabbit polyclonal antibody against AT3 and an anti-rabbit secondary antibody conjugated to phosphatase (Amersham Bioscience, Piscataway, NJ) were used at 1:2000 and 1:5000 dilutions, respectively. The production, use, and specificity of the AT3 antibody were described previously in Stewart et al. (2005). Coomassie staining of replicate gels from the same extractions or of the probed membrane was used to verify equal protein loading. Blots were developed using the ECL kit (Amersham Biosciences, Piscataway, NJ). Chemiluminescence emitted from the filter was detected using X-ray film. Immunoblots were replicated at least twice. Cloning and sequencing Primer sets based on Habanero genomic DNA were used to isolate homologous sequence from NMCA 30036 using PCR. The pGEM-T Easy Vector System I was used for all subcloning and sequencing (Promega, Madison, WI). Sequencing was performed by the Bioresource Center, Cornell University (www.brc.cornell.edu). Results Localization of capsaicinoid accumulation to the epidermis of the Capsicum interlocular septum A cross-section of a ripe C. chinense Habanero fruit with seeds removed, with the pericarp, placenta, and interlocular septum labelled, is shown in Fig. 1b. Capsaicinoids were extracted from Habanero leaf, root, pericarp, interlocular septum, and seed tissues. Capsaicinoid accumulation, monitored by HPLC, was present in the interlocular septum, 52 000±11 000 parts per million (ppm), and absent from all other tissues sampled (Fig. 1c). Residual capsaicinoids were detected in the pericarp, probably due to imperfect separation of the interlocular septum from the pericarp tissues. Close inspection revealed that when the fruit is cut open or handled roughly, capsaicinoids from the blisters (see below) can contaminate nearby seeds. These results are consistent with early radioisotope analysis localizing capsaicinoids to the interlocular septum of pepper fruits (Iwai et al., 1979). Immunolocalization studies using a capsaicinoid antibody were conducted to determine the cellular location of capsaicinoid accumulation within the interlocular septum. Capsaicinoids were not detected in C. chinense NMCA 30036, a non-pungent variety, or negative controls (Fig. 2a–c). Figure 2d shows the immunolocalization of 3-keto-acyl ACP synthase (KAS), a capsaicinoid biosynthetic enzyme, to the epidermal cells consistent with previous reports (Aluru et al., 2003). Similarly, capsaicinoids were localized to the epidermal cells of the interlocular septum (Fig. 2e). Signal was also detected in the cell layer immediately subtending the epidermal cells, but it was not possible to determine whether this was actually due to the presence of capsaicinoids or an artefact of sample preparation. Capsaicinoids and KAS were detected in all the epidermal cells; their expression was not spatially restricted and did not occur in a noticeable pattern in the epidermal cells. Staining of the specimens with crystal violet, a general cytological stain, revealed cellular differentiation between cells under the blisters compared with cells not under the blisters (Fig. 2g). This suggests their involvement in secretion of capsaicinoids into the subcuticular cavity. The results of the organ-specificity of capsaicinoid biosynthesis and immunolocalization studies taken together establish that capsaicinoid accumulation and biosynthesis are localized to the epidermal cells of the interlocular septum in the pepper fruit. Fig. 2. View largeDownload slide Immunolocalization of capsaicinoid biosynthesis to the interlocular septum epidermis in pungent and non-pungent genotypes. (a) Habanero (pungent), rabbit preimmune serum; (b) Habanero, secondary antibody, diluted 1:1500; (c) C. chinense NMCA 30036 (non-pungent), capsaicinoid antibody diluted 1:250; (d) Habanero, KAS antibody, diluted 1:250; (e) Habanero, capsaicinoid antibody, diluted 1:250; (f, g) Habanero interlocular septum sections stained with Crystal Violet. Bar=50 μm (a–e) or 10 μm (f, g). bl, blister; gl, glandular cells. Fig. 2. View largeDownload slide Immunolocalization of capsaicinoid biosynthesis to the interlocular septum epidermis in pungent and non-pungent genotypes. (a) Habanero (pungent), rabbit preimmune serum; (b) Habanero, secondary antibody, diluted 1:1500; (c) C. chinense NMCA 30036 (non-pungent), capsaicinoid antibody diluted 1:250; (d) Habanero, KAS antibody, diluted 1:250; (e) Habanero, capsaicinoid antibody, diluted 1:250; (f, g) Habanero interlocular septum sections stained with Crystal Violet. Bar=50 μm (a–e) or 10 μm (f, g). bl, blister; gl, glandular cells. Association of blisters and presence of capsaicinoids Examination of the blistered structures observed on the interlocular septa of pepper fruit showed that they were only present in the pungent genotype, Habanero, and were entirely absent from the non-pungent genotype, NMCA 30036 (Fig. 3a). To examine the relationship between blisters and pungency more closely, the capsaicinoid accumulation along the interlocular septum was measured before and after brushing the tissue to remove blisters along the surface (Fig. 3b). Similar to studies where exudates are produced in glandular trichomes, it was not possible to remove all blisters from the interlocular septum by brushing (Fridman et al., 2005). Capsaicinoid accumulation in brushed interlocular septa was 20 600±3700 ppm compared with 52 000±6800 ppm in unbrushed samples, a reduction of 60% (Fig. 3). Fig. 3. View largeDownload slide Localization of capsaicinoid accumulation to blisters along the epidermis of the interlocular septum in Habanero. (a) The epidermal surface of the interlocular septum in Habanero (top) and NMCA 30036 (bottom), pungent and non-pungent, respectively, were viewed using a scanning electron microscope. (b) Comparison of the absolute levels of capsaicinoids in Habanero interlocular septa tissue with or without a brushing treatment to remove blisters. Septum tissue was extracted directly with acetonitrile (not brushed) after drying and weighing or gently brushed (brushed) before drying, weighing, and extraction. Values represent the mean ±SD of at least three plants. Fig. 3. View largeDownload slide Localization of capsaicinoid accumulation to blisters along the epidermis of the interlocular septum in Habanero. (a) The epidermal surface of the interlocular septum in Habanero (top) and NMCA 30036 (bottom), pungent and non-pungent, respectively, were viewed using a scanning electron microscope. (b) Comparison of the absolute levels of capsaicinoids in Habanero interlocular septa tissue with or without a brushing treatment to remove blisters. Septum tissue was extracted directly with acetonitrile (not brushed) after drying and weighing or gently brushed (brushed) before drying, weighing, and extraction. Values represent the mean ±SD of at least three plants. The distribution of blisters was examined to understand the relationship between blister development and capsaicinoid accumulation. Blisters were not observed on interlocular septa of ripe non-pungent NMCA 30036 or 10 dpa Habanero fruits (Fig. 4a, b). Blisters were first observed at 20 dpa in Habanero fruit and persisted throughout fruit development (Fig. 4c–f). Generally, the size of the individual blisters increased markedly during fruit development although definitive measurements were hard to make. Consistent with this trend, capsaicinoids did not begin to accumulate in Habanero until 20 dpa, gradually increasing throughout fruit development (Fig. 4g). These results and the previous finding that capsaicinoid biosynthesis is not restricted to cells under the blisters suggest that the blisters act primarily as a site of capsaicinoid storage for materials synthesized in the epidermal cells. Fig. 4. View largeDownload slide Association of blister development and capsaicinoid accumulation. (a) Interlocular septum of a ripe NMCA 30036 fruit. Bar=250 μm. (b–f) Habanero fruits were harvested at 10 d intervals, and the interlocular septa photographed. All images are representative of at least three fruits. Bar=250 μm. (g) HPLC analysis of capsaicinoid accumulation through fruit development in Habanero, NMCA 30036, and C. annuum Maor. Capsaicinoids were extracted from interlocular septa tissue except for 10 d post-anthesis samples, in which whole fruits with seeds removed, were used due to small fruit size. Values represent the mean ±SD for at least three plants. Fig. 4. View largeDownload slide Association of blister development and capsaicinoid accumulation. (a) Interlocular septum of a ripe NMCA 30036 fruit. Bar=250 μm. (b–f) Habanero fruits were harvested at 10 d intervals, and the interlocular septa photographed. All images are representative of at least three fruits. Bar=250 μm. (g) HPLC analysis of capsaicinoid accumulation through fruit development in Habanero, NMCA 30036, and C. annuum Maor. Capsaicinoids were extracted from interlocular septa tissue except for 10 d post-anthesis samples, in which whole fruits with seeds removed, were used due to small fruit size. Values represent the mean ±SD for at least three plants. Genetic analysis of pungency, blisters, and Pun1 An F2 population (n=495) derived from reciprocal crosses of Habanero×NMCA 30036 was generated to confirm an earlier report that the presence/absence of blisters along the interlocular septum is controlled by the single recessive gene, lov, at a second locus controlling pungency (Votava and Bosland, 2002). Habanero is blistered and pungent while NMCA 30036 is non-blistered and non-pungent. The resulting F1 plants were blistered and pungent, confirming the dominance of blisters and pungency. Results for the F2 population, shown in Table 1, indicate that absence of blisters precisely co-segregated with non-pungency. Blisters were never observed in non-pungent plants. In a testcross population, blisters co-segregated exactly with pungency consistent with a 1:1 ratio, confirming monogenic inheritance and perfect association of these traits in C. chinense (Table 1). Table 1. Genetic analysis and co-segregation of blisters, pungency, and Pun1 genotype   n  Pungent  Non-pungent  Expected ratio  Chi-square    Pop. size  Bl  Bl  NB  NB  (Bl:NB)  (P-value)      Pun1  pun12  Pun1  pun12      P1 Habanero  20  20  0  0  0      P2 NMCA 30036  22  0  0  0  22      F1 (P1×P2)  1  1  0  0  0      F1 (P2×P1)  8  8  0  0  0      F2 [(P1×P2)×(P1×P2)]  214  169  0  0  45  3:1  1.80 (0.180)  F2 [(P2×P1)×(P2×P1)]  281  202  0  0  79  3:1  1.45 (0.228)  Pooled F2  495  371  0  0  124  3:1  0.0007 (0.979)  BC1P1 [(P2×P1)×P2)]  85  48  0  0  37  1:1  1.42    n  Pungent  Non-pungent  Expected ratio  Chi-square    Pop. size  Bl  Bl  NB  NB  (Bl:NB)  (P-value)      Pun1  pun12  Pun1  pun12      P1 Habanero  20  20  0  0  0      P2 NMCA 30036  22  0  0  0  22      F1 (P1×P2)  1  1  0  0  0      F1 (P2×P1)  8  8  0  0  0      F2 [(P1×P2)×(P1×P2)]  214  169  0  0  45  3:1  1.80 (0.180)  F2 [(P2×P1)×(P2×P1)]  281  202  0  0  79  3:1  1.45 (0.228)  Pooled F2  495  371  0  0  124  3:1  0.0007 (0.979)  BC1P1 [(P2×P1)×P2)]  85  48  0  0  37  1:1  1.42  A F2 population (n=495) derived from reciprocal crosses Habanero (Pun1/Pun1, pungent, blistered)×NMCA 30036 (pun12/pun12, non-pungent, not blistered) was analysed for the inheritance and co-segregation of blisters, pungency, and Pun1. Bl=blistered; NB=not blistered. View Large The genomic sequence at the Pun1 locus was determined in NMCA 30036 to develop PCR-based markers for testing whether Pun1 segregated independently from the blister phenotype. During the development of these primers, the Pun1 locus from C. chinense was sequenced. A deletion of four base pairs (bp) located centrally in the first exon was identified in C. chinense NMCA 30036 at the Pun1 locus (Fig. 5). This deletion was confirmed by sequencing cDNA from 30 dpa NMCA 30036 fruit. Sequence analysis predicted that this 4 bp deletion causes a frameshift mutation which results in a truncated AT3 protein. The truncated AT3 is not predicted to include the putative active site, HXXXDG (St Pierre and De Luca, 2000). Fig. 5. View largeDownload slide Genomic DNA structure of the Pun1 locus from pungent (Pun1/Pun1) and non-pungent (pun1/pun1; pun12/pun12) genotypes. Schematic diagram of the Pun1, pun1, and pun12 alleles. Exons (closed boxes) were deduced from cDNA sequence. Open boxes depict exons predicted not to be transcribed due to a deletion in the gene. The deletions in the pun1 and pun12 alleles are represented with an inverted triangle. Transcription start/end sites and translation stop codons are also indicated. The putative active site of AT3 is identified by two dots. Primers used for genotyping are indicated with arrows. Fig. 5. View largeDownload slide Genomic DNA structure of the Pun1 locus from pungent (Pun1/Pun1) and non-pungent (pun1/pun1; pun12/pun12) genotypes. Schematic diagram of the Pun1, pun1, and pun12 alleles. Exons (closed boxes) were deduced from cDNA sequence. Open boxes depict exons predicted not to be transcribed due to a deletion in the gene. The deletions in the pun1 and pun12 alleles are represented with an inverted triangle. Transcription start/end sites and translation stop codons are also indicated. The putative active site of AT3 is identified by two dots. Primers used for genotyping are indicated with arrows. Based on this deletion, a co-dominant marker system (Fig. 5) for Pun1 in C. chinense was developed. Three primers were used for genotyping, with primer 3 spanning the deletion found in NMCA 30036. When all the primers are mixed together during PCR, the reaction favours primer 3 as the reverse primer in the NMCA 30036 genotypes, generating a 0.5 kb amplicon (the shorter of two possible amplicons). Alternatively, in Habanero genotypes, primer 2 is the preferred reverse primer and generates a 1.1 kb amplicon. Primer 3 does not generate a significant amplification product in Habanero genotypes due to base pair mismatching between the primer and template. Genotyping of F2 individuals from the segregating population described above revealed that this 4 bp deletion, tentatively named pun12, co-segregated precisely with the absence of blisters and non-pungency in the F2 population shown in Table 1. These results suggest that non-pungency in NMCA 30036 previously attributed to a second locus, lov, is actually perfectly associated with a 4 bp deletion at the Pun1 locus. To confirm this result, genetic complementation analysis was performed to assess whether lov in C. chinense and Pun1 in C. annuum are allelic. Complementation tests to determine allelic diversity of the Pun1 locus To test the hypothesis that the 4 bp deletion identified in the Pun1 locus of NMCA 30036 is the gene previously designated lov, a genetic complementation test was performed by crossing C. chinense NMCA 30036×C. annuum Maor (non-pungent, pun1/pun1). Capsaicinoid accumulation was not detectable in either parent, NMCA 30036 or Maor (Table 2). Capsaicinoid accumulation in all F1 plants was below the lower detection limit of the ELISA assay system (0.50 ppm) indicating that the loss-of-pungency mutations in Maor and NMCA 30036 fail to complement. These data conclusively establish that the locus previously designated lov is actually a second recessive allele of the Pun1 locus, defined by a 4 bp deletion. According to conventions in Capsicum gene nomenclature, it is proposed that this allele be designated pun12, and listed as synonymous with lov. The F1 plants that were recovered from this cross did produce fruit, but did not produce viable seeds and were deformed and exhibited virus-like symptoms, which is often observed in the progeny of such crosses. The reciprocal cross could not be obtained. Table 2. Genetic complementation tests to determine allelism at the Pun1 locus in non-pungent genotypes   Genotype  n  Capsaicinoid accumulation (ppm)  Maor  pun1  8  nda  NMCA 30036  pun12  8  nd  NMCA 30036×Maor  pun12×pun1  2  nd*(<0.50±0.10)b    Genotype  n  Capsaicinoid accumulation (ppm)  Maor  pun1  8  nda  NMCA 30036  pun12  8  nd  NMCA 30036×Maor  pun12×pun1  2  nd*(<0.50±0.10)b  Parents and F1 hybrids were assayed for capsaicinoid accumulation using an ELISA system. a nd, Not detectable. b Asterisk denotes capsaicinoid accumulation was below limits of assay detection. View Large Regulation of AT3, Pal, and Kas gene expression in peppers containing the alleles, Pun1, pun1, and pun12 Transcript accumulation of capsaicinoid biosynthetic genes is hypothesized to correlate with the level and presence of pungency (Curry et al., 1999; Aluru et al., 2003). The expression patterns of AT3, a putative acyltransferase encoded by the Pun1 locus, and phenylalanine ammonia lyase (Pal) and Kas, two structural genes implicated in capsaicinoid biosynthesis, were examined in Habanero (Pun1/Pun1), Maor (pun1/pun1), and NMCA 30036 (pun12/pun12). In Habanero, Pal and Kas expression was similar; both were strongly expressed by 20 dpa before decreasing gradually throughout later fruit development (Fig. 6a). Pal and Kas were specific to the interlocular septum, although a small amount of signal was detected in flowers and seeds. This background probably resulted from the fact that both Pal and Kas are members of gene families and are essential genes in phenylpropanoid and fatty acid metabolism, respectively (Blum et al., 2003). Fig. 6. View largeDownload slide Gene expression patterns of capsaicinoid biosynthetic genes Pal, Kas, and AT3 during fruit development in Pun1, pun1, and pun12 genotypes. (a) Pal, Kas, and AT3 expression was analysed in leaf, flower, seed, pericarp, and interlocular septum tissues through development at 10 d intervals. The full-length cDNA of AT3 was hybridized to blots containing RNA from Habanero, Maor, and NMCA 30036. Whole fruit was used for samples taken at 10 dpa due to their small size. (b) Immunoblot analysis of AT3 expression in the same tissues as described above. Total protein was probed with an AT3 polyclonal antibody (1:2000 dilution) as the primary antibody and horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000 dilution). Whole fruits were used for samples taken at 10 dpa due to their small size. Membranes were Coomassie stained after probing to show equal loading of protein. Fig. 6. View largeDownload slide Gene expression patterns of capsaicinoid biosynthetic genes Pal, Kas, and AT3 during fruit development in Pun1, pun1, and pun12 genotypes. (a) Pal, Kas, and AT3 expression was analysed in leaf, flower, seed, pericarp, and interlocular septum tissues through development at 10 d intervals. The full-length cDNA of AT3 was hybridized to blots containing RNA from Habanero, Maor, and NMCA 30036. Whole fruit was used for samples taken at 10 dpa due to their small size. (b) Immunoblot analysis of AT3 expression in the same tissues as described above. Total protein was probed with an AT3 polyclonal antibody (1:2000 dilution) as the primary antibody and horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000 dilution). Whole fruits were used for samples taken at 10 dpa due to their small size. Membranes were Coomassie stained after probing to show equal loading of protein. AT3 expression was also specific to the interlocular septum and was first detected at 20 dpa. AT3 expression persisted throughout much of fruit development but was not detectable by 50 dpa. The expression patterns of Pal, Kas, and AT3 closely followed that of capsaicinoid accumulation throughout fruit development (Fig. 4g). Capsaicinoids were first detected at 20 dpa and increased through fruit development. At 50 dpa capsaicinoids still accumulated, even though expression of Pal, Kas, and AT3 was not detected. It is plausible that metabolic turnover, for example, a reduction in the degradation of capsaicin, could account for the continued capsaicinoid accumulation at 50 dpa (Bernal et al., 1993; Estrada et al., 2000). In Maor, both Pal and Kas had significantly reduced transcript accumulation throughout much of fruit development relative to Habanero (Fig. 6a). Similar to Habanero, however, there was background expression of both Pal and Kas in leaf, flower, seed, and pericarp tissues. In addition, there was strong expression of Pal and Kas in the seeds, probably due to lignins and fatty acids known to accumulate in seeds (Buchanan et al., 2000). AT3 expression in Maor was not detectable during fruit development. This observation was consistent with the lack of capsaicinoid accumulation and was also consistent with previous results showing that the deletion in AT3 disrupts transcription and translation (Fig. 4g) (Stewart et al., 2005). Pal and Kas had similar expression profiles in NMCA 30036 (Fig. 6a). Both Pal and Kas were strongly expressed early in fruit development, 10 dpa versus 20 dpa in Habanero. At 20 dpa, however, both Pal and Kas were less abundant in NMCA 30036 than in Habanero. Expression levels of Pal and Kas were higher in NMCA 30036 than in Maor, perhaps due to genotype- or species-specific differences. In NMCA 30036, AT3 transcripts were expressed at significantly lower levels when compared with Habanero from 20 dpa through 50 dpa (Fig. 6a). AT3 protein accumulation in Habanero followed the same pattern as transcript accumulation, first detected at 20 dpa, and reduced by 50 dpa (Fig. 6b). AT3 protein was not detectable in Maor or NMCA 30036 genotypes, except in seeds. The absence of AT3 protein accumulation in these genotypes is consistent with their mutations, a 2.5 kb deletion in pun1 and a 4 bp deletion in pun12, which disrupt translation. A cross-hybridizing band was detected in the seed tissues of all genotypes tested. This band persisted despite variations in protein extraction procedures and was consistently found in pungent and non-pungent genotypes. There are probably several acyltransferases involved in the production and storage of fatty acids and oils operating in seed tissues (Buchanan et al., 2000). A similar cross-hybridizing band was reported in the isolation and characterization of DAT, an acyltransferase involved in vindoline biosynthesis (St Pierre et al., 1998). Segregation of gene expression and Pun1 genotype RNA gel blots were made from representative samples of mature green (approximately 40 dpa) fruit from the F2 homozygous progeny classes resulting from the cross of Habanero×NMCA 30036. Pal gene expression did not segregate between the different F2 classes (Fig. 7b). This is consistent with the gene expression studies shown in Fig. 6 which show that Pal is similarly expressed in both Habanero and NMCA 30036 at 40 dpa. Kas appeared to accumulate more in Pun1/Pun1 F2 progeny even though Kas expression at 40 dpa between the two parental genotypes is not significantly different. It is plausible that this difference in Kas gene expression is a result of environmental variation between the parental genotypes shown in (Fig. 6) and F2 progeny (Fig. 7b). AT3 was clearly more abundant in the Pun1/Pun1 progeny than in the pun12/pun12 progeny. Within the Pun1/Pun1 class there were not major differences in AT3 expression even though total capsaicinoid accumulation varied significantly (Fig. 7d). AT3 protein was not detectable in pun12/pun12 progeny, consistent with the genetic lesion that produces the truncated protein (Fig. 7c). Taken together, the results clearly establish a strong relationship between the allelic state of Pun1, pungency, and AT3 and Kas expression. Fig. 7. View largeDownload slide Segregation of Pal, Kas, and AT3 gene expression with the allelic state of Pun1 in a C. chinense Habanero (Pun1/Pun1)×C. chinense NMCA 30036 (pun12/pun12) F2 population. (a) Agarose gel showing PCR bands from Pun1 genotyping of the F2 population. Numbers represent individual plants. 1 kb, P1, and P2 represent the 1 kb DNA ladder, Habanero parent, and NMCA 30036 parent, respectively. (b) RNA gel-blots of Pal, Kas, and AT3 expression in mature green F2 interlocular septum tissue. EtBr staining of rRNA was used as a loading control. Numbers represent individual plants. (c) Immunoblot analysis of AT3 expression in mature green interlocular septum tissue. Total protein was probed with an AT3 polyclonal antibody (1:2000 dilution) as the primary antibody and horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000 dilution). Equal protein loading was confirmed by Coomassie staining the membrane after probing. (d) HPLC analysis of capsaicinoid accumulation in the interlocular septa of mature green Pun1/Pun1 and pun12/pun12 F2 progeny. Values represent the mean ±SD of duplicate extractions. Fig. 7. View largeDownload slide Segregation of Pal, Kas, and AT3 gene expression with the allelic state of Pun1 in a C. chinense Habanero (Pun1/Pun1)×C. chinense NMCA 30036 (pun12/pun12) F2 population. (a) Agarose gel showing PCR bands from Pun1 genotyping of the F2 population. Numbers represent individual plants. 1 kb, P1, and P2 represent the 1 kb DNA ladder, Habanero parent, and NMCA 30036 parent, respectively. (b) RNA gel-blots of Pal, Kas, and AT3 expression in mature green F2 interlocular septum tissue. EtBr staining of rRNA was used as a loading control. Numbers represent individual plants. (c) Immunoblot analysis of AT3 expression in mature green interlocular septum tissue. Total protein was probed with an AT3 polyclonal antibody (1:2000 dilution) as the primary antibody and horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000 dilution). Equal protein loading was confirmed by Coomassie staining the membrane after probing. (d) HPLC analysis of capsaicinoid accumulation in the interlocular septa of mature green Pun1/Pun1 and pun12/pun12 F2 progeny. Values represent the mean ±SD of duplicate extractions. Discussion Due to centuries of human manipulation, taxonomists disagree on the criteria necessary to identify some of the species and varieties of Capsicum. In particular, C. annuum, C. frutescens, and C. chinense are referred to as the C. annuum complex due to the difficulty of demarcating the individual species. Members of this complex include pungent and non-pungent, wild and domesticated varieties which interbreed and are morphologically similar (Walsh and Hoot, 2001). In previous research, it was established that the pun1 allele is responsible for non-pungency within C. annuum as a result of a large deletion at Pun1 that has been conserved and propagated for several centuries (Stewart et al., 2005). In this report, another recessive allele of Pun1, namely pun12, is identified. DNA sequencing of pun12 revealed a 4 bp deletion in the centre of the first exon of AT3. Inheritance studies revealed that pun12 co-segregated with the absence of blisters, non-pungency, and decreased expression of two capsaicinoid biosynthetic genes, Kas and AT3. Genetic complementation tests confirmed that pun12 is allelic to pun1. We have tentatively identified another allele of pun1 in C. frutescens (GM Stellari and M Mazourek, unpublished data). This study establishes that, to date, only mutations in Pun1 define known genetic sources of non-pungency in domesticated Capsicum spp. Results from this study support the long-held assertion that capsaicinoids are synthesized primarily or exclusively in the interlocular septa of pungent fruits. This tissue has independently been referred to as the septum (Ohta, 1962; Rowland et al., 1983), dissepiment (Furuya and Hashimoto, 1954; Balbaa et al., 1968), cross wall (Huffman et al., 1978), or placenta (Iwai et al., 1977a, b, 1979; Fujiwake et al., 1980; Suzuki et al., 1980), generating confusion in the literature. In small-fruited varieties, the distinction between the placenta and interlocular septum is difficult to discern and in other varieties this tissue breaks down during ripening (Rao and Paran, 2003). Huffman et al. (1978) attributed the small amounts of capsaicinoids detected in the seeds to surface contamination during dissection. There has been persistent debate in the literature regarding the existence of glands that accumulate capsaicinoids. At least two reports concluded that the existence of specialized glands containing capsaicinoids could not be verified (Erwin, 1932; Huffman et al., 1978). Conversely, other reports have documented the presence of capsaicin-secreting glands, sometimes called receptacles, vesicles, or blisters, along the interlocular septum of pungent genotypes (Furuya and Hashimoto, 1954; Ohta, 1962; Suzuki et al., 1980; Rowland et al., 1983; Zamski et al., 1987; Votava and Bosland, 2002). In botany, the term ‘gland’ is used broadly, often having the connotation of specialized secretory structures (e.g. glandular trichomes of tomato or mint). However, any cells secreting specialized metabolites can be referred to as glandular even if they lack glandular trichomes (Fahn, 1979). For example, the papillae cells of the stigma surface are called glandular based on their secretion of carbohydrates and lipids to aid pollen tube growth, yet they lack formal secretory structures (Buchanan et al., 2000). The term blister has been used to describe the epidermal swellings produced by the filling of subcuticular cavities along the interlocular septum of pungent peppers (Rowland et al., 1983; Zamski et al., 1987). In an analogous system, the resin exudate of epidermal cells along young leaves and stipules in Populus spp. (Salicaceae) is secreted into the space between the outer wall of the cells and the cuticle, forming a bulge or blister (Langehheim, 2003). In this article, the term blister was used in reference to such epidermal swellings in pungent pepper fruits and the term glandular cells in broad reference to any cells that secrete capsaicin. Blisters in Habanero begin forming at 20 dpa concomitant with capsaicinoid accumulation. This link between specialized morphological features, blisters, and their specialized metabolites, capsaicinoids, is consistent with studies in sage and thyme (Lamiaceae), showing that the development of the glandular trichomes is correlated with essential oil production (Croteau et al., 1981; Yamaura et al., 1989). Immunolocalization of capsaicinoids and Kas, a subunit of the fatty acid synthase complex involved in capsaicinoid biosynthesis, showed that the epidermal cells are the site of capsaicinoid biosynthesis. Capsaicinoid biosynthesis is not restricted to the cells immediately subtending the blisters but occurs along the entire epidermis. Based on differential staining observed during microscopic analysis, the cells immediately under the blister are glandular cells, or idioblasts, that secrete capsaicinoids. Capsaicinoids are secreted into spaces between the cell wall and the cuticle and, as the pressure builds, the cuticle is separated and bulges to form a blister with a subcuticular cavity (Furuya and Hashimoto, 1954; Ohta, 1962; Rowland et al., 1983; Zamski et al., 1987). Similar to the blisters on resin-secreting tissues in Populus spp., the pepper blisters are formed from the exudate secreted from multiple cells; however, it is not known whether capsaicinoids are transported intercellularly from nearby cells not immediately under a blister (Langehheim, 2003). These results are consistent with the conclusion of Rowland et al. (1983), who reported that capsaicinoids were only found in the glandular areas along the interlocular septum of Jalapeño peppers. Several reports in the literature describe blisters in non-pungent Capsicum varieties (Rowland et al., 1983; Zamski et al., 1987). Such a phenomenon was not observed in this analysis. A possible source of these observations may be that intense yellow-pigmented areas occur in both pungent and non-pungent varieties; however, careful examination reveals no association with pungency. This is consistent with previous work that concluded that the presence of the yellow pigmentation does not necessarily indicate the presence of capsaicinoids (Huffman et al., 1978). A previous report stated that the presence/absence of blisters constituted a second locus controlling pungency, lov, in the non-pungent C. chinense genotype NMCA 30036 (Votava and Bosland, 2002). Analysis of F2 and testcross populations indicated that the absence of blisters and non-pungency perfectly co-segregate in every population examined, consistent with the possibility that a single defect has pleiotropic effects on capsaicinoid biosynthesis and blisters. This implies that capsaicinoid biosynthesis is required for blister formation. Genetic complementation tests reported here definitively prove that non-pungency in NMCA 30036 is due to a recessive allele at the Pun1 locus herein designated pun12. Our results also support the hypothesis that the transcriptional state of some genes in the capsaicinoid biosynthetic pathway is positively correlated with pungency (Curry et al., 1999; Aluru et al., 2003). The complete absence of AT3 protein accumulation in pun1 and pun12 genotypes is consistent with a lack of capsaicinoid accumulation. The pun1 allele is characterized by the absence of AT3 expression, owing to a 2.5 kb deletion of AT3, and residual levels of Pal and Kas expression (Stewart et al., 2005). In pun12 genotypes, a small 4 bp frameshift mutation results in the absence of AT3 protein accumulation. Also within pun12 both Pal and Kas are expressed at levels greater than pun1 but less than Pun1. A strong up-regulation of several capsaicinoid biosynthetic genes (pAMT, Pal, Kas, BCAT, FatA) occurs 20 dpa after flowering coinciding with capsaicinoid accumulation in pungent varieties of both C. annuum and C. chinense (Stewart et al., 2005). In addition, the transcript accumulation of AT3 and Kas at 20 dpa was higher in pungent genotypes than in non-pungent genotypes from both C. annuum and C. chinense. This pattern of expression was confirmed in an F2 population. This suggests that the association of elevated transcript levels of genes involved in capsaicinoid biosynthesis with capsaicinoid biosynthesis, itself, is consistent across multiple Capsicum species. While there were small differences in Pal expression between pungent and non-pungent varieties, its expression did not segregate in an F2 population. This indicates that coordinated transcriptional regulation of capsaicinoid biosynthesis occurs downstream of Pal with genes that are specific to this metabolic pathway. Current research efforts aim to characterize relevant gene expression in non-pungent Capsicum accessions drawn from diverse species and to analyse the genetic basis for variation in the expression of genes implicated in this biosynthetic pathway. The identification of a new recessive allele of Pun1 and the development of co-dominant markers will be useful for marker-assisted selection in breeding programmes and studies of allelic diversity at loci relevant for agriculture. Growing evidence suggests that dramatic changes in phenotypes during plant domestication can result from perturbations in single dominant genes, such as Pun1. The gain and loss of strawberry flavour, the architecture of maize inflorescence, and the liberating of maize kernels from the hardened fruitcases of the maize progenitor teosinte are all traits controlled by single dominant genes (Aharoni et al., 2004; Gallavotti et al., 2004; Wang et al., 2005). Manipulation of such genes will be useful in the metabolic engineering of natural products for the benefit of humanity. We would like to acknowledge Mary Kreitinger, George Moriarity, Matthew Falise, Arnon Ben-Chaim, Steve Czaplewski (Syngenta Seeds), Lynn Bohs, Soumitra Goshroy, and Dominick Paolillo for technical assistance and useful discussions. We would also like to thank Robert Jarret, USDA Capsicum Germplasm Collection, for assistance. We would like to acknowledge Beacon Analytical Systems, Inc. for donation of the capsaicinoid antibody. This work was supported in part by USDA IFAFS Plant Genome Award No. 2001-52100-11347, NSF Metabolic Biochemistry Award No. 0412056 and supplemental awards, NSF Plant Genome 0218166 and NSF MCB 0417056, to CS and MMJ and NIH GMS06-08136 to MOC. 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Zocchi, Graziano;De Nisi, Patrizia;Dell'Orto, Marta;Espen, Luca;Gallina, Pietro Marino

doi: 10.1093/jxb/erl259pmid: 17229758

Abstract Iron deficiency responses were investigated in roots of soybean, a Strategy I plant species. Soybean responds to iron deficiency by decreasing growth, both at the root and shoot level. Chlorotic symptoms in younger leaves were evident after a few days of iron deficiency, with chlorophyll content being dramatically decreased. Moreover, several important differences were found as compared with other species belonging to the same Strategy I. The main differences are (i) a lower capacity to acidify the hydroponic culture medium, that was also reflected by a lower H+-ATPase activity as determined in a plasma membrane-enriched fraction isolated from the roots; (ii) a drastically reduced activity of the phosphoenolpyruvate carboxylase enzyme; (iii) a decrease in both cytosolic and vacuolar pHs; (iv) an increase in the vacuolar phosphate concentration, and (v) an increased exudation of organic carbon, particularly citrate, phenolics, and amino acids. Apparently, in soybean roots, some of the responses to iron deficiency, such as the acidification of the rhizosphere and other related processes, do not occur or occur only at a lower degree. These results suggest that the biochemical mechanisms induced by this nutritional disorder are differently regulated in this plant. A possible role of inorganic phosphate in the balance of intracellular pHs is also discussed. Fe(III)-chelate reductase, Glycine max L., intracellular pH, iron deficiency, PM H+-ATPase Introduction Iron is an essential oligo-element for all living organisms, including plants, since, as a transition element, it takes part in fundamental biological redox processes, such as respiration and photosynthesis, and in chlorophyll biosynthesis (Marschner, 1995). Soils are normally well furnished with iron, which is the fourth element in the Earth's crust, but in well-aerated and in calcareous soils it is found in oxide and hydroxide compounds with a very low solubility (Lindsay and Schwab, 1982), so that, in these conditions, plants often have to face an iron availability that is limited. Plants respond to shortage of iron by inducing responses directed towards the acquisition of the element from the rhizosphere, and, according to the way they respond, they have been divided in Strategy I (dicots and non-Graminaceous monocots) and Strategy II (Graminaceae) (Marschner and Römheld, 1994) plants. The most complex responses are induced in Strategy I plants, that base their iron supply on a reduction-based mechanism (Schmidt, 1999), while Strategy II plants base their iron acquisition on the release of phytosiderophores and the subsequent uptake of the Fe3+-phytosiderophore complex (Curie and Briat, 2003). The reduction mechanism implies the activation of a Fe(III)-chelate reductase (FC-R), that is the ‘conditio sine qua non’ for the acquisition of the ion, since only the ferrous form is transported into the root (Chaney et al., 1972; Yi and Guerinot, 1996). The putative redox chain has not yet been identified in plasma membrane, but two sequences coding FC-R activities, named FRO2 and FRO1 have recently been cloned from A. thaliana, pea, and tomato, respectively (Robinson et al., 1999; Waters et al., 2002; Li et al., 2004). However, the whole response to Fe-deficiency is not only limited to this mechanism, since there are other important events that come along with it. First of all, the induction of a specific transporter belonging to the ZIP family, named IRT1 (Iron-Regulated Transporter) (Guerinot, 2000) localized on the root plasma membrane, has been shown to be co-regulated with FRO2 in A. thaliana (Vert et al., 2003). The gene for IRT1 has been cloned from A. thaliana (Eide et al., 1996; Vert et al., 2002) and its activity identified through a functional expression in the yeast double mutant fet3fet4 (Eide et al., 1996). IRT1 isologues have also been found in pea (Cohen et al., 1998) and tomato (Eckhardt et al., 2001). As stated before, the major problem plants have to cope with regarding iron acquisition is its scarce availability largely due to its low solubility. In fact, in well-aerated soils the predominant form is the ferric form and its solubility is very scarce in the physiological pH range (Marschner, 1995). To solve this problem, plants have developed, under low-iron conditions, the capacity to decrease the rhizospheric pH by increasing proton extrusion. It has been shown that this process is linked to the activation of a specific plasma membrane H+-ATPase of the root epidermal cells (Zocchi and Cocucci, 1990; Rabotti and Zocchi, 1994; Dell'Orto et al., 2000), with the aim of increasing the solubility of the sparingly insoluble iron forms by decreasing the pH of the rhizosphere, in order to generate a driving force for the uptake of the ion (Zocchi and Cocucci, 1990) and to facilitate the access of Fe-chelates to the site of reduction by neutralizing the negative charges, thereby decreasing the repulsion effect. As well as these activities, which are all located on the root plasma membrane, it has been found that the metabolism is strongly involved in order to sustain the production of reducing equivalents [NAD(P)H] and ATP (Rabotti et al., 1995; Espen et al., 2000; Zocchi, 2006). In particular, it has been shown that the activity of the PEPC is increased up to several fold (De Nisi and Zocchi, 2000; Lopéz-Millán et al., 2000). This increase could be linked with the production of substrates for the FC-R and H+-ATPase activities and generation of H+ for the cytosolic pH-stat (Espen et al., 2000). The activation of these processes makes a plant more or less efficient in the acquisition of iron. In this work it is shown that, in soybean, the mechanisms supporting the Fe-deficiency response are differently activated with respect to other Strategy I plants already studied. Materials and methods Plant material and growth conditions Seeds of soybean (Glycine max L. cv. A2012 from Asgrow, Italy) were used in this work. According to the company this cultivar ranks excellent for the tolerance to the Fe-deficiency chlorosis. Seeds were surface-sterilized, sown in agriperlite, watered with 0.1 mM CaSO4, allowed to germinate in the dark at 26 °C for 6 d, and then transferred to a nutrient solution with the following composition: 2 mM Ca(NO)3, 0.75 mM K2SO4, 0.65 mM MgSO4, 0.5 mM KH2PO4, 10 μM H3BO3, 1 μM MnSO4, 0.5 μM CuSO4, 0.5 μM ZnSO4, 0.05 μM (NH4)Mo7O24, and 0.1 mM Fe-EDTA (when added). The pH was adjusted to 6.0–6.2 with NaOH. Aerated hydroponic cultures were maintained in a growth chamber with a day/night regime of 16/8 h and a PPFD of 200 μmol m−2 s−1 at the plant level. The temperature was 18 °C in the dark and 24 °C in the light. Culture medium was changed weekly. Plants showed chlorotic symptoms after approximately 10 d of culture in the absence of Fe. Previous experiments carried out with agar-embedded roots in the presence of a pH indicator (bromocresol purple) and of BPDS had shown that the responses (acidification and Fe(III) reduction, respectively) were localized in the first 3–4 cm of the root apices (not shown). For this reason all the in vivo and in vitro analyses were carried out on 4-cm-long root apical segments. Leaf chlorophyll determination Leaf chlorophyll was extracted in 80% (v/v) acetone from fully expanded youngest leaves; solutions were centrifuged at 10 000 g for 10 min prior to measuring the absorbance in a spectrophotometer (model V550, Jasco). Chlorophyll content was determined according to Lichtenthaler (1987). Measurement of the acidification capacity of the nutrient solution Acidification of the medium was determined directly in the nutrient solution by measuring the pH every day with a pHM64 (Radiometer, Copenhagen) pH-meter. In vivo FC-R activity Fe(III)-reductase activity was measured in excised roots by using bathophenanthrolinedisulphonate (BPDS) (Chaney et al., 1972). Ten apical root segments, about 4 cm long, were incubated in 5 ml of a solution with the following composition: 0.5 mM CaSO4, 0.1 mM Fe(III)-EDTA, 0.25 mM BPDS, 10 mM MES-NaOH pH 5.5 in the dark at 25 °C. After 3 h 2 ml of the solution were withdrawn and the absorbance at 535 nm was determined spectrophotometrically. The results are linear over the experimental period. The amount of reduced Fe was calculated by the concentration of the Fe(II)- (BPDS)3 complex formed (ϵ of BPDS is 22.1 mM−1 cm−1). Contribution of the material released by the roots on the reduction of Fe3+ was less than 5% of the total. Isolation of plasma membrane vesicles Plasma membrane-enriched vesicles from 14-d-old plant root apical segments grown in the presence or absence of Fe were prepared by the two-phase partitioning procedure as previously described (Rabotti and Zocchi, 1994). Final pellet collected at 80 000 g for 30 min was resuspended in a medium containing 250 mM sucrose, 2 mM MES-BTP (pH 7.0) and 4 mM PMSF. Determination of H+-ATPase activity The H+-ATPase activity of plasma membrane (PM) vesicles was determined with a spectrophotometric method (Palmgren et al., 1990), coupling ATP hydrolysis to NADH oxidation at 25 °C in 1.5 ml final volume as already reported (Rabotti and Zocchi, 1994). Reaction was started by addition of 20–50 μl of PM preparation. NADH oxidation was followed at 340 nm and the absorbance changes monitored over a 5 min period. Determination of FC-R activity The NADH-dependent FC-R activity of PM vesicles was assayed in a medium containing 250 mM sucrose, 15 mM MOPS-BTP (pH 6.0), 0.25 mM K3Fe(CN)6, 0.25 mM NADH, and 0.01% Lubrol (Sigma-Aldrich). Reaction was started by the addition of 20–50 μl of PM preparation. NADH oxidation was followed at 340 nm and the absorbance changes monitored over a 5 min period. Enzyme assays Cytosolic soluble enzymes were extracted from 14-d-old plant root apical segments grown in the presence or absence of Fe as already reported (Rabotti et al., 1995). Hexokinase (HK) (EC 2.7.1.1) activity was determined in a buffer containing 82.5 mM triethanolamine-NaOH (pH 7.6), 2 mM MgSO4, 1 mM EDTA, 0.5 mM PEP, 0.2 mM NADH, 1 mM ATP, 5 mM glucose, 30 μg ml−1 PK (EC 2.7.1.40), and 15 μg ml−1 LDH (EC 1.1.1.27). Phosphofructokinase 1 (PFK-1) (EC 2.7.1.11) and pyruvate kinase (PK) were determined as already reported by Espen et al. (2000) while phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) was determined as reported by De Nisi and Zocchi (2000). Reaction was started by adding aliquots of protein extracts and the enzymatic assay was performed at 25 °C in 1.5 ml final volume. Oxidation of NADH was followed spectrophotometrically at 340 nm. Proteins were determined by the Bradford method using γ-globulin as the standard (Bradford, 1976). Collection of root exudates After 14 d of hydroponic culture with or without Fe, plants were rinsed and transferred to vessels containing 300 ml of distilled water. Root exudates were collected for a period of 4 h. After the collection, Micropur® was added to the exudates to prevent microbial degradation of organic solutes. The samples were then cooled to 0 °C and filtered through filter paper. Solutions containing root exudates were then concentrated by evaporation and again filtered on a 0.22 μm cellulose acetate filters. This final solution was used for quantification of citrate, amino acids, phenols, and total organic carbon. Quantification of citrate, amino acids, phenols and total soluble carbon in root exudates Citrate was quantified enzymatically, using a specific kit from Boehringer-Mannheim; the assay was performed according to the manufacturer's instructions. Total amino acids were quantified by the ninhydrin method according to Hirs et al. (1954). Total phenolics in the root exudates were estimated by Folin–Ciocalteau method, as described by Singleton and Rossi (1965), measuring the absorbance at 750 nm. Phenolics concentration was calculated from the calibration curve using caffeic acid as a standard. Finally, soluble organic carbon was determined by using a modified procedure according to Von Wirén et al. (1995) through oxidation with K2Cr2O7, and the Cr3+ formation was determined spectrophotometrically at 578 nm. Nuclear magnetic resonance spectrometry The 31P-NMR spectra were recorded on a standard broad-band 10 mm probe on a Bruker AMX 600 spectrometer (Bruker Analytische Messtechnik, Rheinstetten-Forchheim, Germany) equipped with an X32 data system, running UXNMR software, version 920801. The 31P-NMR spectra were recorded at 242.9 MHz without lock, with a Waltz-based broad-band proton decoupling and a spectral window of 16 kHz. The spectra were acquired using a 90 °C pulse angle and a 6 s recycle time to give fully relaxed resonance (except for vacuolar phosphate). In vivo31P-NMR experiments were carried out by packing about 35 root segments (up to 4 cm, excised from the tip) obtained from 14-d-old plants grown in the presence or absence of Fe, into a 10-mm-diameter NMR tube equipped with a perfusion system connected to a peristaltic pump in which the aerated, thermoregulated (26 °C) medium [0.5 mM CaSO4, 1 mM MDP, 1 mM MES-BTP (pH 6.1)] flowed at 10 ml min−1, as described by Espen et al. (2000). Cytoplasmic and vacuolar pHs were estimated from the chemical shift of Pi resonance after construction of a standard titration curve (Roberts et al., 1980, 1981). Pi content was estimated measuring the area of the 31Pi resonances by Lorential line-shape analysis and the values obtained were referred to the percentage volume of the tissue in the NMR tube (Spickett et al., 1992; Espen et al., 2000). Measurement of Pi levels Roots from plants grown in the presence or in the absence of Fe were homogenized in 4 vols of 10% (v/v) ice-cold trichloroacetic acid and centrifuged at 13 000 g for 15 min. Inorganic phosphate was determined in the supernatant using the Fiske and Subbarow (1925) method. Statistical analyses Values are the means ±SE of three independent experiments in triplicate. One-way analysis of variance (ANOVA) was used for all the tested parameters and the means were compared by Student t test at P ≤0.05. Data reported in Table 8 were treated with a two-way analysis of variance (ANOVA) by using the Tukey test at P ≤0.05. Results Soybean plants responded to Fe shortage by greatly decreasing growth both at the root and shoot level (Table 1). Interveinal chlorosis of the younger leaves is a classical symptom of iron deficiency and in these leaves the chlorophyll content was dramatically decreased after 14 d of Fe deficiency (Table 2). Table 1. Root and shoot fresh weight of 14-d-old soybean plants grown in the presence (control) or in the absence (−Fe) of Fe   Root  Shoot  Control  1.18±0.03 b  3.07±0.08 b  −Fe  0.80±0.03 a  1.89±0.02 a    Root  Shoot  Control  1.18±0.03 b  3.07±0.08 b  −Fe  0.80±0.03 a  1.89±0.02 a  Plants were harvested and roots and shoot separated and the fresh weight determined. Data are expressed as g−1 FW plant and represent mean values ±SE from three independent experiments in triplicate. Different letters correspond to significant difference at P ≤0.05. View Large Table 2. Change in the chlorophyll content of 14-d-old plants grown in the presence (control) or in the absence (−Fe) of Fe Chlorophyll    Control  2.03±0.09 b  −Fe  0.19±0.07 a  Chlorophyll    Control  2.03±0.09 b  −Fe  0.19±0.07 a  Data are expressed as mg chlorophyll g−1 FW and represent mean values ±SE from three independent experiments in triplicate. Different letters correspond to significant difference at P ≤0.05. View Large The time-course of the medium acidification by the soybean plant roots grown in hydroponic culture in the presence and in the absence of iron, along with their capacity to reduce the iron chelate Fe(III)-EDTA in vivo, are shown in Fig. 1. The acidification of the medium occurs, but at a very low degree, less than 1.0 pH unit, even after 16 d of Fe deficiency. When compared with other plants like cucumber, for instance, where the acidification reaches a difference of about 2.5 pH units in a few h (Zocchi and Cocucci, 1990; Rabotti and Zocchi, 1994), pH changes occurring with soybean are very poor. Conversely, the activity of the Fe3+ reduction in vivo is efficient and reaches a maximum after 12 d of hydroponic culture. This is the reason why the choice was made to use 14-d-old plants to perform all the measurements both in vivo and in vitro. The assay of the enzymatic activities involved in the iron reduction and in the acidification of the medium was also determined on a plasma membrane-enriched fraction isolated from roots. As determined in vivo, the activities of the two enzymes involved were enhanced under Fe deficiency also in vitro, with the increase in FC-R being roughly around 100%. The increase in H+-ATPase activity was small, only around 24% (Table 3), confirming data obtained in vivo. The lower H+-ATPase activity under Fe deficiency is also coherent with a very low transmembrane electrical potential difference determined between +Fe and −Fe roots, less than 10 mV (not shown), as compared to values found in cucumber which were around 40 mV (Zocchi and Cocucci, 1990). Table 3. Activities of plasma membrane enzymes in soybean roots   FC-R  H+-ATPase  Control  122.9±7.8 a  37.6±2.2 a  −Fe  252.2±15.8 b  46.5±3.0 b    FC-R  H+-ATPase  Control  122.9±7.8 a  37.6±2.2 a  −Fe  252.2±15.8 b  46.5±3.0 b  Fe(III)-chelate reductase and H+-ATPase activities were determined in 14-d-old plants grown in the presence (control) or in the absence (−Fe) of Fe. Data are expressed as nmol NADH mg−1 protein min−1 and represent the mean ±SE of three independent experiments in triplicate. Different letters correspond to significant difference at P ≤0.05. View Large Fig. 1. View largeDownload slide Effect of iron deficiency on the acidification of the nutrient solution and on the Fe(III)-EDTA reduction in vivo. The induction of FC-R activity (open symbols) and acidification of medium solution (closed symbols) was compared under Fe-sufficient (square) and Fe-deficient (circles) conditions. Data are representative of one typical experiment repeated three times with similar results. Fig. 1. View largeDownload slide Effect of iron deficiency on the acidification of the nutrient solution and on the Fe(III)-EDTA reduction in vivo. The induction of FC-R activity (open symbols) and acidification of medium solution (closed symbols) was compared under Fe-sufficient (square) and Fe-deficient (circles) conditions. Data are representative of one typical experiment repeated three times with similar results. Other metabolic activities that are usually increased with Fe deficiency, and it has been assumed to be important for the whole response to Fe deficiency (Espen et al., 2000; Zocchi, 2005). These include phosphoenolpyruvate carboxylase, that has been shown to increase in cucumber and sugar beet roots (De Nisi and Zocchi, 2000; Lopéz-Millán et al., 2000). The determination of some glycolytic enzyme activities also confirms such an increase for the soybean plants, while, on the contrary, it was quite surprising to find that the PEPC activity was decreased under the same condition (Table 4). Table 4. Activities of soluble enzymes in soybean roots   HK  PFK1  PK  PEPC  Control  346±18 a  321±19 a  142±9 a  170±4 b  −Fe  445±21 b  418±18 b  224±11 b  131±6 a    HK  PFK1  PK  PEPC  Control  346±18 a  321±19 a  142±9 a  170±4 b  −Fe  445±21 b  418±18 b  224±11 b  131±6 a  Hexokinase (HK), phosphofructokinase-1 (PFK-1), pyruvate kinase (PK), and phosphoenolpyruvate carboxylase (PEPC) activities were determined in 14-d-old plants grown in the presence (control) or in the absence (−Fe) of Fe. Data are expressed as nmol NADH mg−1 protein min−1 and represent the mean ±SE of three independent experiments in triplicate. Different letters correspond to significant difference at P ≤0.05. View Large The total organic carbon in exudates from soybean roots under Fe-deficiency was almost doubled with respect to the control; the analysis of the specific compounds in exudates (citrate, amino acids, and phenolics) shows that their increase follows more or less what occurs for the total organic carbon (Table 5). On a per cent basis, these compounds account for 6.0, 7.5, and 4.8% of the total exuded carbon, respectively, for the −Fe condition. These values are of the same order of magnitude of those found by Römheld and Marschner (1983) for Fe-deficient peanut plants. Table 5. Effect of iron deficiency on the release of citrate, amino acids, phenolics, and total soluble organic carbon from 14-d-old roots grown in the presence (control) or in the absence (−Fe) of Fe   Citrate  Amino acids  Phenolics  Organic carbon  Control  1.9±0.06 a  2.3±0.07 a  1.6±0.04 a  32±0.1 a  −Fe  3.3±0.03 b  4.2±0.05 b  2.7±0.04 b  56±0.4 b    Citrate  Amino acids  Phenolics  Organic carbon  Control  1.9±0.06 a  2.3±0.07 a  1.6±0.04 a  32±0.1 a  −Fe  3.3±0.03 b  4.2±0.05 b  2.7±0.04 b  56±0.4 b  Data are expressed as μg g−1 FW h−1 and represent mean values ±SE from three independent experiments in triplicate. Different letters correspond to significant difference at P ≤0.05. Phenolics are expressed as caffeic acid-equivalents. View Large More information on the biochemical changes that occur in soybean roots grown in the presence and in the absence of iron were obtained by a 31P-NMR analysis. The in vivo spectra obtained with roots of 14-d-old plants are shown in Fig. 2. The spectra are characteristic of a well oxygenated plant tissue and were stable over several hours of data acquisition. The well-defined peaks of resonance allowed for the definition of the intracellular pHs and a quantitative determination of the Pi. Iron starvation induced several changes and the most evident was the increase in the vacuolar phosphate content and the increase in the sugar phosphate and NTP (more likely ATP) peaks (Fig. 2). A quantification of the intracellular Pi concentration is shown in Table 6, where an increase in the vacuolar Pi content (+83%) is evident. Values of the pHc and pHv are reported in Table 7. Iron-deficiency induces a slight, but still significant decrease, both in the pHc and to a greater extent in the pHv. These data are consistently different from those determined in a similar study conducted with cucumber roots, where it was found that the vacuolar phosphate was almost completely depleted under Fe-deficiency and the intracellular pHs were slightly increased (Espen et al., 2000). The concentration of Pi in TCA extracts from roots at different growing times is reported in Table 8. The results show that the Pi level in the tissue was in agreement with the increase seen in the in vivo31P-NMR experiments. Table 6. Effect of iron deficiency on intracellular cytoplasmic and vacuolar Pi content   Intracellular Pi    Cytosol  Vacuole  Control  0.77  12.43  −Fe  0.90  22.70    Intracellular Pi    Cytosol  Vacuole  Control  0.77  12.43  −Fe  0.90  22.70  Intracellular and vacuolar Pi content in 14-d-old plants grown in the presence (control) or in the absence (−Fe) of Fe. Pi concentration was calculated from the intensities of resonances of the 31P-NMR spectra, by comparison with those of standard solutions previously calibrated against 33 mM MDP contained in a capillary tube, and were referred to the per cent volume of the tissue in the NMR tube. Data are expressed as μmol ml−1 tissue volume and represent the mean of three independent experiments. SE never exceeded 5%. View Large Table 7. Changes in intracellular pHs   pHc  pHv  Control  7.55  5.76  −Fe  7.50  5.66    pHc  pHv  Control  7.55  5.76  −Fe  7.50  5.66  Determination of pHc and pHv was carried out on root segments excised from 14-d-old plants grown in the presence (control) or in the absence (−Fe) of Fe. The pH values were calculated from the chemical shift of Pi after construction of a standard titration curve and are the average of three independent experiments. The accuracy was calculated as three times the maximum variation observed in the experiments and is equal to ±0.02 and ±0.04 pH units for the cytoplasm and the vacuole, respectively. View Large Table 8. Concentration of total soluble Pi Plant age (d)  Soluble Pi    Control  −Fe  8  15.8±0.5 c  13.5±0.4 d  10  15.2±0.4 c  19.1±0.7 b  14  11.0±0.5 e  27.6±0.7 a  16  11.7±0.7 e  27.2±0.6 a  Plant age (d)  Soluble Pi    Control  −Fe  8  15.8±0.5 c  13.5±0.4 d  10  15.2±0.4 c  19.1±0.7 b  14  11.0±0.5 e  27.6±0.7 a  16  11.7±0.7 e  27.2±0.6 a  Pi concentration was determined on extracts from soybean root segments of plants grown in the presence (control) or in the absence (−Fe) of Fe. Data are expressed as μmol Pi g−1 FW and represent mean values ±SE from at least three independent experiments in triplicate. In the case of significant interactions between plant age and treatment, values followed by different letters, are statistically different (P ≤0.05). View Large Fig. 2. View largeDownload slide 31P-NMR spectra of excised soybean root segments obtained from 14-d-old plants grown in the absence (−Fe) or in the presence (+Fe) of iron. Chemical shifts are quoted relative to 85% H3PO4. The resonance assignments are as follows: 1, Glc6P; 2, Fru6P; 3, phosphocholine; 4, cytosolic Pi; 5, vacuolar Pi; 6, γ-phosphate of NTP and β-phosphate of NDP; 7, α-phosphate of NTP and NDP, UDP-Glc and NAD(P)H; 8, UDP-Glc; 9, β-phosphate of NTP. In three independent experiments the resonance intensities differed by 12% at the most. The nucleotide region is shown on an expanded (×6) scale. Fig. 2. View largeDownload slide 31P-NMR spectra of excised soybean root segments obtained from 14-d-old plants grown in the absence (−Fe) or in the presence (+Fe) of iron. Chemical shifts are quoted relative to 85% H3PO4. The resonance assignments are as follows: 1, Glc6P; 2, Fru6P; 3, phosphocholine; 4, cytosolic Pi; 5, vacuolar Pi; 6, γ-phosphate of NTP and β-phosphate of NDP; 7, α-phosphate of NTP and NDP, UDP-Glc and NAD(P)H; 8, UDP-Glc; 9, β-phosphate of NTP. In three independent experiments the resonance intensities differed by 12% at the most. The nucleotide region is shown on an expanded (×6) scale. Discussion Strategy I plants have been classified on the basis of their biochemical responses to Fe deficiency (Marschner and Römheld, 1994), the main process being a reduction-based mechanism that reduces apoplastic Fe3+ to Fe2+, the form necessary for uptake. In the strategy adopted by dicots and non-graminaceous monocots, every species can modulate the activation of biochemical mechanisms as a function of specific environmental conditions or according to intrinsic characteristics, such as the capacity to modify the metabolism in order to sustain the energetic effort or to extrude inorganic (ions, protons) or organic (amino acids, organic acids, phenolics, flavins, etc.) compounds (Zocchi, 2006, and references therein). To understand the whole response to Fe deficiency it is important to know not only which mechanisms are induced by the Fe-deficient plants but, also, how plants may regulate those mechanisms. Alkaline and calcareous soils represent a serious concern for iron acquisition by plants, since in these conditions the range of inorganic iron availability is around 0.1–10% of the normal requirement for optimal plant growth (Römheld and Marschner, 1986). One-third of the world's soil are below the level of iron bioavailability, limiting plant growth and productivity. All plant species belonging to the Strategy I growing on calcareous soils suffer from Fe deficiency, but there is an inter- and an intraspecific variability in their susceptibility to this nutritional disorder, despite a similar demand for iron. These genotypic differences are strongly related to the ability of plants to mobilize iron in the rizosphere and to their capacity to take up the ion. Soybean plants are particularly sensitive to the presence of bicarbonate and/or to elevated soil pH, since even tolerant genotypes greatly suffer under these conditions (M Dell'Orto, personal communication). If the results obtained with other plant species, such as cucumber, are compared, distinctive differences in the responses to Fe deficiency can be noticed. Rizosphere acidification has been recognized for many years as an important response to iron deficiency for Strategy I plants (Römheld and Marschner, 1986; Schmidt, 1999), and this response has been associated with the activity of a plasmalemma-localized H+-ATPase (Zocchi and Cocucci, 1990; Dell'Orto et al., 2000). However, unlike the FC-R, increased H+-ATPase activity has not always been observed, and the extent of such stimulation seemed to differ considerably among plant species and genotypes, ranging from very low values to about 100% (Schmidt, 1999, and references therein). For instance, in cucumber roots, one of the most active acidifying species, it was shown that a higher activity under iron deficiency was associated with an enhancement in the level of the enzyme (Dell'Orto et al., 2000) and of its transcript (Dell'Orto et al., 2002). However, for many other dicots, including soybean, this capacity is strongly limited even in the efficient genotypes (Brown and Jones, 1976; Landsberg, 1982), although there is evidence for localized acidification of the rhizosphere (Römheld and Marschner, 1984). In the literature only one report (Yi and Guerinot, 1996) has clearly shown acidification under iron deficiency by Arabidopsis roots. This could explain why in Arabidopsis, Thimm et al. (2001), using microarray analysis, did not find any change in the expression of the gene encoding the root H+-ATPase. A similar scenario seems to occur in soybean, where the increase in the H+-ATPase activity is quite low both in vivo and in vitro (Fig. 1; Table 3). Another striking difference is the development of transfer cells in the apical root portion. Some authors correlated the increase in the medium acidification with the development of transfer cells. However, these morphological modifications do not occur in soybean roots (Landsberg, 1982; this work, not shown). Therefore it could be hypothesized that, beyond the fundamental step of iron reduction, the efficiency responses rely on other reactions that could be differently regulated among different species. The 31P-NMR study has shown that both pHc and pHv, as determined by the chemical shift of Pi, are decreased in soybean plant roots grown under Fe deficiency. This was quite surprising, since, working with cucumber, it had previously been found that both pHc and pHv had increased (Espen et al., 2000). From Fig. 2 and from Table 6 further important information is apparent. When comparing the results obtained with those found in cucumber plants (Espen et al., 2000), a major difference exists with regard to the relative amount of vacuolar Pi, which is enhanced in soybean whereas it is almost completely depleted in cucumber (Espen et al, 2000). How could this different compartmentalization of Pi be explained? It was claimed (Davies, 1973) that the plasmalemma H+-ATPase and the PEPC activities are part of the plant pH-stat mechanism. Plants might have developed different extra-help systems to balance the pHc, one of these could involve the Pi movements in the cell. As suggested by Kurkdjian and Guern (1989) the vacuole could actively participate in the physiological responses and to the poise of pHc and, as hypothesized by Espen et al. (2000), Pi could also be involved in a mechanism of pH-stat. Two different scenarios might be predicted. In the first one, suitable for species active in the acidification of the rhizosphere, increased PM-H+-ATPase activity corresponds with the alkalinization of the pHc which, in turn, activates PEPC. The major need of H+ in the cytosol to keep the pH at its physiological level might come from increasing the rate of glycolysis (Sakano, 1998) and/or from a de-protonation of H2PO4− released from the vacuole (Espen et al., 2000). In the case of soybean a second picture might be hypothesized. The lower activity of the PM H+-ATPase (Fig. 1; Table 3) is associated with a decrease in the pHc (Table 7) also as the result of a more active glycolytic pathway producing H+ (Table 4). Decrease in the pHc would, as a consequence, decrease the activity of the PEPC (Table 4). In soybean, H+ could be pumped into the vacuole to re-equilibrate the pHc to its physiological value (in fact the pHv decreases, Table 7) by a tonoplast-associated H+-ATPase and/or H+-translocating PPase, which would facilitate the Pi transport into the vacuole (Table 6). There is evidence for ATP-dependent Pi transport across the tonoplast (Sakano et al., 1995). The last two mechanisms could be stimulated by the increased amount of the cytosolic ATP (Fig. 2) and/or PPi as the consequence, from either a lower utilization by the PM-H+-ATPase, or from active catabolism. A simple model to explain the movement of Pi across the tonoplast and its possible involvement in the pH-stat mechanisms are depicted in Fig. 3. At present, no biochemical or molecular information are available for the Pi transporters nor on the activity of the H+ translocating mechanisms at the tonoplast, and further work will be necessary to elucidate these mechanisms and their implication in the mechanism of intracellular pH poise. In conclusion, from this work it emerges that the efficient response to iron starvation comes from many different responses; the more a plant is able to activate such activities the more efficient it will be in iron acquisition. Expression and regulation of these responses could be different among species or genotypes providing them with a different level of efficiency. Fig. 3. View largeDownload slide Model for intracellular Pi movements at the tonoplast level. 1, ATP or PPi H+ pumping; 2, Pi influx mechanism(s); 3, Pi efflux mechanism(s). Fig. 3. View largeDownload slide Model for intracellular Pi movements at the tonoplast level. 1, ATP or PPi H+ pumping; 2, Pi influx mechanism(s); 3, Pi efflux mechanism(s). Abbreviations Abbreviations BPDS bathophenanthrolinedisulphonate BTP 1, 3-bis[tris(hydroxymethyl)-methylamino]-propane FC-R Fe(III)-chelate reductase MDP methylenediphosphonate MOPS 4-morpholinopropanesulphonic acid PEP phosphoenolpyruvate PEPC phosphoenolpyruvate carboxylase pHc cytosolic pH pHv vacuolar pH PM plasmalemma PMSF phenylmethylsulphonyl fluoride PPFD photosynthetic photon flux density This work was supported by grants from the Italian Ministry of Education (Cofin 2004) and FIRST 2004 to GZ. We thank Dr Silvia Donnini for her help in the statistical analysis. 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Jiménez, Carlos; Cossío, Belén R.; Rivard, Christopher J.; Berl, Tomás; Capasso, Juan M.

doi: 10.1093/jxb/erl260pmid: 17220513

Abstract In mammalian cells, MAPKs are involved in both stress response (JNK and p38 pathways) and cell proliferation and differentiation [extracellular signal-regulated kinase (ERK)] through protein kinase cascades. Exposure of Dunaliella viridis cell cultures to PD98059, a very specific inhibitor of the ERK signalling pathway, resulted in a total arrest of cell proliferation and a complete dephosphorylation of ERK. As shown by flow cytometry analysis of propidium iodide-stained cells, PD98059 stopped mitosis at the G2 phase after the S phase has been completed. Multiple physiological parameters such as cell motility and reducing power generation (NADPH) clearly indicate that the treated cells are wholly viable. Exposure of D. viridis to environmental stresses that impair cell division, such as hyperosmotic shock, nitrogen starvation, or sublethal UV irradiation, caused a marked decrease in the phospho-ERK levels as detected by western blot. Two 400 bp polynucleotides from D. viridis with high homologies to published sequences of ERK1 and ERK2 were cloned, sequenced, and submitted to GenBank. Northern blot analysis revealed two mRNA bands of ∼1.9 kb, consistent with the expected size of ERK proteins (∼40 kDa). Sequence analysis showed that they contained several mitogen-activated protein kinase (MAPK) conserved domains, including II, III, VIb, VII, and the double phosphorylation motif. Interestingly, in D. viridis, this motif was T*DY* instead of the canonic T*EY*. Based on this finding, ERK plant sequences can be divided into two groups, one termed the T*DY* branch and the other termed the T*EY* branch. The molecular and functional data presented here suggest that ERK is a very ancient signalling pathway and that it was already present in the last common ancestor of all eukaryotic cells. Cell division, Dunaliella, ERK, environmental stress Introduction Animals have three well-characterized mitogen-activated protein kinase (MAPK) cascades that participate in the cellular response to a wide variety of stress factors. These cascades consist of a series of protein kinases that phosphorylate and activate in a sequential fashion the associated downstream protein kinase. In animals, the p38 and the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) cascades are responsible for stress adaptation, while the extracellular signal-regulated kinase (ERK) cascade is involved in mitogenic stimuli and differentiation (Widmann et al., 1999). In agreement with Hirt (1997) and Tena and Renaudin (1998), MAPKs might also be involved in signal transduction of several environmental factors in plants; thus their survival in a permanently changing environment has required development of sophisticated defence and adaptation mechanisms. In a previous work (Jiménez et al., 2004), the presence of p38-like and SAPK/JNK-like MAPK signalling pathway components was demonstrated in the unicellular microalga Dunaliella viridis, and it was shown that operation of these cascades is crucial for adaptation and survival of this microalga upon hyperosmotic stress. MAPKs are involved in signalling both cell proliferation and differentiation in mammals through the ERK phosphorylation pathway (Marshall, 1995). This pathway is structurally similar to, but functionally distinct from the other two MAPK cascades. It has been reported that in animal cells many different stimuli (e.g. growth and neurotrophic factors, cytokins, hormones and neurotransmitters, virus infection, transforming agents, and carcinogens) activate both the ERK1 and ERK2 pathways. For example, senescent cells do not phosphorylate ERK1/2 in human fibroblasts (Park et al., 2002), and activation (phosphorylation) of ERK is required for induced lens cell proliferation and fibre differentiation (Lovicu and McAvoy, 2001); moreover, inhibition of ERK signalling can block the morphological changes associated with lens fibre differentiation. Inhibition of ERK1/2 phosphorylation also reduces mitosis in fetal lung explants, diminishing branching morphogenesis and epithelial proliferation (Kling et al., 2002). Inhibition of the specific upstream MAPK kinase (MAPKK) of the ERK (MEK) reduced the serum-stimulated DNA synthesis and proliferation of Swiss 3T3 cells (Willard and Crouch, 2001). In plants, various proteins with characteristics of MAPKs have been identified as being involved in cell proliferation and developmental processes (Jonak et al., 1993; Mizoguchi et al., 1994; Wilson et al., 1997; Préstamo et al., 1999; Huang et al., 2002; Mishra et al., 2006; Zhang et al., 2006). Furthermore, cellular differentiation and proliferation were found to be accompanied by an increase in the expression of ERKs (Coronado et al., 2002). ERK homologues have been identified in tobacco cell suspensions, as a consequence of activation in plant pathogen defence (Lebrun-García et al., 1998). These ERK homologues have apparent molecular weights of 46 kDa and 50 kDa, slightly larger than their human homologues (p42/p44), and react with anti-human ERK1/2 antibodies. In addition, results showed that the progression of both differentiation and proliferation processes was accompanied by an increase in the expression of ERKs together with the nuclear targeting of these proteins. The relative proportion of ERKs in the nucleus versus the cytoplasm was higher in cells undergoing proliferation (Coronado et al., 2002), suggesting that the translocation of ERKs to the nucleus could be associated with the initiation of proliferation, as indicated for other MAPKs in mammalian (Chen et al., 1992) and meristematic plant cells (Préstamo et al., 1999). Duerr et al. (1993) reported the recovery of a full-length cDNA clone encoding a MAPK from alfalfa of 44 kDa with characteristics of an ERK. Bögre et al. (1999, 2000) detected the alfalfa MKK3 and tobacco NTF6 proteins and their associated activity only in dividing cells. Novikova et al. (2000) found that in Arabidopsis thaliana both ERK1 and phosphotyrosine antibodies immunoprecipitated MBP (myelin basic protein)-phosphorylating activity, detecting a polypeptide band of 47 kDa with characteristics of mammalian ERK-like MAPKs (Mishra et al., 2006; Zhang et al., 2006). ERK activation occurs through phosphorylation of threonine and tyrosine (e.g. 202 and 204 of human MAPK ERK, or 183 and 185 of rat ERK2) at the sequence T*EY*, by a single upstream MAPKK. In this work, it is demonstrated that MAPKs of the ERK family are present in the unicellular microalga D. viridis, and that its phosphorylation is required for cell division. Moreover, for the first time, the sequence of a cDNA of an ERK from a unicellular plant is presented. Materials and methods Algae culture Dunaliella viridis Teodoresco was grown axenically at 2 M NaCl as previously described (Jiménez et al., 2004). Cell density was determined by means of counting viable cells using a haemocytometer. Experimental condition Cells in their mid-exponential growth phase were used in experiments, and were subjected to various stresses including: (i) osmotic stress; (ii) UV irradiation; and (iii) nutritional stress. Osmotic stress involved increasing the osmotic pressure of the medium by the addition of NaCl to the culture medium to a final concentration of 4 M NaCl. The influence of UV irradiation was studied by exposing cultures of D. viridis to non-lethal doses of 40 mJ cm−2 of UV light in the range 200–400 nm using a GS Gene Linker UV chamber (Bio-Rad). Cultures were placed in Petri dishes of 14 cm in diameter, and after UV exposure they were kept under continuous orbital shaking at an irradiance of 150 μmol m−2 s−1 of visible light (400–700 nm). Samples were withdrawn at the described times. For nutritional stress, cells were centrifuged at 1500 g for 10 min, and resuspended in nitrate-free medium; this procedure was repeated twice. Treatment with inhibitors Appropriate volumes of concentrated PD98059 (Calbiochem, La Jolla, CA, USA) inhibitor solution in dimethylsulphoxide (DMSO) (>1000×) were added to a final concentration in the culture medium of 20 μM. This is a selective and cell-permeable inhibitor of the ERK MAPKK (MEK), that acts by inhibiting the phosphorylation of the ERK and the subsequent phosphorylation of downstream substrates. Western blot analysis At fixed times, 50 ml of culture were centrifuged at 1500 g for 10 min. The pellets were resuspended in 1 ml of MAPK lysis buffer (Capasso et al., 2001), and treated as previously described (Jiménez et al., 2004). Protein concentration was determined by the bicinchoninic acid method (Pierce, Rockford, IL, USA). SDS-PAGE, electroblot to poly(vinylidene difluoride) membrane, and immunodetection were performed as previously described (Capasso et al., 2001; Jiménez et al., 2004). Antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Phospho-p44/p42 MAP kinase (Thr202/Tyr204) antibody detects endogenous levels of p44 and p42 MAP kinase (ERK1 and ERK2) only when catalytically activated by phosphorylation at Thr202 and Tyr204 of human ERK, or Thr183 and Tyr185 of rat ERK2. The antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAPK. Band analysis was performed as previously described (Capasso et al., 2001). Cell viability Cell viability measurements were performed by means of the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium] colorimetric assay, as previously described (Capasso et al., 2003). RNA purification and cDNA synthesis Mid log cultures of D. viridis were harvested by low speed centrifugation (1500 g, 10 min) and cell pellets were lysed in 10 ml of TRIZOL (Life Technologies Inc.) with a Polytron (Dispersing and Mixing Technology); phase separation was obtained by the addition of chloroform (1:5 v/v) and centrifugation at 12 000 g for 15 min at 4 °C. The aqueous phase was collected, precipitated with isopropanol (1:2 v/v), and centrifuged. The resulting pellet was washed twice with 75% ethanol and air-dried. RNA was further purified using the RNeasy protocol (Qiagen) as per the manufacturers' protocol. Nucleic acid quantity and purity were assessed by UV spectrometry (using the absorbance at 260 nm and the 260/232 nm ratio, respectively). RNA integrity was assessed by capillary electrophoresis using the 28S:18S rRNA ratio. Total RNA (5 μg) was reverse transcribed to cDNA with Superscript (Life Technologies Inc.) using random hexamers. Primer design To design the ERK1 amplification primers, protein and nucleotide sequences from the following organisms were retrieved from GenBank and analysed by protein CrustalW alignment: Medicago sativa (accession no. J224336), Blumeria graminis (accession no. AF301165), Giardia intestinalis (accession no. AY149274), and Mus musculus (accession no. NM_011952). Several conserved regions were detected, in particular, the amino acid sequences VAIKKI and TRWYRAPE. Using these data, the following degenerated primers were designed: ERK1-F1 5′-GTKGCBATMAAGAAGAT-3′ and ERK1-R1 5′-GGSGCKCKRTACCARCGHGT-3′. For the ERK2 amplification primers, the following GenBank data were utilized: Giardia intestinalis (accession no. AY149275), Plasmodium falciparum (accession no. X82646), and Homo sapiens (accession no. NM_139021). The conserved amino acid sequences TDAQRTFREI and YVATRWYR were used to design the following degenerated polymerase chain reaction (PCR) primers: ERK2-F1 5′-ACMGATGCYCARMGMAC-3′ and ERK2-R1 5′-TACCAKCGHGTKGCHACRTA-3′. PCR and cloning cDNA samples were amplified using the above-described primers and the Advantage cDNA PCR kit (Clontech) in a Mastercycler gradient apparatus (Eppendorf). Products were analysed by 2% agarose gel electrophoresis, and bands of interest were detected by ethidium bromide staining and excised. DNA was extracted with the Geneclean II Kit (BIO 101), ligated to the pGEM-TEasy vector (Promega) and transfected into JM109 competent cells (Promega). Insert-containing clones were selected by the ampicillin resistance and blue/white test. Plasmid DNA from white colonies was obtained using a QIAprep system (Qiagen) and analysed by agarose gel electrophoresis upon digestion with NotI. Relevant clones were sequenced with the T7 and SP6 primers. Northern blot analysis RNA isolated as described above was separated by agarose gel electrophoresis as described by Liu and Chou (1990) and transferred to a nylon membrane by using the Genie blotter (Idea Scientific) as suggested by the manufacturer. The primer DERK1Sp-R 5′-GTCTGTCCAGAAGATGGGGGATGTTGCTGG-3′ was used as an ERK1-specific probe; this sequence is complementary to the partially cloned D. viridis ERK1 mRNA (see Results). The primer DERK2-Prb 5′- CTGGGACACTGAGCGAGCAAGACCATAGTC-3′ was used as an ERK2-specific probe; this sequence is complementary to the partially cloned D. viridis ERK2 mRNA (see Results). Primers were radiolabelled with [γ-32P]ATP using the enzyme polynucleotide kinase and purified by spin desalting on Sephadex G-25 columns. Pre-hybridization and hybridization were carried out in rapid hybridization buffer (Amersham) at 42 °C for 2 h. Blots were washed three times in 1.5× sodium chloride–sodium citrate–EDTA (SSPE)–0.1% SDS at room temperature, and three times in 0.75× SSPE–0.1% SDS at 42 °C, exposed to X-ray film, and the results were analysed as described above for western blot. Equal loading in northern blots was verified by staining with ethidium bromide and analysis of rRNA band intensity. Detection of cell cycle phase by means of flow cytometry The cell cycle phase of D. viridis was detected by means of propidium iodide staining and flow cytometry analysis. Cells were harvested by centrifugation (1500 g, 10 min) and the pellets resuspended in 4% paraformaldehyde prepared in 2 M NaCl. Cells were kept in this solution for at least 2 h at 4 °C. Paraformaldehyde was removed by means of centrifugation and the cells were washed twice in 2 M NaCl and once in Na-citrate (50 mM). Pellets were then resuspended in 0.5 ml of Na-citrate (50 mM)+50 mg of RNase+saponin (0.1% v/v), and incubated for 2 h at 37 °C. After this period, 0.5 ml of Na-citrate (50 mM)+2 μg of propidium iodide+saponin (0.1% v/v) were added, and incubated for 30 min at room temperature. Flow cytometry analysis was performed as described in Sazer and Sherwood (1990) using a Becton Dickinson FAC-Scan system. Statistical analysis All data are present as mean ±SD. One-way analyis of variance (ANOVA) test (SPSS 11.5) was performed to evaluate statistically significant changes in metabolites between the study groups. P-values <0.5 were recognized as statistically significant. Results Cell division is abolished in the presence of ERK inhibitor The first objective of this study was to evaluate the possible physiological implications of an active ERK cascade in D. viridis. For this, cultures of the microalga were inoculated in either the presence or absence of the specific inhibitor of ERK phosphorylation, PD98059. Growth of D. viridis was almost completely inhibited in the presence of 20 μM of the inhibitor (Fig. 1), both when it was added to the growth medium upon dilution (Fig. 1A), and when it was added 48 h after inoculation of the cultures, at the onset of the exponential phase of growth (Fig. 1B). As can be seen, only one division cycle of the cells occurred after the addition of the inhibitor. Similar results were obtained at different initial culture densities (not shown). Cell density was much lower in the cell cultures treated with the ERK inhibitor. However, the inhibitor did not affect cell viability, since cell motility (not shown) and cell capacity for generating reducing power (in the form of NADH and NADPH) were not affected (Fig. 2). Interestingly, while Dunaliella cells were not dividing in the presence of the ERK inhibitor, they continued to generate reducing power. A significant drop in cell viability was detected after 4–5 d in the presence of the inhibitor, that might be related to the average life span of non-dividing Dunaliella cells. It is noteworthy that PD98059 is a very selective and cell-permeable inhibitor of the ERK pathway and at the concentration used does not appear to affect alternative signalling pathways. Fig. 1. Open in new tabDownload slide Effect of the ERK inhibitor, PD98059 (20 μM final) on growth of D. viridis. The inhibitor was added either at the time of inoculation of fresh medium (A) or at the onset of the exponential growth phase (B). Data represent the mean and SEM of two independent experiments. Fig. 1. Open in new tabDownload slide Effect of the ERK inhibitor, PD98059 (20 μM final) on growth of D. viridis. The inhibitor was added either at the time of inoculation of fresh medium (A) or at the onset of the exponential growth phase (B). Data represent the mean and SEM of two independent experiments. Fig. 2. Open in new tabDownload slide Effect of the addition of ERK inhibitor on cell viability in cultures of D. viridis. Cell viability was evaluated by reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium] (MTS) as previously described (Capasso et al., 2003) per ml of culture. Data represent the mean and SEM of two independent experiments. Fig. 2. Open in new tabDownload slide Effect of the addition of ERK inhibitor on cell viability in cultures of D. viridis. Cell viability was evaluated by reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium] (MTS) as previously described (Capasso et al., 2003) per ml of culture. Data represent the mean and SEM of two independent experiments. Propidium iodide staining was employed for DNA-specific fluorescence by means of flow cytometry and showed that cells incubated in the presence of 20 μM of the ERK inhibitor developed twice the amount of DNA as compared with the control cells (Fig. 3). The analysis of control cultures of D. viridis demonstrates that the majority of the cells contain a single copy of DNA and indicates that the G1 phase is the longest duration in the cell cycle. Dunaliella cells exposed to ERK inhibitor double their DNA content during S phase, and cell cycle progression is arrested at G2 phase and does not progress further into mitosis. Therefore, ERK phosphorylation is crucial for the continuation of cell division in Dunaliella cells. Fig. 3. Open in new tabDownload slide Evaluation of cellular DNA content in cultures of D. viridis in the presence and absence of the ERK inhibitor, PD98059 by propidium iodide staining and flow cytometry. The horizontal and vertical axes show relative DNA content and cell number, respectively. Cultures were inoculated at a cell density of 3.0×105 cell ml−1 in either the presence or absence of 20 μM of PD98059 inhibitor. Data are the mean of a representative experiment. Analyses performed in five independent experiments were reproducible. Fig. 3. Open in new tabDownload slide Evaluation of cellular DNA content in cultures of D. viridis in the presence and absence of the ERK inhibitor, PD98059 by propidium iodide staining and flow cytometry. The horizontal and vertical axes show relative DNA content and cell number, respectively. Cultures were inoculated at a cell density of 3.0×105 cell ml−1 in either the presence or absence of 20 μM of PD98059 inhibitor. Data are the mean of a representative experiment. Analyses performed in five independent experiments were reproducible. p44 and p42 proteins are phosphorylated in dividing cells Once the possible implication of an ERK-type MAPK in Dunaliella cell division was shown, the second objective of this work was to detect the presence of ERK-like proteins in this microalga, and to investigate its relationship to cell division. For this purpose, cell extracts were assayed with specific antibodies that detect endogenous levels of p44 and p42 MAPK (ERK1 and ERK2) only when catalytically activated by phosphorylation at the threonine and tyrosine in the amino acid sequence T*E/DY*. This sequence is absolutely conserved in all organisms in which ERK has been cloned and sequenced. Figure 4A shows two proteins of 42 kDa and 44 kDa that were phosphorylated in cells of D. viridis while actively dividing. The apparent molecular weights of these bands are similar to those of the phosphorylated ERK1/2 from mammals. Band analysis indicated that maximal phosphorylation occurred during days 1–5, representing the exponential phase of growth of the cultures. Phosphorylation was highly reduced at the stationary phase of growth and when cultures entered a clear decline including cell death (days 9 and 12, respectively). Specific antibodies against the non-phosphorylated form of ERK1/2 (p42 and p44) did not cross-react with any proteins from D. viridis extracts (data not shown). Fig. 4. Open in new tabDownload slide (A) Correlation between phosphorylation of ERK-like MAPKs and cell density during proliferation of D. viridis cultures. (B) Cell growth arrest induced by the inhibitor PD98059 is correlated with dephosphorylation of ERK-like proteins. Western blots were prepared using 50 mg of protein per lane and assayed with antibodies specific to the phosphorylated form of mammalian ERK1/2 containing the sequence T*EY*. Arrows indicate a very strong band of 44 kDa and a very faint 42 kDa band. Cell density was measured as described in the Materials and methods, and data points represent the mean and SEM, n=6. A representative western blot (50 mg of protein per lane) and densitometry measurements of three independent experiments are also depicted. Fig. 4. Open in new tabDownload slide (A) Correlation between phosphorylation of ERK-like MAPKs and cell density during proliferation of D. viridis cultures. (B) Cell growth arrest induced by the inhibitor PD98059 is correlated with dephosphorylation of ERK-like proteins. Western blots were prepared using 50 mg of protein per lane and assayed with antibodies specific to the phosphorylated form of mammalian ERK1/2 containing the sequence T*EY*. Arrows indicate a very strong band of 44 kDa and a very faint 42 kDa band. Cell density was measured as described in the Materials and methods, and data points represent the mean and SEM, n=6. A representative western blot (50 mg of protein per lane) and densitometry measurements of three independent experiments are also depicted. Phosphorylation of p44 and p42 proteins is abolished in the presence of ERK inhibitor Cell extracts of cultures of D. viridis inoculated in either the presence or absence of the specific inhibitor of mammalian ERK phosphorylation—PD98059, were assayed with the antibodies against the mammalian phosphorylated form of ERK1 and ERK2. It should be noted that the inhibitor PD98059 acts to inhibit the upstream kinase (MKK1/2) from ERK1/2 and thereby prevents phosphorylation of ERK1/2. Figure 4B demonstrates that in the presence of the ERK inhibitor, dephosphorylation of both p44 and p42 proteins occurs in cell cultures. These ERK-like proteins were strongly phosphorylated at day 1 (after inoculation with cultures in their mid-log phase of growth); however, no phosphorylated protein was detected in Dunaliella cell extracts at day 4. In contrast, increasing phosphorylation occurred between day 1 and day 5 in control cultures, coinciding with the exponential growth phase (Figs 1A, 4A). Thus, there is clearly a direct correlation between the inhibition of cell division and the level of phosphorylation of ERK-like proteins in D. viridis. Dephosphorylation of the ERK-like proteins is induced by stress Several kinds of environmental stresses are known to inhibit cell division transiently in Dunaliella (e.g. hyperosmotic stress). In this study, the influence of specific stresses on phosphorylation of p44 and p42 in D. viridis was evaluated. Figure 5A shows that the level of phosphorylation of both proteins was significantly reduced after hyperosmotic stress (2 M→4 M NaCl) and in response to non-lethal UV stress (Fig. 5B). Moreover, nitrogen starvation resulted in an almost total dephosphorylation of both proteins over a more extended period of time (Fig. 6). Dephosphorylation of the p44 and p42 ERK-like proteins coincided with cell division arrest. Fig. 5. Open in new tabDownload slide Time dependence of ERK phosphorylation level and cell density following hyperosmotic and UV stress. Both (A) hyperosmotic stress (2 M→4 M NaCl) and (B) non-lethal UV stress (40 mJ cm−2) clearly reduced the level of phosphorylation of ERK-like proteins in D. viridis cultures. This reduction in phosphorylated ERK proteins is correlated with cell division arrest. Data points represent the mean and SEM, n=6. Shown is a representative western blot (50 mg of protein per lane), and densitometry measurements of two independent experiments are also depicted. Fig. 5. Open in new tabDownload slide Time dependence of ERK phosphorylation level and cell density following hyperosmotic and UV stress. Both (A) hyperosmotic stress (2 M→4 M NaCl) and (B) non-lethal UV stress (40 mJ cm−2) clearly reduced the level of phosphorylation of ERK-like proteins in D. viridis cultures. This reduction in phosphorylated ERK proteins is correlated with cell division arrest. Data points represent the mean and SEM, n=6. Shown is a representative western blot (50 mg of protein per lane), and densitometry measurements of two independent experiments are also depicted. Fig. 6. Open in new tabDownload slide Comparison of the effect of nitrogen starvation stress on the phosphorylation of ERK-like proteins and cell density in D. viridis as a function of incubation time. Nitrogen depletion from the medium resulted in an almost total dephosphorylation of both ERK proteins, coinciding with total cell division arrest. Data points represent the mean and SEM, n=6. Shown is a representative western blot (50 mg of protein per lane), and densitometry measurements of three independent experiments are also depicted. Fig. 6. Open in new tabDownload slide Comparison of the effect of nitrogen starvation stress on the phosphorylation of ERK-like proteins and cell density in D. viridis as a function of incubation time. Nitrogen depletion from the medium resulted in an almost total dephosphorylation of both ERK proteins, coinciding with total cell division arrest. Data points represent the mean and SEM, n=6. Shown is a representative western blot (50 mg of protein per lane), and densitometry measurements of three independent experiments are also depicted. Cloning of ERK1- and ERK2-like mRNAs When total RNA extracted from Dunaliella cultures was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with the ERK1-specific primers described in the Materials and methods, an amplicon of the expected size (∼450 bp) was detected upon agarose gel electrophoresis. Similarly, an amplicon of ∼400 bp was detected after RT-PCR with the ERK2-specific primers. These fragments were extracted from the gel, ligated in the pGEM-Teasy vector, and cloned. Multiple plasmid preparations were obtained and those containing the expected size insert were sequenced from both ends using T7 and SP6 primers. A 446 bp and a 395 bp polynucleotide sequence were obtained that unambiguously code for a 148 polypeptide and a 131 polypeptide sequence. These sequences have been submitted to GenBank under accession numbers AY628422 and AY630341 for ERK1 and ERK2, respectively. As shown in Fig. 7, these sequences have a high degree of homology with ERKs from a wide array of organisms. As depicted in Fig. 8, several canonical serine, threonine, or tyrosine protein kinase domains (Hanks and Quinn, 1991) were identified, in particular, domains II, III, VIb, and VII. Most importantly, the Dunaliella ERK sequences contain the double phosphorylation motif characteristic of MAPKs in general and of ERK in particular TE/DYVATRW (JM Capasso, unpublished results). To verify the existence of these mRNAs, RNA was extracted from algal cultures and analysed by northern blot using specific D. viridis ERK1 and ERK2 oligonucleotide probes. As shown in Fig. 9, both probes detected bands of similar size (1.9±0.2 kb) that are consistent with a 400 amino acid protein. However, the band intensity of ERK1 is at least one order of magnitude stronger than that of ERK2, corroborating the western blot results. Fig. 7. Open in new tabDownload slide Depiction of the CrustalW alignments of D. viridis ERK1 and ERK2 with known amino acid sequences from diverse organisms show a high degree of homology from mammals to algae. ERK1 comparison abbreviations and GenBank accession numbers: Dv, Dunaliella viridis, AY628422; At, Arabidopsis thaliana, NM_112686 (49–195); Tv, Triticum vulgaris, AJ606018 (131–277); Cr, Chlamydomonas reinhartiiAB035141 (84–228), Mm: Mus musculusNM_011952 (69–213). ERK2 comparison abbreviations and GenBank accession numbers: Dv, Dunaliella viridis, AY630341; Dd, Dictyostelium discoideum, A56492 (53–183); Pf, Plasmodium falciparum, X82646 (62–191); Gi, Giardia intestinalisAY149275 (54–184); Hs, Homo sapiens, AY065978 (52–182). Sequences with a black background indicate identity, and sequences with a grey background indicate conserved substitution. Fig. 7. Open in new tabDownload slide Depiction of the CrustalW alignments of D. viridis ERK1 and ERK2 with known amino acid sequences from diverse organisms show a high degree of homology from mammals to algae. ERK1 comparison abbreviations and GenBank accession numbers: Dv, Dunaliella viridis, AY628422; At, Arabidopsis thaliana, NM_112686 (49–195); Tv, Triticum vulgaris, AJ606018 (131–277); Cr, Chlamydomonas reinhartiiAB035141 (84–228), Mm: Mus musculusNM_011952 (69–213). ERK2 comparison abbreviations and GenBank accession numbers: Dv, Dunaliella viridis, AY630341; Dd, Dictyostelium discoideum, A56492 (53–183); Pf, Plasmodium falciparum, X82646 (62–191); Gi, Giardia intestinalisAY149275 (54–184); Hs, Homo sapiens, AY065978 (52–182). Sequences with a black background indicate identity, and sequences with a grey background indicate conserved substitution. Fig. 8. Open in new tabDownload slide Sequence analysis of D. viridis ERK1 and ERK2 reveals the presence of several canonical kinase domains (Hanks and Quinn, 1991) including the double phosphorylation motif (TDY). Fig. 8. Open in new tabDownload slide Sequence analysis of D. viridis ERK1 and ERK2 reveals the presence of several canonical kinase domains (Hanks and Quinn, 1991) including the double phosphorylation motif (TDY). Fig. 9. Open in new tabDownload slide Northern blot analysis of D. viridis-specific ERK1/2 mRNA in cell extracts using oligonucleotide probes developed from sequence analysis. Both probes detected bands of 1.9±0.2 kb that are consistent with a 400 amino acid protein. Northern blot data depicted were representative of four independent experiments. Fig. 9. Open in new tabDownload slide Northern blot analysis of D. viridis-specific ERK1/2 mRNA in cell extracts using oligonucleotide probes developed from sequence analysis. Both probes detected bands of 1.9±0.2 kb that are consistent with a 400 amino acid protein. Northern blot data depicted were representative of four independent experiments. Levels of ERK1 mRNA do not change Even though it was not possible to establish potential variations in the non-phosphorylated ERK proteins in Dunaliella as a result of a lack of cross-reactivity with the mammalian derived antibodies, the data resulting from cloning of both MAPKs in Dunaliella clearly show that the levels of ERK mRNA do not change in batch cultures over a period of 21 d (Fig. 10); this is found in the face of a clear decline in culture viability in the latter stages. Fig. 10. Open in new tabDownload slide Northern blot analysis of D. viridis-specific ERK1/2 mRNA in cell extracts over a 21 d growth period. Densitometry of two independent experiments demonstrates that the ERK1/2 mRNA levels did not change significantly over the incubation period. Fig. 10. Open in new tabDownload slide Northern blot analysis of D. viridis-specific ERK1/2 mRNA in cell extracts over a 21 d growth period. Densitometry of two independent experiments demonstrates that the ERK1/2 mRNA levels did not change significantly over the incubation period. Discussion In this study, it was demonstrated that cell division in the unicellular microalga D. viridis is almost completely inhibited by a very specific and selective inhibitor of ERK phosphorylation in mammals. Under these conditions, cell viability was not affected. These results clearly indicate that an ERK-type MAPK pathway is involved in cell proliferation in D. viridis. Moreover, the use of specific antibodies against the dual-phosphorylated form of human ERK (ERK1 and ERK2) reveals the presence of two proteins of 44 kDa and 42 kDa in actively dividing cells of D. viridis similar in molecular weight to ERK1 and ERK2 from mammals. Maximal phosphorylation occurred during the exponential phase of growth of the cultures. Moreover, the ERK inhibitor clearly eliminated additional phosphorylation of ERK, resulting in dephosphorylation of existing ERK proteins during continued incubation of D. viridis. Therefore, it may be concluded that phosphorylation of an ERK-type MAPK in the unicellular microalga D. viridis is crucial for cell division. In addition, it was demonstrated that various stress conditions, which inhibit cell division, also significantly reduce the level of phosphorylation of ERK proteins. To our knowledge, this is the first report that relates cell division and MAPK phosphorylation in photosynthetic unicellular organisms. ERKs have been reported to be involved in proliferation and differentiation in different mammalian cells (Marshall, 1995), and in plants there is ample evidence for their role in stress response (Coronado et al., 2002). Based on sequence analysis, the presently known plant MAPKs are most similar to ERKs (Mishra et al., 2006; Zhang et al., 2006), even though increasing evidence indicates that those kinases are also involved in various forms of biotic and abiotic stress responses, and not only in cell proliferation and division control. In a previous work (Jiménez et al., 2004), the involvement of p38-like and JNK-like MAPKs in stress response in D. viridis was demonstrated; however, no sequence analysis of those kinases has yet been reported. A variety of genes encoding MAPKs have been identified in plants (e.g. alfalfa, Arabidopsis, parsley, pea, petunia, and tobacco), and all amino acid sequences of the presently known plant homologues are most similar to the ERKs. However, some expressed sequence tags and sequences derived from the Arabidopsis genome project are clearly distinct from ERKs (Ligterink, 2000). From an analysis of the sequence homology of the predicted amino acid sequences, plant ERK-like MAPKs can be further divided into four distinct subgroups (Ligterink, 2000), and at least some of the MAPKs of subgroup III are involved in cell cycle regulation (Bögre et al., 1999, 2000). The first evidence that protein phosphorylation regulates mitosis in plant cells was the finding that metaphase was prolonged when cells were treated with the protein kinase inhibitor K252-a (Wolniak and Larsen, 1995). Several recent works provide evidence of the activity of ERK-like MAPKs in plants also involved in cell division and differentiation. Jonak et al. (1993) found that a plant homologue of MAPK is expressed in alfalfa proliferating cells, while Duerr et al. (1993) reported the recovery of a full-length cDNA clone encoding a MAPK from alfalfa of 44 kDa with characteristics of ERK that may play a role in the mitogenic induction of symbiotic root nodules on alfalfa by Rhizobium signal molecules. Treatment of Arabidopsis roots with auxin (a plant hormone involved in plant growth and development) transiently induced increases in protein kinase activity with characteristics of mammalian ERK-like MAPKs (Mockaitis and Howell, 2000). In addition, several studies were aimed at investigating whether MAPKs are involved in G1 phase control. Mizoguchi et al. (1994) found that cell cycle progression in cultured tobacco cells was blocked by omitting auxin from the medium, while Wilson et al. (1997) reported that cultured tobacco BY2 cells were arrested in the G1 phase of the cell cycle by omitting phosphate from the medium. After adding back auxin or phosphate, the cells resumed proliferation. In this work, it has been shown that ERK phosphorylation is crucial for cell cycle progression in D. viridis, and that non-dividing cells have their cell cycle blocked in G2 phase, and demonstrate twice the amount of DNA as compared with control, dividing cells. Interestingly, other researchers have shown that activation of an ERK-type MAPK in plants occurs in response to stress; however, no direct correlation of phosphorylation of those proteins and cell proliferation was shown. Samuel et al. (2000) reported that brief exposure to ozone led within minutes to activation of an ERK-type MAPK (∼46 kDa) in tobacco. Novikova et al. (2000) found increased MBP phosphorylation in wild type A. thaliana treated with ethylene (a plant hormone involved in fruit ripening and in stress response), and that a polypeptide band of 47 kDa cross-reacted with both ERK1 and phosphotyrosine antibodies. Huang et al. (2002) report isolation of a MAPK from rice that may function both in the stress signalling pathway and in panicle development. All these works help to understand that ERK phosphorylation is crucial for plants' response to stress. In contrast, in the unicellular microalga D. viridis, ERK1 and ERK2 were rapidly dephosphorylated in response to stress, coinciding with cell division arrest. Other authors have demonstrated that ERKs are also mostly responsible for cell proliferation control in plant cells. The present data reinforce this hypothesis, since phosphorylation of ERK-type proteins is necessary for cell division in a unicellular microalga that evolved from a common ancestor some 1.4–1.6 billion years ago. Multicellularity evolved independently in plants and animals, but the rise in signalling complexity is also characteristic of plants (at least 15 Arabidopsis MAPKs have been described, compared with only six in yeast). It appears that both plants and animals attained a basic tool kit for signalling from a common unicellular ancestor, which then evolved independently as these organisms attained multicellularity (Bögre et al., 2000). The response of the ERK-type proteins detected in D. viridis clearly reflects that of mammalian ERKs. As previously detailed, Park et al. (2002) reported that senescent cells of human fibroblasts do not phosphorylate ERK1/2, while Lovicu and McAvoy (2001) concluded that ERK phosphorylation is absolutely necessary for lens cell proliferation and fibre differentiation. Many other reports (see Introduction) on mammalian cells are coincident with those presented in this work, indicating a similar function of the ERK pathway both in a unicellular microalga and in mammalian cells. Cloning of ERKs from D. viridis showed a high degree of identity with those from a very wide variety of organisms, ranging from protozoa to human. The sequence of ERK1 from Dunaliella indicates that the characteristic TEY motif is not universal among all organisms. In fact, the TEY motif is found in all animals, protozoa, fungi, and most plants in which this MAPK has been cloned and sequenced. However, a group of plants shows the TDY motif, among them Dunaliella, Zea mays, Selaginella, Arabidopsis, Triticum, and Oryza. The present data enable a cladogram to be proposed based on the sequence of ERK1 (Fig. 11), in which two different branches for plants appear, one containing the characteristic motif TEY and including the microalga Chlamydomonas (as well as animals, fungi, and protozoa) and another plant branch characterized by the presence of the TDY motif that includes Dunaliella. The apparent evolutionary divergence between the two closely related green algae, Chlamydomonas and Dunaliella, is suprising. However, since this comparison is based on about one-third of the ERK1 or ERK2 coding sequence (even thought they are the most conserved part of the mRNAs), it is likely that the complete sequence of the corresponding mRNAs may change the cladogram. On the other hand, the database has been searched and the sequence TEY in Arabidopsis and TDY in Chlamydomonas sequences was not found. Since the complete genome of these organisms is not available, the possibility cannot be discarded that there is another set of ERK-like genes with this motif (i.e. TEY versus TDY). One explanation may be that the switch from TEY to TDY involves only a single nucleotide substitution in the third position of the codon. Since that mutation will lead to a conserved amino acid substitution, it is possible that this single nucleotide substitution may have occurred during plant evolution. Fig. 11. Open in new tabDownload slide Cladogram analysis of ERK1 sequences from the literature with D. viridis. This comparison was performed using MacVector Software (version 6.5, Oxford Molecular Ltd, Madison, WI, USA) employing only the relevant partial sequences as indicated by the numbers between the parentheses. Fig. 11. Open in new tabDownload slide Cladogram analysis of ERK1 sequences from the literature with D. viridis. This comparison was performed using MacVector Software (version 6.5, Oxford Molecular Ltd, Madison, WI, USA) employing only the relevant partial sequences as indicated by the numbers between the parentheses. In conclusion, cell division in the microalga D. viridis is crucially dependent on phosphorylation of ERK MAPKs. Cell division control seems to follow similar patterns of signal transduction in unicellular and multicellular organisms. Analysis of ERK genes indicates that ERK sequences are strongly conserved from unicellular microalgae to higher plants and mammals, suggesting that the origin of the ERK pathway pre-dates the divergence of the plant and the fungi/animal lineages. Abbreviations Abbreviations ERK extracellular signal-regulated kinase MAPK mitogen-activated protein kinase MBP myelin basic protein The authors are indebted to the Department of Genetics of the University of Málaga for providing access to the flow cytometer and for technical help, and thank Diana Capasso and Professor Lynn Heasley for expert review of the manuscript. CJ was supported by Research Projects REN2002-00340/MAR and CGL2005-01071/BOS of the Spanish Ministry of Science and Technology, and by two Research Fellowships from the University of Málaga and the Spanish Ministry of Education, Culture and Sport (PR2003-110). This work was supported by National Institutes of Health Grants DK-19928 and DK-66544 to TB. References Bögre L , Calderini O , Binarova P , et al. 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Mazzitelli, Luca;Hancock, Robert D.;Haupt, Sophie;Walker, Paul G.;Pont, Simon D. A.;McNicol, Jim;Cardle, Linda;Morris, Jenny;Viola, Roberto;Brennan, Rex;Hedley, Peter E.;Taylor, Mark A.

doi: 10.1093/jxb/erl266