Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Abscisic acid is involved in the iron‐induced synthesis of maize ferritin.

Abscisic acid is involved in the iron‐induced synthesis of maize ferritin. The EMBO Journal vol.12 no.2 pp.651 -657, 1993 Abscisic acid is involved in the iron-induced synthesis of maize ferritin St6phane Lobr6aux1, Thierry Hardy2 and atoms in their central cavity in a soluble, non-toxic bioavailable Jean-Francois Briat1' 3 form (Theil, 1987; Harrison et al., 1989; Crichton, 1990; Andrews et al., 1992). 'Laboratoire de Biologie Vegetale, Centre National de la Moleculaire Eukaryotic ferritins (plant and animal) arise from a Recherche Scientifique et Joseph Fourier BP 53X, F-38041 Universiti common ancestral gene which is illustrated by their high Grenoble Cedex and 2Laboratoire de Biologie Moleculaire et Cellulaire sequence homology (Ragland et al., 1990; Lescure et Vegetale, Rh6ne-Poulenc Agrochimie, 14-20 rue al., Pierre Baizet, BP 9163, F-69263 Lyon, 09, Cedex France 1991; Spence et al., 1991; Andrews et al., 1992; Lobreaux et al., 1992). This homology implies an extraordinary 3Corresponding author conservation of their three-dimensional structure (Andrews Communicated by J.-D.Rochaix et al., 1992). Plant and animal ferritins, however, do not share the same cytological location and the control of their The ubiquitous iron storage protein ferritin has a highly synthesis in response to iron overload does not take place conserved structure in plants and animals, but a distinct by the same mechanism. First, plant ferritins are found cytological location and a different level of control in within plastids (chloroplasts and non-green plastids), while response to iron excess. Plant ferritins are plastid- animal ferritins are cytoplasmic proteins (Seckbach, 1982; localized and transcriptionally regulated in response to Van der Mark et al., 1983b; Ragland et al., 1990; Lescure iron, while animal ferritins are found in the cytoplasm et al., 1991). Secondly, iron induction of ferritin synthesis and have their expression mainly controlled at the is at the translational level mainly controlled in animals, translational level. In order to understand the basis of while transcription has been shown to be the principal target these differences, we developed hydroponic cultures of of the iron response in plants (Zahringer et al., Van 1976; maize plantlets which allowed an increase in the intra- der Mark et al., 1983a; Klausner and Harford, 1989; cellular iron concentration, leading to a transient accumu- Proudhon et al., 1989; Theil, 1990; Lescure et al., 1991). lation of ferritin mRNA and protein (Lobreaux,S., These differences need to be explained. Massenet,O. and Briat,J.F., 1992, Plant Mol. Biol., 19, Concerning the transcriptional control of plant ferritin 563-575). Here, it is shown that iron induces ferritin in synthesis response to iron, two possibilities have to be and RAB (Responsive to Abscisic Acid) mRNA accumula- considered. First, iron by itself could be an effector turning tion relatively with abscisic acid (ABA) accumulation. on and off ferritin plant gene transcription by direct inter- Ferritin mRNA also accumulates in response to action with some unknown trans-acting factors. For example, exogenous ABA. Synergistic experiments demonstrate in of yeast, transcription activation the metallothionein gene that the ABA and iron responses are lUnked, although CUPI occurs only when copper binds to ACEl (Furst et al., full expression of the ferritin genes cannot be entirely in Escherichia aerobactin 1988) and, coli, the operon is explained by an increase in ABA concentration. when it binds repressed by the Fur protein Fe2+ (Bagg and Inducibility of ferritin mRNA accumulation by iron is environmental Neilands, 1987). Alternately, high iron dramatically decreased in the maize ABA-deficient a stress concentrations could generate response inducing mutant vp2 and can be rescued by addition of exogenous under the control ferritin gene transcription, of an integrated ABA, confirming the involvement of ABA in the iron transduction Control of of plant pathway. expression genes response in plants. Therefore, it is concluded that a major in to stress is often mediated a transduction response by part of the iron-induced biosynthesis of ferritin is which involves the hormone abscisic pathway plant acid achieved a an in through pathway involving increase the see Skriver and Under (ABA) (for a review Mundy, 1990). level of the plant hormone ABA. The general conclusion standard iron nutrition ferritins conditions, plant are not of this work is that the synthesis of in the same protein detected in and vegetative organs (roots leaves), they response to the same environmental signal can be accumulate in seeds their maturation and are during degraded controlled by separate and distinct mechanisms in plants in the first days of growth following germination (Lobreaux and animals. and the iron Briat, 1991). Furthermore, manipulating Key words: abscisic acid/ferritin/iron stress/maize of the culture medium concentrations of cell plant suspen- of results in sions and hydroponically grown plantlets iron of ferritin loading, and in transient induction in synthesis et as well as in roots these cells (Lescure and al., 1991), Introduction of et leaves plantlets (Lobreaux al., 1992). This of an essential element for almost all and environmental control of The use iron, developmental plant ferritin living is on molecules which is reminiscent of the of RAB organisms, dependent biological synthesis expression pattern the of and of iron in to Abscisic and led us to prevent problems insolubility toxicity genes (Responsive Acid) postulate be involved in the control the of these that ABA could of ferritin presence oxygen. Among synthesis molecules, ferritins, a role iron in It should be noted in a class of multimeric in to ubiquitous proteins, play key response plants. that, plants, to several thousand iron because of their RAB are ability sequester genes specifically expressed during embryogenesis Oxford Press University S.Lobr6aux, T.Hardy and J.-F.Briat it is already strongly induced, and close to its while and can be induced in vegetative organs under various stress maximum accumulation, in leaves. It is interesting to note water and salt stresses are the best conditions, among which that at zero time, ferritin mRNA is already present in both an inducible of ferritin documented. Using system synthesis organs and that its concentration in roots can even be higher et 1992), we tested this hypothesis in maize (Lobreaux al., at 3 h after iron treatment than what is observed (Figure 1). that ABA is involved in the iron-stress and demonstrated be the case. This This appears to not always apparent drop to the accumulation of this plastid-located response leading mRNA levels occurs when are in ferritin only plants iron storage protein. harvested at the beginning of the photoperiod. Ferritin mRNA is much less abundant at zero time if samples are Results collected during the photoperiod (see lane 1 in Figures 3 and and Figure 4 in Lobreaux et al., 1992). In such a mRNA accumulation 5, Iron induces RAB and ferritin the amount of ferritin mRNA detected in roots is be induced ABA situation, RAB genes are known to by during the same at zero time and after 3 h post-iron treatment (not various stress conditions embryogenesis and under (Skriver This will be developed further in if iron induction of ferritin shown). particular point and Mundy, 1990). Therefore, mRNA accumulation in the Discussion. Ferritin response is at least in ABA, as we synthesis mediated, part, by and has decreased or it is conceivable that RAB genes could also to iron is transient already to, below, postulate, in both leaves and roots 24 h after iron treat- In order to test this hypothesis, we initial levels respond to iron stress. 1). pMAH9 mRNA also accumulates trans- and two RAB ment (Figure measured the level of ferritin mRNAs, namely iently in leaves, where it reaches its maximum at 6 h, et et and pMAH9 (Gomez al., 1988; Didierjean al., 1992) the whereas its level gradually increased in roots throughout et al., 1990), immediately following iron pMA12 (Villardel was first 24 h after treatment (Figure 1). pMA12 transcript of maize treatment hydroponically grown plantlets (Lobreaux not induced in roots. However, in leaves, pMA12 mRNA et Northern analysis of root and leaf RNA al., 1992). reached a maximum 15 h after the treatment, followed by indicated that ferritin mRNA is rapidly induced after iron 24 h It is to note that treatment with maximum accumulation of ferritin mRNA a decrease at (Figure 1). important the same RNA has been used to perform the h As an internal the RNAs from preparation at 6 (Figure 1). control, Northern blot analysis presented in Figure 1. Thus, two were with a cytoplasmic the same preparation probed different maize RAB as well as ferritin, are rapidly ribosomal protein (CS1 1) riboprobe, known to be genes, induced by an iron stress. It is important to note that each constitutively expressed at the same level in roots and leaves in 1 that the CS transcript displayed a unique pattern of accumulation. (Thompson et al., 1992). It is clear Figure 11 for the same in roots and leaves of maize Furthermore, this pattern can also vary, mRNA level is constant plantlets of iron treatment. Kinetics of ferritin transcript, between roots and leaves. The significance at different times after mRNA are rather different in roots than in will be discussed later (see the accumulation these observations Discussion). leaves. In roots, the amount of transcript is very low at 3 h Iron induces ABA synthesis L EAvES ROOTS The above observation has shown that the expression of FE dS 4 Timelhi 0 3 6 15 ,24 L-: 7. -- known RAB genes is induced by an iron stress in maize plantlets. Such a result could imply that the iron treatment pMFli leads to ABA synthesis and accumulation, correlatively to an increase in ferritin mRNA. In order to address this point, pcsll :i ", .X'- :.. -::: .*Z! 7 CO .0 0 Ii ..: ... .1.1% 2iD 7 00. C 5300 0) pMA12 Im 300' -a) Fig. 1. Effect of an iron stress on the accumulation of ferritin and RAB mRNA in maize plantlets. 10 of total RNA purified from Ag roots or leaves at 0 (plantlets were harvested during the first 30 min of the photoperiod), 3, 6, 15 and 24 h post-iron treatment were loaded in _-4Roots each lane (zero time was omitted in the blots probed with pMAH9 and pMA12). As an internal control, RNAs were also probed with a Leaves X c Hours cytoplasmic ribosomal protein (CS1 1) riboprobe. RNA probes were synthesized as described in Materials and methods from pMFli [SacI-StuI fragment of ferritin FMl cDNA from Lobreaux et al. Fig. 2. Concentrations of ABA in roots and leaves of maize plantlets (1992) cloned in pKSII], pMAH9 [EcoRI fragment from Gomez et al. at different times after the addition of iron. Preparation of samples was (1988) cloned in pKSII], pMA12 [PstI fragment from Villardel et al. as described in Materials and methods. ABA dosage used the (1990) cloned in pKSII] or [EcoRI-RsaI fragment from pCSI1 Phytodetek (Sunnyvale, CA) ABA determination kit. Each value Lebrun and Freyssinet (1991) cloned in pKSII] and used as indicated. results from the mean of six measurements performed with organs RNAs from the same preparation were used in this experiment. harvested from two independent cultures. Standard error was <10%. 652 ABA-mediated regulation of plant ferritin synthesis the ABA concentration was determined by an immunoassay 11 cytoplasmic ribosomal mRNA remains constant the CS using S-ABA monoclonal antibody (Gomez et al., 1988; in and leaves of maize plantlets after a treatment of roots Yamaguchi-Shinozaki et al., 1990). 24 h Determination of the ABA 3, 6, 15 and (Figure 1). First, the ABA concentration in roots and leaves was in leaves, 3 h after iron addition, revealed an concentration determined at different times after iron treatment (Figure 2). increase of 4.7-fold over the phytohormone level of the It is clear that iron stress can lead to the same transitory sample for which no iron was added (Figure 3). A good increase in ABA concentration, in both organs, with a correlation appears to exist between ferritin mRNA maximum observed 3 -6 h after iron addition in the culture accumulation and ABA concentration increases. Therefore, medium. This increase has been estimated to be 4.5-fold. it may be concluded that an iron-dependent transient Secondly, the iron concentration dependence of ferritin accumulation of both ABA and ferritin mRNA occurs in our synthesis has been assessed by Northern analysis of total leaf system. RNA prepared 12 h after the addition of increasing amounts Exogenous ABA induces ferritin synthesis of iron to the plantlet culture medium (Figure 3). It is clearly These results do not prove that ABA, by itself, can induce apparent that the amount of ferritin mRNA increases when ferritin In order to provide proof of this point, synthesis. iron-EDTA concentrations are increased from 0 to 250 IM. 10 ktM ABA were added, after iron starvation, to the culture A further increase of iron-EDTA to 500 does not lead /M medium instead of the iron mixture. The level of ferritin to another increase in ferritin mRNA level. This indicates mRNA was then monitored by Northern analysis at different that 2 250 ,uM iron is saturating this system during a treat- times after hormone addition. The pMAH9 probe was used ment of 12 h. At a saturating concentration of iron (500 ytM), as a positive control for ABA treatment. Results of such an experiment are shown in Figure 4A. Ferritin mRNA was 1 2 3 4 induced both in leaves and roots of ABA-treated plants. The maximum level was reached 15 h after treatment. Further- more, it is shown in Figure 5 that the level of leaf ferritin mRNA in response to a 12 h treatment with increases of exogenous ABA. In such an increasing concentration the amount of the CS 1 1 cytoplasmic ribosomal experiment, protein mRNA remains constant, whatever the concentra- transcripts tion of exogenous ABA used (Figure 5). pMAH9 were also induced in roots and leaves after ABA treatment 410 730 80' A 170 280 4A). The profile of pMAH9 mRNA accumulation (Figure was identical to that observed for ferritin. A difference is 3. Iron dependence of ferritin mRNA and ABA accumulation. Fig. observed between the kinetics of accumulation for both Maize plantlets were induced for ferritin synthesis by using increasing mRNA leaves and roots. In roots, the species between h iron treatment amounts of iron. Leaves were then collected 3 after pMAH9 mRNAs decreases only 12 h treatment for ferritin mRNA amount of ferritin and for ABA determination and after blot. Lane 1: no iron added (plantlets were visualization by Northern 24 while a dramatic drop occurs in leaves at slightly at h, h the had begun); lane 2: 50 harvested >3 after photoperiod AM time. ABA able to this particular Exogenous is, therefore, / lane 3: 100 iron-EDTA / iron -EDTA 7.5 iron-citrate; zM itM induce ferritin mRNA accumulation in iron-starved maize 15 lane 4: 250 iron-EDTA / 37.5 iron-citrate; iron-citrate; yM AM AM plantlets. lane 5: 500 iron-EDTA / 75 iron-citrate. ABA concentration AM izM is the mean of three standard error was < 10%. In order to examine the fate of the ferritin protein measurements; ROOTS LEAVES 2 4 3 15 24 6 15 6 Time(h) 3 pMFli GAPEP AL-....A .hA pMAH9 ABA. Accumulation of ferritin and transcripts from maize roots and leaves in pMAH9 4. ferritin synthesis by exogenous (A) Fig. Induction of were as described in Materials and methods that 10 ABA were added to the culture medium instead Culture conditions except to ABA. zM response and 24 RNA was extracted from roots and leaves, and 10 RNA from each sample were analysed by blot After 15 Northern of iron. 3, 6, h, ztg RNA either from (ferritin) or pMAH9 (RAB). (B) Ferritin and glyceraldehyde 3-phosphate hybridization using probes produced pMFli subunit accumulation as determined by immunodetection using polyclonal antibodies raised either against maize seed dehydrogenase (GAPDH, GapC) after of 40 of total extracted from maize leaves. Lane 1: no iron added; lane 2: 24 h after the or maize protein ferritin GAPDH, blotting ytg iron-EDTA 75 uM iron-citrate; lane 3: 24 h after the addition of 200 AM ABA; lane 4: 20 ng pure maize seed ferritin. This of 500 / addition ztM three the same result. was times, experiment repeated giving 653 S.Lobr6aux, T.Hardy and J.-F.Briat I e,., ?. .* w - :-~~~~~~.. -. pMFli :1 :i ABA BA F .-r lIII Fig. 5. Synergistic effect of iron and ABA on ferritin mRNA accumulation. blots were Northern after of 10 of (A) performed electrophoresis /g total RNA from either purified plantlets untreated 1: were harvested >3 h after (lane plantlets the had or treated for 12 h photoperiod begun) with increasing concentrations of ABA from 0 to 500 with 500 iron-EDTA (lanes 2-6), uM / 75 iron-citrate and with a mixture ltM ptM (lane 7) of 200 ABA 500 iron-EDTA plus / 75 iron-citrate RNAs from the same were (lane 8). with both ferritin IAM AM /iM preparation probed riboprobe and CS11 ribosomal (pMFli) cytoplasmic of the blots with the ferritin riboprobe (pCS11). (B) Quantification Northern hybridized riboprobe was after performed by computer image the Numbers indicate units of the amount analysis scanning of ferritin autoradiographs. arbitrary RNA. accumulation under such an 2 200 conditions, immunodetection ABA is this ktM exogenous saturating system during experiment was total extracted a performed using protein from treatment of 12 to the h, leading maximum steady-state leaves 24 h after treatment with either ABA level of exogenous or ferritin mRNA observed. maize Next, plantlets were iron. shown in As lane iron induces Figure 4B, 2, ferritin exposed to either iron alone or simultaneously to iron and protein accumulation in leaves 24 h after its 200 as ABA. The amount of ferritin addition, transcript accumulated AM previously reported et in to (Lobrdaux al., 1992). response both treatments varied 3 Exogenous only % (Figure SB, ABA treatment of iron-starved is also lanes 7 plantlets responsible and which is not 8), significant. This observation for an increase in the amount of ferritin indicates that protein subunit there is no additivity of exogenous ABA and (Figure 4B, lane 3); this relative increase much iron is, however, responses on ferritin mRNA accumulation. We can, lower than that in iron response to treatment. the Probing conclude that the iron stress therefore, performed saturates same protein extracts Western by blot, a the using polyclonal transduction to pathway, leading ferritin mRNA raised the maize antibody against cytoplasmic accumulation in glyceraldehyde to ABA. It response strongly suggests the 3-phosphate dehydrogenase has shown that the involvement of this (GapC), hormone in the iron-induced synthesis amount of this was protein not affected iron or of maize ferritin. by exogenous However, when are plantlets incubated in ABA treatment (Figure 4B). 200 the ABA, accumulation of ferritin mRNA observed 1sM is 68% of only the level measured when maize is treated Exogenous ABA and iron responses are not additive with iron. This result indicates that an event other than an All the results presented above strongly support the increase in ABA level is involved during iron stress, and hypothesis that ABA is involved during ferritin gene activa- is responsible for 32% of the ferritin mRNA accumulation tion in response to iron overload. To further test this observed 12 h after treatment. hypothesis, an experiment was performed to investigate if there is any synergistic effect between iron and ABA Genetic treat- evidence of involvement of ABA in the iron- ment. In this experiment, the steady-state level of the CS 11 induced synthesis of maize ferritin cytoplasmic ribosomal mRNA has been determined as an All these results strongly support the hypothesis that ABA internal control and does not change is involved significantly as an hormonal relay during iron-induced ferritin (Figure SA). In a first step, we determined the ABA biosynthesis. concen- In order to prove that ABA is necessary for tration which is necessary to induce a maximal accumulation maximal accumulation of ferritin mRNA during iron stress, of ferritin mRNA in response to this hormone, when applied we performed an iron induction experiment in a maize to maize plantlets for 12 h. The mutant ferritin mRNA level from deficient in ABA. For this purpose, we used leaves of iron-starved maize plantlets incubated with various viviparous 2 plantlets; this mutant has a defect in the ABA concentrations was measured by Northern analysis and carotenoid biosynthetic pathway, which produces the quantified by scanning of the autoradiographs (Figure 5). precursors for ABA synthesis (Zeevaart and Creelman, It is clearly apparent that the amount of ferritin mRNA Pla 1988; et al., 1989). Homozygous vp2 plantlets were increases when ABA concentrations are increased from 0 obtained in vitro and transferred to hydroponic culture for to 200 ktM in the culture medium. For an unknown reason, iron starvation, followed by a 12 h iron treatment. The the amount of ferritin mRNA induced by 100 ABA is inducibility of ferritin transcript accumulation in leaves was AtM lower 50 slightly than that at When the hormone quantified by Northern analysis in vp2 and wild-type AM. concentration is further increased to 500 ktM, the level of plantlets. In the ABA-deficient mutant, a 1.4-fold increase ferritin mRNA detected does not increase any more. Thus, in ferritin mRNA level is observed in response to iron 654 ABA-mediated regulation of plant ferritin synthesis and Briat, 1991; et al., 1992), we (Lobreaux Lobreaux WT postulate that ABA could be involved in the control of ferritin ABA - .. synthesis. Indeed, exogenous ABA is able to induce ferritin Fe - _-Fe +Fe mRNA accumulation (Figures 4A and 5). Concentrations of exogenous ABA giving this response are in the range of pMF1 of those described for other systems (Mundy and Chua, 1988; Pla et al., 1989). The decrease in this response at 24 h, mainly in leaves, could be due to ABA degradation during pCS11 its transport and has already been observed (Mundy and Chua, 1988). At the protein level, exogenous ABA allows accumulation of the ferritin subunit, but to a lower extent than is observed after iron treatment (Figure 4B). It makes sense because treatment with exogenous ABA does not allow full accumulation of ferritin mRNA, as does iron treatment (Figure 5 and see below for discussion). Furthermore, it is important to be reminded that iron or ABA addition are achieved after 9 days of iron starvation, therefore the response to exogenous ABA occurs at a very low intracellular iron concentration, which has been previously reported (Lobreaux et al., 1992). It has been known for a long time that, in animal systems, iron is necessary to 'Y increase the stability of ferritin molecules (Crichton, 1971; Drysdale and Shafritz, 1975). It could be the same in plants and it would explain the relatively low ferritin protein accumulation in response to exogenous ABA under condi- tions of iron Fig. 6. Steady-state level of ferritin mRNA in response to iron in starvation. maize leaves from wild-type (WT) and viviparous 2 (vp2) mutant. Because iron and ABA are both able to induce ferritin (A) 10 of total leaf RNA purified from wild-type and lp2 plantlets, Ag accumulation, the possibility that the iron and the ABA grown without iron or with 500 iron-EDTA 75 ytM iron-citrate AM responses are linked was a legitimate question to answer. for 12 h, were probed on the same Northern blot by using the ferritin In 10 yg of total leaf RNA purified from Iron treatment leads to an increase in ABA concentration riboprobe (pMFli). addition, iron or control vp2 plantlets (grown without ABA) and vp2 plantlets which is dependent on the iron concentration in the culture harvested 12 h after the addition of 500 exogenous ABA were also AM medium (Figures 2 and 3). RAB genes are also induced by probed by Northern blots using the ferritin riboprobe (pMFli). As an iron treatment (Figure 1). However, differences in the internal control, the various RNA samples purified from vp2 plantlets kinetics of ferritin and RAB mRNA accumulation in response were also cytoplasmic ribosomal riboprobe probed using the CSl (pCS 1). The RNA probes were synthesized as described in Materials to iron (Figure 1) indicate that multiple events are probably and from and from 15 h at methods pMFli pCSl Exposure was for in This is involved regulating expression of these genes. X film. of -70°C using a Kodak Royal 0-Mat (B) Quantification from particularly evident the differences in mRNA accumula- Northern blots from and leaf RNAs from wild-type vp2 purified tion for between roots and leaves the same transcript. iron or after the addition of 500 plantlets grown without (-Fe) zM it does not imply that entirely different pathways iron-EDTA / 75 iron-citrate for 12 and with the However, (+Fe) h, probed AM This was ferritin riboprobe. quantification performed by computer control the of these genes. For example, pMA12 expression Numbers indicate image analysis after scanning the autoradiographs. and pMAH9, which have very different kinetics of of of ferritin RNA. The same result was arbitrary units the amount accumulation in response to iron (Figure 1), are both in two obtained independent experiments. ABA (Gomez et al., 1988; Villardel et al., regulated by More than one pathway could be involved in the 1990). in the amount of ferritin while wild-type plantlets (Figure 6), control of each The same common pathway could be gene. 7.5 times in iron-treated maize mRNA is higher transcript utilized until a certain step preceding branching of second- in iron-starved The failure of iron induction than plantlets. for each tissue. ary pathways, specific for each gene and/or mutant cannot be attributed to an in the vp2 independent for ferritin Such a complexity is clearly demonstrated to ABA since addition of defect additional deficiency, mRNA. As a first to link the iron and ABA attempt induces ferritin mRNA ABA to exogenous vp2 plantlets an iron treatment was in the responses, performed presence The level of the CS accumulation (Figure 6A). 11 cytoplasmic known of RAB of gibberellic acid (GA3), a antagonist gene remains constant in mRNA concentration ribosomal vp2 et In such an activation (Pena-Cortez al., 1989). experiment addition of iron or ABA with or without plantlets we observed that GA3 the iron-induced partially suppressed to note that after iron starva- It is (Figure 6A). interesting accumulation of ferritin mRNA in 6 h after the iron leaves, the amount of the and to iron tion, prior induction, transcript for unknown treatment an reason, (not shown). However, in than in The is 2.1-fold higher vp2 wild-type plantlets. be variable from culture to this suppression was found to observed in the of ferritin decrease mRNA strong inducibility A ABA/iron culture. experiment (Figure 5) synergistic in that ABA is essential for accumulation vp2 plants proves that ABA and iron are linked, but reveals, indeed, pathways of ferritin induced iron. maximum synthesis by of ferritin mRNA accumulation in that the regulation iron cannot be the response to entirely explained by pathway since full an increase in ABA involving concentration, Discussion cannot be obtained a of the iron expression response by concentration. Such an observ- of ferritin in ABA Based on the of saturating exogenous pattern expression plant the difference in ferritin mRNA and ation could response to iron and during growth development explain 655 S.Lobr4aux, T.Hardy and J.-F.Briat during iron stress, remains to be determined. It has to be accumulation between roots and leaves (Figure 1). The ABA are very similar in both organs at the various remembered that environmental and developmental controls concentrations are not necessarily mediated by the same pathway. In the times analyzed after iron addition, and peak between 3 and case of the proteinase inhibitor pin2 gene, ABA is involved 6 h (Figure 2). However, it is important to notice that after in the wounding response, but not during developmental 3 h ferritinmRNA has not yet accumulated in roots, while control (Pena-Cortez et al., 1991). it is already close to its maximum in leaves (Figure 1). This It has been reported that the bulk of ABA in plants is found indicates that a second ABA-independent pathway could within plastids, where it may be synthesized by the cleavage operate at 3 h in leaves. Alternately, an increase in the ABA of leaf cells, mediated by iron, cannot be ruled of violaxanthin to give xanthoxin as a first step (for a review sensitivity Such an effect has recently been reported by Bostok see Zeevaart and Creelman, 1988). In plants, ferritins are out. also localized within plastids (Seckbach, 1982). In this and Quatrano (1992), showing that salt can increase the context, it is tempting to postulate that during iron stress, sensitivity of rice cells to ABA, affecting Em gene which can be a natural toxicity problem in flooded acidic expression. soils, an increase in iron uptake by plastids could occur, During the course of these kinetic experiments, we leading to an oxidative stress that raises the ABA concen- observed variations in the level of ferritin mRNA at zero time, according to the particular moment when samples were tration through photochemical and/or enzymatic cleavage of xanthophylls. As a consequence ABA, through a transduc- collected (beginning versus a few hours of photoperiod). This tion pathway, could activate ferritin gene expression. Ferritin could be explained by circadian regulation of the expres- is then targeted to plastids where it is assembled in order sion of ferritin genes and/or by differences in the compart- mentalization of ABA, which is known to change according to store excess iron. Experiments are in progress to test this hypothesis. to the light/dark status of the plant (Zeevaart and Creelman, 1988). Further evidence of ABA involvement in the iron- induced ferritin synthesis was gained by testing the iron Materials and methods response in a maize ABA-deficient mutant. Inducibility of Plant cultures ferritin mRNA accumulation by iron in vp2 homozygous Maize plantlets (Zeamays, var. MO17) were grown under hydroponic culture plantlets, which lack ABA due to blocking in the carotenoid in an iron-free medium as previously described (Lobreaux et al., 1992). biosynthesis pathway (Zeevaart and Creelman, 1988), is After 9 days of iron starvation, different treatments were performed. Iron 5.4-fold less than in the wild type (Figure 6B). Interestingly, induction was achieved by adding 500zM Fe-EDTA, 150 Na3-citrate /lM addition of exogenous ABA to vp2 plantlets induces ferritin and 75zM FeSO4 in the culture medium. For ABA treatment, ABA (Sigma) was added to the medium, instead of the iron mixture, by dilution mRNA accumulation (Figure 6A). This result is in agree- of a 100 mM stock solution in ethanol. Root and leaf samples were harvested ment with the fact that the failure of iron induction in the at different times after treatment, frozen in liquid nitrogen and stored at vp2 mutant is due to ABA deficiency and not to an addi- -700C. tional independent defect. In vp2, iron treatment still induces Viviparous 2 homozygous plantlets can only be obtained by selfing of ferritin mRNA accumulation, but to a much lower extent heterozygous plants. Therefore, plants were first obtained from heterozygous seeds (kindly provided by Dr M.Pages, Barcelona) in the greenhouse. The than in wild type (Figure 6). This observation could be growth conditions were 24°C during the day and 18°C at night, with natural related to the fact that part of the iron response could be light supplemented with artificial light to ensure a minimum of 300 1tE/m2/s independent of the ABA response (Figure 5). Although the during a 16 h photoperiod. At -30 days after pollination, ears with white ferritin mRNA level, before adding iron, is always higher kernels were harvested, surface sterilized with a 6.25% sodium hypochlorite solution containing a small amount of detergent as a surfactant, and then ( -2-fold) in vp2 than in wild-type plantlets, making a direct rinsed in three changes of sterile deionized water. White kernels were isolated comparison between the two genetic backgrounds difficult. and put in germination in individual plastic jars with 30 ml of N6 medium So far, developmental and environmental regulation of (Chu et al., 1975). In vitro conditions were 24°C constant, a 16 h RAB genes have, as a common effector, variations in photoperiod (80 fluorescent lighting 'Fluora', Osram) and 80% lE/m2/s osmotic pressure due to either dessication or salt and water relative humidity. When vp2 plantlets were -10 cm high, they were transferred to hydroponic culture medium without iron for 7 days prior to stress (Skriver and Mundy, 1990). The observation that iron treatment with 500 Fe-EDTA, 150yM Na3-citrate, and 75 ytM ferritin gene expression responds to ABA could be relevant piM FeSO4. for understanding the regulation of ferritin synthesis during RNA extraction and analysis development. Plant ferritins, under normal conditions of iron Total RNA extraction was performed as previously described (Lobreaux nutrition, are iron storage proteins which accumulate during et al., 1992). Northern blot analysis of total RNA samples was achieved seed formation and are degraded during seed germination using RNA probes, according to Lobreaux et al. (1992). For ferritin mRNA (Lobreaux and Briat, 1991). This is analogous to the detection, the pMFli ferritin probe was used (Lobreaux et al., 1992). Clones pMAH9 (Gomez et al., 1988; Didierjean et al., 1992), pMA12 (Villardel behaviour of known RAB protein. However, iron stress is et al., 1990) and pCSl 1 (Lebrun and Freyssinet, 1991) were gifts, respect- not likely to be related to an osmotic stress (Mundy and ively, from Dr G.Burkard (Strasbourg), Dr M.Pages (Barcelona) and Dr Chua, 1988; Skriver and Mundy, 1990) because of the M.Lebrun (Lyon). EcoRI insert from pMAH9, PstI insert from pMA12 micromolar range of iron salt we used. It is more likely to and the EcoRI-RsaI fragment from pCSl 1 were subcloned in the appropriate be related to an oxidative stress because of the role metals, restriction sites of Bluescript II KS. For quantification of mRNA levels, autoradiographs were scanned using a 256 grey level scanner (Apple) and such as iron, can have in the conversion of reduced oxygen images were analysed with the software Image 1.3.7. (NIH, Bethesda, USA). into hydroxyl radicals, one of the most reactive species known, through Haber-Weiss reaction (Halliwell, 1987; Protein extraction and analysis The preparation of pure maize seed ferritin and rabbit polyclonal antibodies Imlay and Linn, 1988). Consistent with this point is the raised against its subunit have already been reported (Lobreaux et al., 1992). report that an oxidative stress generated by radiation raises Polyclonal antibodies raised against maize cytoplasmic glyceraldehyde the ABA concentration in wheat seedlings (Degani and Itai, 3-phosphate dehydrogenase (GapC) were a generous gift of Pr. R.Cerff 1978). Whether ferritin synthesis during seed development (Brunschweig University, Germany). Total protein preparation from leaves, protein concentration measurements. is regulated both by ABA and an additional pathway, as 656 ABA-mediated regulation of plant ferritin synthesis SDS-polyacrylamide gel electrophoresis and immunodetection were as Van der Mark,F., Van der Briel,W. and Huisman,H.G. (1983b) Biochem. already described (Lobreaux et al., 1992). J., 214, 943-950. Villardel,J., Freire,M.A., Torrent,M., Martinez,M.C., Goday,A., ABA dosage Plant Mol. 423 Biol., 14, -432. and (1990) Plants were grown hydroponically as described above. At various times TorneaJM. PagMs,M. Mc. BiSo., 15, post-iron treatment, roots and leaves were ground Extracts 905-912. in liquid nitrogen. were prepared according to Yamaguchi-Shinozaki et al. (1990) and the ABA Z inr., Bi,B USA, 73, concentration was determined using the Phytodetek-ABA (Idetek Inc., 857B-861. kit CA) according to the manufacturer's instructions. Zeevaart,J.A.D. and Sunnyvale, (1988) Annu. Rev. Physiol. Creelman,R.A. Plant Plant Mol. Biol., 39, 439-473. Received on on Acknowledgements October 1992 8, 1992; revised 26, July, We gratefully acknowledge Dr M.Pages (CID-CSIC Barcelona, Spain) for the gift of pMA12 and of maize vp2 seeds, Dr G.Burkard (CNRS Strasbourg, France) for the gift of p23.2 (pMAH9), Dr Lebrun Poulenc, (Rh6ne Lyon, France) for the gift of pCSl 1 and Pr. R.Cerff (Brunschweig University, Germany) for the gift of maize GAPDH polyclonal antibody. We thank Dr De Rose (Rh6ne Poulenc, Lyon, France) for comments and critical reading of this manuscript. This work was supported by the Centre National de la Recherche Scientifique (URA 1178) and by the Ministere de la Recherche et de la Technologie (grant 89 C 0869) to J.F.B. References Andrews,S.C., Arosio,P., Bottke,W., Briat,J.F., von Darl,M., Harrison,P.M., Laulhere,J.P., Levi,S., Lobreaux,S. and Yewdall,S.J. (1992) J. Biochem., 47, Inorg. 161-174. Bagg,A. and Neilands,J.B. (1987) 26, 5471-5477. Biochemistry, Bostock,R.M. and Quatrano,R.S. (1992) Plant Physiol., 98, 1356-1363. Chu,C.C., Wang,C.C., Sun,C.S., Hsu,C., Yin,K.C., Chu,C.Y. and Bi,F.Y. (1975) Sci. Sin., 659-668. 18, Crichton,R.R. (1971) Biochim. Acta, 229, 75-82. Biophys. Crichton,R.R. (1990) Adv. Protein Chem., 40, 281-353. Degani,N. and Itai,C. (1978) Exp. Bot., 18, 113-115. Environ. Didierjean,L., Frendo,P. and Burkard,G. (1992) Plant Mol. Biol., 18, -849. Drysdale,J.W. and Shafritz,D.A. (1975) Biochem. Acta, 383, Biophys. 97-105. Hu,S., Hackett,R. and Hamer,D. (1988) Fiirst,P., Cell, 55, 705-717. Gomez,J., Sanchez-Martinez,D., Stiefel,V., Rigau,J., Puigdomnech,P. and Pages,M. (1988) Nature, 334, 262-264. Halliwell,B. (1987) FASEB J., 1, 358-364. Harrison,P.M., Artymiuk,P.J., Ford,G.C., Lawson,D.M., Smith,J.M.A., Treffry,A. and White,J.L. (1989) In Mann,S., Webb,J. and Williams,R.J.P. (eds), Biomineralization: Chemical and Biomedical Perspectives. VCH, Weinheim, pp. 257-294. Imlay,J.A. and Linn,S. (1988) Science, 240, 1302-1309. Klausner,R.D. and Harford,J.B. (1989) Science, 246, 870-872. Lebrun,M. and Freyssinet,G. (1991) Plant Mol. Biol., 17, 265-268. Lescure,A.M., Proudhon,D., Pesey,H., Ragland,M., Theil,E.C. and Briat,J.F. (1991) Proc. Natl. Acad. Sci. USA, 88, 8222-8226. Lobredaux,S. and Briat,J.F. (1991) Biochem. J., 274, 601-606. Lobreaux,S., Massenet,O. and Briat,J.F. (1992) Plant Mol. Biol., 19, 563 -575. Mundy,J. and Chua,N.H. (1988) EMBO J., 7, 2279-2286. Pena-Cortez,H., Sanchez-Serrano,J.J., Mertens,R., Willmitzer,L. and Prat,S. (1989) Proc. Natl. Acad. Sci. USA, 86, 9851-9855. Pena-Cortez,H., Willmitzer,L. and Sanchez-Serrano,J.J. (1991) Plant Cell, 3, 963-972. Pla,M., Goday,A., Vilardell,J., Gomez,J. and Pages,M. (1989) Plant Mol. Biol., 13, 385-394. Proudhon,D., Briat,J.F. and Lescure,A.M. (1989) Plant Phvsiol., 90, 586-590. Ragland,M., Briat,J.F., Laulhere,J.P., Massenet,O. and Gagnon,J., Theil,E.C. (1990) J. Biol. Chem., 265, 18339-18344. Seckbach,S. (1982) J. Plant. Nutr., 5, 369-394. and Mundy,J. (1990) Plant Cell, 2, 503-512. Skriver,K. Spence,M.J., Henzl,M.T. and Lammers,P.J. (1991) Plant Mol. Biol., 17, 499-504. Theil,E.C. (1987) Annu. Rev. Biochem., 56, 289-315. Theil,E.C. (1990) J. Biol. Chem., 265, 4771-4774. Thompson,M.D., Jacks,C.M., Lenvik,T.R. and Gantt,J.S. (1992) Plant Mol. Biol., 18, 931-944. Van der Mark,F., Bienfait,H.F. and Van der (1983a) Biochem. Ende,H. Biophys. Res. Commun., 115, 463-469. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Abscisic acid is involved in the iron‐induced synthesis of maize ferritin.

Lobréaux, S.; Hardy, T.; Briat, J.F.
The EMBO Journal , Volume 12 (2) – Feb 1, 1993
Download PDF
7 pages

Loading next page...
 
/lp/springer-journals/abscisic-acid-is-involved-in-the-iron-induced-synthesis-of-maize-QHkjsaKtey

References (5)

Publisher
Springer Journals
Copyright
Copyright © European Molecular Biology Organization 1993
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1002/j.1460-2075.1993.tb05698.x
Publisher site
See Article on Publisher Site

Abstract

The EMBO Journal vol.12 no.2 pp.651 -657, 1993 Abscisic acid is involved in the iron-induced synthesis of maize ferritin St6phane Lobr6aux1, Thierry Hardy2 and atoms in their central cavity in a soluble, non-toxic bioavailable Jean-Francois Briat1' 3 form (Theil, 1987; Harrison et al., 1989; Crichton, 1990; Andrews et al., 1992). 'Laboratoire de Biologie Vegetale, Centre National de la Moleculaire Eukaryotic ferritins (plant and animal) arise from a Recherche Scientifique et Joseph Fourier BP 53X, F-38041 Universiti common ancestral gene which is illustrated by their high Grenoble Cedex and 2Laboratoire de Biologie Moleculaire et Cellulaire sequence homology (Ragland et al., 1990; Lescure et Vegetale, Rh6ne-Poulenc Agrochimie, 14-20 rue al., Pierre Baizet, BP 9163, F-69263 Lyon, 09, Cedex France 1991; Spence et al., 1991; Andrews et al., 1992; Lobreaux et al., 1992). This homology implies an extraordinary 3Corresponding author conservation of their three-dimensional structure (Andrews Communicated by J.-D.Rochaix et al., 1992). Plant and animal ferritins, however, do not share the same cytological location and the control of their The ubiquitous iron storage protein ferritin has a highly synthesis in response to iron overload does not take place conserved structure in plants and animals, but a distinct by the same mechanism. First, plant ferritins are found cytological location and a different level of control in within plastids (chloroplasts and non-green plastids), while response to iron excess. Plant ferritins are plastid- animal ferritins are cytoplasmic proteins (Seckbach, 1982; localized and transcriptionally regulated in response to Van der Mark et al., 1983b; Ragland et al., 1990; Lescure iron, while animal ferritins are found in the cytoplasm et al., 1991). Secondly, iron induction of ferritin synthesis and have their expression mainly controlled at the is at the translational level mainly controlled in animals, translational level. In order to understand the basis of while transcription has been shown to be the principal target these differences, we developed hydroponic cultures of of the iron response in plants (Zahringer et al., Van 1976; maize plantlets which allowed an increase in the intra- der Mark et al., 1983a; Klausner and Harford, 1989; cellular iron concentration, leading to a transient accumu- Proudhon et al., 1989; Theil, 1990; Lescure et al., 1991). lation of ferritin mRNA and protein (Lobreaux,S., These differences need to be explained. Massenet,O. and Briat,J.F., 1992, Plant Mol. Biol., 19, Concerning the transcriptional control of plant ferritin 563-575). Here, it is shown that iron induces ferritin in synthesis response to iron, two possibilities have to be and RAB (Responsive to Abscisic Acid) mRNA accumula- considered. First, iron by itself could be an effector turning tion relatively with abscisic acid (ABA) accumulation. on and off ferritin plant gene transcription by direct inter- Ferritin mRNA also accumulates in response to action with some unknown trans-acting factors. For example, exogenous ABA. Synergistic experiments demonstrate in of yeast, transcription activation the metallothionein gene that the ABA and iron responses are lUnked, although CUPI occurs only when copper binds to ACEl (Furst et al., full expression of the ferritin genes cannot be entirely in Escherichia aerobactin 1988) and, coli, the operon is explained by an increase in ABA concentration. when it binds repressed by the Fur protein Fe2+ (Bagg and Inducibility of ferritin mRNA accumulation by iron is environmental Neilands, 1987). Alternately, high iron dramatically decreased in the maize ABA-deficient a stress concentrations could generate response inducing mutant vp2 and can be rescued by addition of exogenous under the control ferritin gene transcription, of an integrated ABA, confirming the involvement of ABA in the iron transduction Control of of plant pathway. expression genes response in plants. Therefore, it is concluded that a major in to stress is often mediated a transduction response by part of the iron-induced biosynthesis of ferritin is which involves the hormone abscisic pathway plant acid achieved a an in through pathway involving increase the see Skriver and Under (ABA) (for a review Mundy, 1990). level of the plant hormone ABA. The general conclusion standard iron nutrition ferritins conditions, plant are not of this work is that the synthesis of in the same protein detected in and vegetative organs (roots leaves), they response to the same environmental signal can be accumulate in seeds their maturation and are during degraded controlled by separate and distinct mechanisms in plants in the first days of growth following germination (Lobreaux and animals. and the iron Briat, 1991). Furthermore, manipulating Key words: abscisic acid/ferritin/iron stress/maize of the culture medium concentrations of cell plant suspen- of results in sions and hydroponically grown plantlets iron of ferritin loading, and in transient induction in synthesis et as well as in roots these cells (Lescure and al., 1991), Introduction of et leaves plantlets (Lobreaux al., 1992). This of an essential element for almost all and environmental control of The use iron, developmental plant ferritin living is on molecules which is reminiscent of the of RAB organisms, dependent biological synthesis expression pattern the of and of iron in to Abscisic and led us to prevent problems insolubility toxicity genes (Responsive Acid) postulate be involved in the control the of these that ABA could of ferritin presence oxygen. Among synthesis molecules, ferritins, a role iron in It should be noted in a class of multimeric in to ubiquitous proteins, play key response plants. that, plants, to several thousand iron because of their RAB are ability sequester genes specifically expressed during embryogenesis Oxford Press University S.Lobr6aux, T.Hardy and J.-F.Briat it is already strongly induced, and close to its while and can be induced in vegetative organs under various stress maximum accumulation, in leaves. It is interesting to note water and salt stresses are the best conditions, among which that at zero time, ferritin mRNA is already present in both an inducible of ferritin documented. Using system synthesis organs and that its concentration in roots can even be higher et 1992), we tested this hypothesis in maize (Lobreaux al., at 3 h after iron treatment than what is observed (Figure 1). that ABA is involved in the iron-stress and demonstrated be the case. This This appears to not always apparent drop to the accumulation of this plastid-located response leading mRNA levels occurs when are in ferritin only plants iron storage protein. harvested at the beginning of the photoperiod. Ferritin mRNA is much less abundant at zero time if samples are Results collected during the photoperiod (see lane 1 in Figures 3 and and Figure 4 in Lobreaux et al., 1992). In such a mRNA accumulation 5, Iron induces RAB and ferritin the amount of ferritin mRNA detected in roots is be induced ABA situation, RAB genes are known to by during the same at zero time and after 3 h post-iron treatment (not various stress conditions embryogenesis and under (Skriver This will be developed further in if iron induction of ferritin shown). particular point and Mundy, 1990). Therefore, mRNA accumulation in the Discussion. Ferritin response is at least in ABA, as we synthesis mediated, part, by and has decreased or it is conceivable that RAB genes could also to iron is transient already to, below, postulate, in both leaves and roots 24 h after iron treat- In order to test this hypothesis, we initial levels respond to iron stress. 1). pMAH9 mRNA also accumulates trans- and two RAB ment (Figure measured the level of ferritin mRNAs, namely iently in leaves, where it reaches its maximum at 6 h, et et and pMAH9 (Gomez al., 1988; Didierjean al., 1992) the whereas its level gradually increased in roots throughout et al., 1990), immediately following iron pMA12 (Villardel was first 24 h after treatment (Figure 1). pMA12 transcript of maize treatment hydroponically grown plantlets (Lobreaux not induced in roots. However, in leaves, pMA12 mRNA et Northern analysis of root and leaf RNA al., 1992). reached a maximum 15 h after the treatment, followed by indicated that ferritin mRNA is rapidly induced after iron 24 h It is to note that treatment with maximum accumulation of ferritin mRNA a decrease at (Figure 1). important the same RNA has been used to perform the h As an internal the RNAs from preparation at 6 (Figure 1). control, Northern blot analysis presented in Figure 1. Thus, two were with a cytoplasmic the same preparation probed different maize RAB as well as ferritin, are rapidly ribosomal protein (CS1 1) riboprobe, known to be genes, induced by an iron stress. It is important to note that each constitutively expressed at the same level in roots and leaves in 1 that the CS transcript displayed a unique pattern of accumulation. (Thompson et al., 1992). It is clear Figure 11 for the same in roots and leaves of maize Furthermore, this pattern can also vary, mRNA level is constant plantlets of iron treatment. Kinetics of ferritin transcript, between roots and leaves. The significance at different times after mRNA are rather different in roots than in will be discussed later (see the accumulation these observations Discussion). leaves. In roots, the amount of transcript is very low at 3 h Iron induces ABA synthesis L EAvES ROOTS The above observation has shown that the expression of FE dS 4 Timelhi 0 3 6 15 ,24 L-: 7. -- known RAB genes is induced by an iron stress in maize plantlets. Such a result could imply that the iron treatment pMFli leads to ABA synthesis and accumulation, correlatively to an increase in ferritin mRNA. In order to address this point, pcsll :i ", .X'- :.. -::: .*Z! 7 CO .0 0 Ii ..: ... .1.1% 2iD 7 00. C 5300 0) pMA12 Im 300' -a) Fig. 1. Effect of an iron stress on the accumulation of ferritin and RAB mRNA in maize plantlets. 10 of total RNA purified from Ag roots or leaves at 0 (plantlets were harvested during the first 30 min of the photoperiod), 3, 6, 15 and 24 h post-iron treatment were loaded in _-4Roots each lane (zero time was omitted in the blots probed with pMAH9 and pMA12). As an internal control, RNAs were also probed with a Leaves X c Hours cytoplasmic ribosomal protein (CS1 1) riboprobe. RNA probes were synthesized as described in Materials and methods from pMFli [SacI-StuI fragment of ferritin FMl cDNA from Lobreaux et al. Fig. 2. Concentrations of ABA in roots and leaves of maize plantlets (1992) cloned in pKSII], pMAH9 [EcoRI fragment from Gomez et al. at different times after the addition of iron. Preparation of samples was (1988) cloned in pKSII], pMA12 [PstI fragment from Villardel et al. as described in Materials and methods. ABA dosage used the (1990) cloned in pKSII] or [EcoRI-RsaI fragment from pCSI1 Phytodetek (Sunnyvale, CA) ABA determination kit. Each value Lebrun and Freyssinet (1991) cloned in pKSII] and used as indicated. results from the mean of six measurements performed with organs RNAs from the same preparation were used in this experiment. harvested from two independent cultures. Standard error was <10%. 652 ABA-mediated regulation of plant ferritin synthesis the ABA concentration was determined by an immunoassay 11 cytoplasmic ribosomal mRNA remains constant the CS using S-ABA monoclonal antibody (Gomez et al., 1988; in and leaves of maize plantlets after a treatment of roots Yamaguchi-Shinozaki et al., 1990). 24 h Determination of the ABA 3, 6, 15 and (Figure 1). First, the ABA concentration in roots and leaves was in leaves, 3 h after iron addition, revealed an concentration determined at different times after iron treatment (Figure 2). increase of 4.7-fold over the phytohormone level of the It is clear that iron stress can lead to the same transitory sample for which no iron was added (Figure 3). A good increase in ABA concentration, in both organs, with a correlation appears to exist between ferritin mRNA maximum observed 3 -6 h after iron addition in the culture accumulation and ABA concentration increases. Therefore, medium. This increase has been estimated to be 4.5-fold. it may be concluded that an iron-dependent transient Secondly, the iron concentration dependence of ferritin accumulation of both ABA and ferritin mRNA occurs in our synthesis has been assessed by Northern analysis of total leaf system. RNA prepared 12 h after the addition of increasing amounts Exogenous ABA induces ferritin synthesis of iron to the plantlet culture medium (Figure 3). It is clearly These results do not prove that ABA, by itself, can induce apparent that the amount of ferritin mRNA increases when ferritin In order to provide proof of this point, synthesis. iron-EDTA concentrations are increased from 0 to 250 IM. 10 ktM ABA were added, after iron starvation, to the culture A further increase of iron-EDTA to 500 does not lead /M medium instead of the iron mixture. The level of ferritin to another increase in ferritin mRNA level. This indicates mRNA was then monitored by Northern analysis at different that 2 250 ,uM iron is saturating this system during a treat- times after hormone addition. The pMAH9 probe was used ment of 12 h. At a saturating concentration of iron (500 ytM), as a positive control for ABA treatment. Results of such an experiment are shown in Figure 4A. Ferritin mRNA was 1 2 3 4 induced both in leaves and roots of ABA-treated plants. The maximum level was reached 15 h after treatment. Further- more, it is shown in Figure 5 that the level of leaf ferritin mRNA in response to a 12 h treatment with increases of exogenous ABA. In such an increasing concentration the amount of the CS 1 1 cytoplasmic ribosomal experiment, protein mRNA remains constant, whatever the concentra- transcripts tion of exogenous ABA used (Figure 5). pMAH9 were also induced in roots and leaves after ABA treatment 410 730 80' A 170 280 4A). The profile of pMAH9 mRNA accumulation (Figure was identical to that observed for ferritin. A difference is 3. Iron dependence of ferritin mRNA and ABA accumulation. Fig. observed between the kinetics of accumulation for both Maize plantlets were induced for ferritin synthesis by using increasing mRNA leaves and roots. In roots, the species between h iron treatment amounts of iron. Leaves were then collected 3 after pMAH9 mRNAs decreases only 12 h treatment for ferritin mRNA amount of ferritin and for ABA determination and after blot. Lane 1: no iron added (plantlets were visualization by Northern 24 while a dramatic drop occurs in leaves at slightly at h, h the had begun); lane 2: 50 harvested >3 after photoperiod AM time. ABA able to this particular Exogenous is, therefore, / lane 3: 100 iron-EDTA / iron -EDTA 7.5 iron-citrate; zM itM induce ferritin mRNA accumulation in iron-starved maize 15 lane 4: 250 iron-EDTA / 37.5 iron-citrate; iron-citrate; yM AM AM plantlets. lane 5: 500 iron-EDTA / 75 iron-citrate. ABA concentration AM izM is the mean of three standard error was < 10%. In order to examine the fate of the ferritin protein measurements; ROOTS LEAVES 2 4 3 15 24 6 15 6 Time(h) 3 pMFli GAPEP AL-....A .hA pMAH9 ABA. Accumulation of ferritin and transcripts from maize roots and leaves in pMAH9 4. ferritin synthesis by exogenous (A) Fig. Induction of were as described in Materials and methods that 10 ABA were added to the culture medium instead Culture conditions except to ABA. zM response and 24 RNA was extracted from roots and leaves, and 10 RNA from each sample were analysed by blot After 15 Northern of iron. 3, 6, h, ztg RNA either from (ferritin) or pMAH9 (RAB). (B) Ferritin and glyceraldehyde 3-phosphate hybridization using probes produced pMFli subunit accumulation as determined by immunodetection using polyclonal antibodies raised either against maize seed dehydrogenase (GAPDH, GapC) after of 40 of total extracted from maize leaves. Lane 1: no iron added; lane 2: 24 h after the or maize protein ferritin GAPDH, blotting ytg iron-EDTA 75 uM iron-citrate; lane 3: 24 h after the addition of 200 AM ABA; lane 4: 20 ng pure maize seed ferritin. This of 500 / addition ztM three the same result. was times, experiment repeated giving 653 S.Lobr6aux, T.Hardy and J.-F.Briat I e,., ?. .* w - :-~~~~~~.. -. pMFli :1 :i ABA BA F .-r lIII Fig. 5. Synergistic effect of iron and ABA on ferritin mRNA accumulation. blots were Northern after of 10 of (A) performed electrophoresis /g total RNA from either purified plantlets untreated 1: were harvested >3 h after (lane plantlets the had or treated for 12 h photoperiod begun) with increasing concentrations of ABA from 0 to 500 with 500 iron-EDTA (lanes 2-6), uM / 75 iron-citrate and with a mixture ltM ptM (lane 7) of 200 ABA 500 iron-EDTA plus / 75 iron-citrate RNAs from the same were (lane 8). with both ferritin IAM AM /iM preparation probed riboprobe and CS11 ribosomal (pMFli) cytoplasmic of the blots with the ferritin riboprobe (pCS11). (B) Quantification Northern hybridized riboprobe was after performed by computer image the Numbers indicate units of the amount analysis scanning of ferritin autoradiographs. arbitrary RNA. accumulation under such an 2 200 conditions, immunodetection ABA is this ktM exogenous saturating system during experiment was total extracted a performed using protein from treatment of 12 to the h, leading maximum steady-state leaves 24 h after treatment with either ABA level of exogenous or ferritin mRNA observed. maize Next, plantlets were iron. shown in As lane iron induces Figure 4B, 2, ferritin exposed to either iron alone or simultaneously to iron and protein accumulation in leaves 24 h after its 200 as ABA. The amount of ferritin addition, transcript accumulated AM previously reported et in to (Lobrdaux al., 1992). response both treatments varied 3 Exogenous only % (Figure SB, ABA treatment of iron-starved is also lanes 7 plantlets responsible and which is not 8), significant. This observation for an increase in the amount of ferritin indicates that protein subunit there is no additivity of exogenous ABA and (Figure 4B, lane 3); this relative increase much iron is, however, responses on ferritin mRNA accumulation. We can, lower than that in iron response to treatment. the Probing conclude that the iron stress therefore, performed saturates same protein extracts Western by blot, a the using polyclonal transduction to pathway, leading ferritin mRNA raised the maize antibody against cytoplasmic accumulation in glyceraldehyde to ABA. It response strongly suggests the 3-phosphate dehydrogenase has shown that the involvement of this (GapC), hormone in the iron-induced synthesis amount of this was protein not affected iron or of maize ferritin. by exogenous However, when are plantlets incubated in ABA treatment (Figure 4B). 200 the ABA, accumulation of ferritin mRNA observed 1sM is 68% of only the level measured when maize is treated Exogenous ABA and iron responses are not additive with iron. This result indicates that an event other than an All the results presented above strongly support the increase in ABA level is involved during iron stress, and hypothesis that ABA is involved during ferritin gene activa- is responsible for 32% of the ferritin mRNA accumulation tion in response to iron overload. To further test this observed 12 h after treatment. hypothesis, an experiment was performed to investigate if there is any synergistic effect between iron and ABA Genetic treat- evidence of involvement of ABA in the iron- ment. In this experiment, the steady-state level of the CS 11 induced synthesis of maize ferritin cytoplasmic ribosomal mRNA has been determined as an All these results strongly support the hypothesis that ABA internal control and does not change is involved significantly as an hormonal relay during iron-induced ferritin (Figure SA). In a first step, we determined the ABA biosynthesis. concen- In order to prove that ABA is necessary for tration which is necessary to induce a maximal accumulation maximal accumulation of ferritin mRNA during iron stress, of ferritin mRNA in response to this hormone, when applied we performed an iron induction experiment in a maize to maize plantlets for 12 h. The mutant ferritin mRNA level from deficient in ABA. For this purpose, we used leaves of iron-starved maize plantlets incubated with various viviparous 2 plantlets; this mutant has a defect in the ABA concentrations was measured by Northern analysis and carotenoid biosynthetic pathway, which produces the quantified by scanning of the autoradiographs (Figure 5). precursors for ABA synthesis (Zeevaart and Creelman, It is clearly apparent that the amount of ferritin mRNA Pla 1988; et al., 1989). Homozygous vp2 plantlets were increases when ABA concentrations are increased from 0 obtained in vitro and transferred to hydroponic culture for to 200 ktM in the culture medium. For an unknown reason, iron starvation, followed by a 12 h iron treatment. The the amount of ferritin mRNA induced by 100 ABA is inducibility of ferritin transcript accumulation in leaves was AtM lower 50 slightly than that at When the hormone quantified by Northern analysis in vp2 and wild-type AM. concentration is further increased to 500 ktM, the level of plantlets. In the ABA-deficient mutant, a 1.4-fold increase ferritin mRNA detected does not increase any more. Thus, in ferritin mRNA level is observed in response to iron 654 ABA-mediated regulation of plant ferritin synthesis and Briat, 1991; et al., 1992), we (Lobreaux Lobreaux WT postulate that ABA could be involved in the control of ferritin ABA - .. synthesis. Indeed, exogenous ABA is able to induce ferritin Fe - _-Fe +Fe mRNA accumulation (Figures 4A and 5). Concentrations of exogenous ABA giving this response are in the range of pMF1 of those described for other systems (Mundy and Chua, 1988; Pla et al., 1989). The decrease in this response at 24 h, mainly in leaves, could be due to ABA degradation during pCS11 its transport and has already been observed (Mundy and Chua, 1988). At the protein level, exogenous ABA allows accumulation of the ferritin subunit, but to a lower extent than is observed after iron treatment (Figure 4B). It makes sense because treatment with exogenous ABA does not allow full accumulation of ferritin mRNA, as does iron treatment (Figure 5 and see below for discussion). Furthermore, it is important to be reminded that iron or ABA addition are achieved after 9 days of iron starvation, therefore the response to exogenous ABA occurs at a very low intracellular iron concentration, which has been previously reported (Lobreaux et al., 1992). It has been known for a long time that, in animal systems, iron is necessary to 'Y increase the stability of ferritin molecules (Crichton, 1971; Drysdale and Shafritz, 1975). It could be the same in plants and it would explain the relatively low ferritin protein accumulation in response to exogenous ABA under condi- tions of iron Fig. 6. Steady-state level of ferritin mRNA in response to iron in starvation. maize leaves from wild-type (WT) and viviparous 2 (vp2) mutant. Because iron and ABA are both able to induce ferritin (A) 10 of total leaf RNA purified from wild-type and lp2 plantlets, Ag accumulation, the possibility that the iron and the ABA grown without iron or with 500 iron-EDTA 75 ytM iron-citrate AM responses are linked was a legitimate question to answer. for 12 h, were probed on the same Northern blot by using the ferritin In 10 yg of total leaf RNA purified from Iron treatment leads to an increase in ABA concentration riboprobe (pMFli). addition, iron or control vp2 plantlets (grown without ABA) and vp2 plantlets which is dependent on the iron concentration in the culture harvested 12 h after the addition of 500 exogenous ABA were also AM medium (Figures 2 and 3). RAB genes are also induced by probed by Northern blots using the ferritin riboprobe (pMFli). As an iron treatment (Figure 1). However, differences in the internal control, the various RNA samples purified from vp2 plantlets kinetics of ferritin and RAB mRNA accumulation in response were also cytoplasmic ribosomal riboprobe probed using the CSl (pCS 1). The RNA probes were synthesized as described in Materials to iron (Figure 1) indicate that multiple events are probably and from and from 15 h at methods pMFli pCSl Exposure was for in This is involved regulating expression of these genes. X film. of -70°C using a Kodak Royal 0-Mat (B) Quantification from particularly evident the differences in mRNA accumula- Northern blots from and leaf RNAs from wild-type vp2 purified tion for between roots and leaves the same transcript. iron or after the addition of 500 plantlets grown without (-Fe) zM it does not imply that entirely different pathways iron-EDTA / 75 iron-citrate for 12 and with the However, (+Fe) h, probed AM This was ferritin riboprobe. quantification performed by computer control the of these genes. For example, pMA12 expression Numbers indicate image analysis after scanning the autoradiographs. and pMAH9, which have very different kinetics of of of ferritin RNA. The same result was arbitrary units the amount accumulation in response to iron (Figure 1), are both in two obtained independent experiments. ABA (Gomez et al., 1988; Villardel et al., regulated by More than one pathway could be involved in the 1990). in the amount of ferritin while wild-type plantlets (Figure 6), control of each The same common pathway could be gene. 7.5 times in iron-treated maize mRNA is higher transcript utilized until a certain step preceding branching of second- in iron-starved The failure of iron induction than plantlets. for each tissue. ary pathways, specific for each gene and/or mutant cannot be attributed to an in the vp2 independent for ferritin Such a complexity is clearly demonstrated to ABA since addition of defect additional deficiency, mRNA. As a first to link the iron and ABA attempt induces ferritin mRNA ABA to exogenous vp2 plantlets an iron treatment was in the responses, performed presence The level of the CS accumulation (Figure 6A). 11 cytoplasmic known of RAB of gibberellic acid (GA3), a antagonist gene remains constant in mRNA concentration ribosomal vp2 et In such an activation (Pena-Cortez al., 1989). experiment addition of iron or ABA with or without plantlets we observed that GA3 the iron-induced partially suppressed to note that after iron starva- It is (Figure 6A). interesting accumulation of ferritin mRNA in 6 h after the iron leaves, the amount of the and to iron tion, prior induction, transcript for unknown treatment an reason, (not shown). However, in than in The is 2.1-fold higher vp2 wild-type plantlets. be variable from culture to this suppression was found to observed in the of ferritin decrease mRNA strong inducibility A ABA/iron culture. experiment (Figure 5) synergistic in that ABA is essential for accumulation vp2 plants proves that ABA and iron are linked, but reveals, indeed, pathways of ferritin induced iron. maximum synthesis by of ferritin mRNA accumulation in that the regulation iron cannot be the response to entirely explained by pathway since full an increase in ABA involving concentration, Discussion cannot be obtained a of the iron expression response by concentration. Such an observ- of ferritin in ABA Based on the of saturating exogenous pattern expression plant the difference in ferritin mRNA and ation could response to iron and during growth development explain 655 S.Lobr4aux, T.Hardy and J.-F.Briat during iron stress, remains to be determined. It has to be accumulation between roots and leaves (Figure 1). The ABA are very similar in both organs at the various remembered that environmental and developmental controls concentrations are not necessarily mediated by the same pathway. In the times analyzed after iron addition, and peak between 3 and case of the proteinase inhibitor pin2 gene, ABA is involved 6 h (Figure 2). However, it is important to notice that after in the wounding response, but not during developmental 3 h ferritinmRNA has not yet accumulated in roots, while control (Pena-Cortez et al., 1991). it is already close to its maximum in leaves (Figure 1). This It has been reported that the bulk of ABA in plants is found indicates that a second ABA-independent pathway could within plastids, where it may be synthesized by the cleavage operate at 3 h in leaves. Alternately, an increase in the ABA of leaf cells, mediated by iron, cannot be ruled of violaxanthin to give xanthoxin as a first step (for a review sensitivity Such an effect has recently been reported by Bostok see Zeevaart and Creelman, 1988). In plants, ferritins are out. also localized within plastids (Seckbach, 1982). In this and Quatrano (1992), showing that salt can increase the context, it is tempting to postulate that during iron stress, sensitivity of rice cells to ABA, affecting Em gene which can be a natural toxicity problem in flooded acidic expression. soils, an increase in iron uptake by plastids could occur, During the course of these kinetic experiments, we leading to an oxidative stress that raises the ABA concen- observed variations in the level of ferritin mRNA at zero time, according to the particular moment when samples were tration through photochemical and/or enzymatic cleavage of xanthophylls. As a consequence ABA, through a transduc- collected (beginning versus a few hours of photoperiod). This tion pathway, could activate ferritin gene expression. Ferritin could be explained by circadian regulation of the expres- is then targeted to plastids where it is assembled in order sion of ferritin genes and/or by differences in the compart- mentalization of ABA, which is known to change according to store excess iron. Experiments are in progress to test this hypothesis. to the light/dark status of the plant (Zeevaart and Creelman, 1988). Further evidence of ABA involvement in the iron- induced ferritin synthesis was gained by testing the iron Materials and methods response in a maize ABA-deficient mutant. Inducibility of Plant cultures ferritin mRNA accumulation by iron in vp2 homozygous Maize plantlets (Zeamays, var. MO17) were grown under hydroponic culture plantlets, which lack ABA due to blocking in the carotenoid in an iron-free medium as previously described (Lobreaux et al., 1992). biosynthesis pathway (Zeevaart and Creelman, 1988), is After 9 days of iron starvation, different treatments were performed. Iron 5.4-fold less than in the wild type (Figure 6B). Interestingly, induction was achieved by adding 500zM Fe-EDTA, 150 Na3-citrate /lM addition of exogenous ABA to vp2 plantlets induces ferritin and 75zM FeSO4 in the culture medium. For ABA treatment, ABA (Sigma) was added to the medium, instead of the iron mixture, by dilution mRNA accumulation (Figure 6A). This result is in agree- of a 100 mM stock solution in ethanol. Root and leaf samples were harvested ment with the fact that the failure of iron induction in the at different times after treatment, frozen in liquid nitrogen and stored at vp2 mutant is due to ABA deficiency and not to an addi- -700C. tional independent defect. In vp2, iron treatment still induces Viviparous 2 homozygous plantlets can only be obtained by selfing of ferritin mRNA accumulation, but to a much lower extent heterozygous plants. Therefore, plants were first obtained from heterozygous seeds (kindly provided by Dr M.Pages, Barcelona) in the greenhouse. The than in wild type (Figure 6). This observation could be growth conditions were 24°C during the day and 18°C at night, with natural related to the fact that part of the iron response could be light supplemented with artificial light to ensure a minimum of 300 1tE/m2/s independent of the ABA response (Figure 5). Although the during a 16 h photoperiod. At -30 days after pollination, ears with white ferritin mRNA level, before adding iron, is always higher kernels were harvested, surface sterilized with a 6.25% sodium hypochlorite solution containing a small amount of detergent as a surfactant, and then ( -2-fold) in vp2 than in wild-type plantlets, making a direct rinsed in three changes of sterile deionized water. White kernels were isolated comparison between the two genetic backgrounds difficult. and put in germination in individual plastic jars with 30 ml of N6 medium So far, developmental and environmental regulation of (Chu et al., 1975). In vitro conditions were 24°C constant, a 16 h RAB genes have, as a common effector, variations in photoperiod (80 fluorescent lighting 'Fluora', Osram) and 80% lE/m2/s osmotic pressure due to either dessication or salt and water relative humidity. When vp2 plantlets were -10 cm high, they were transferred to hydroponic culture medium without iron for 7 days prior to stress (Skriver and Mundy, 1990). The observation that iron treatment with 500 Fe-EDTA, 150yM Na3-citrate, and 75 ytM ferritin gene expression responds to ABA could be relevant piM FeSO4. for understanding the regulation of ferritin synthesis during RNA extraction and analysis development. Plant ferritins, under normal conditions of iron Total RNA extraction was performed as previously described (Lobreaux nutrition, are iron storage proteins which accumulate during et al., 1992). Northern blot analysis of total RNA samples was achieved seed formation and are degraded during seed germination using RNA probes, according to Lobreaux et al. (1992). For ferritin mRNA (Lobreaux and Briat, 1991). This is analogous to the detection, the pMFli ferritin probe was used (Lobreaux et al., 1992). Clones pMAH9 (Gomez et al., 1988; Didierjean et al., 1992), pMA12 (Villardel behaviour of known RAB protein. However, iron stress is et al., 1990) and pCSl 1 (Lebrun and Freyssinet, 1991) were gifts, respect- not likely to be related to an osmotic stress (Mundy and ively, from Dr G.Burkard (Strasbourg), Dr M.Pages (Barcelona) and Dr Chua, 1988; Skriver and Mundy, 1990) because of the M.Lebrun (Lyon). EcoRI insert from pMAH9, PstI insert from pMA12 micromolar range of iron salt we used. It is more likely to and the EcoRI-RsaI fragment from pCSl 1 were subcloned in the appropriate be related to an oxidative stress because of the role metals, restriction sites of Bluescript II KS. For quantification of mRNA levels, autoradiographs were scanned using a 256 grey level scanner (Apple) and such as iron, can have in the conversion of reduced oxygen images were analysed with the software Image 1.3.7. (NIH, Bethesda, USA). into hydroxyl radicals, one of the most reactive species known, through Haber-Weiss reaction (Halliwell, 1987; Protein extraction and analysis The preparation of pure maize seed ferritin and rabbit polyclonal antibodies Imlay and Linn, 1988). Consistent with this point is the raised against its subunit have already been reported (Lobreaux et al., 1992). report that an oxidative stress generated by radiation raises Polyclonal antibodies raised against maize cytoplasmic glyceraldehyde the ABA concentration in wheat seedlings (Degani and Itai, 3-phosphate dehydrogenase (GapC) were a generous gift of Pr. R.Cerff 1978). Whether ferritin synthesis during seed development (Brunschweig University, Germany). Total protein preparation from leaves, protein concentration measurements. is regulated both by ABA and an additional pathway, as 656 ABA-mediated regulation of plant ferritin synthesis SDS-polyacrylamide gel electrophoresis and immunodetection were as Van der Mark,F., Van der Briel,W. and Huisman,H.G. (1983b) Biochem. already described (Lobreaux et al., 1992). J., 214, 943-950. Villardel,J., Freire,M.A., Torrent,M., Martinez,M.C., Goday,A., ABA dosage Plant Mol. 423 Biol., 14, -432. and (1990) Plants were grown hydroponically as described above. At various times TorneaJM. PagMs,M. Mc. BiSo., 15, post-iron treatment, roots and leaves were ground Extracts 905-912. in liquid nitrogen. were prepared according to Yamaguchi-Shinozaki et al. (1990) and the ABA Z inr., Bi,B USA, 73, concentration was determined using the Phytodetek-ABA (Idetek Inc., 857B-861. kit CA) according to the manufacturer's instructions. Zeevaart,J.A.D. and Sunnyvale, (1988) Annu. Rev. Physiol. Creelman,R.A. Plant Plant Mol. Biol., 39, 439-473. Received on on Acknowledgements October 1992 8, 1992; revised 26, July, We gratefully acknowledge Dr M.Pages (CID-CSIC Barcelona, Spain) for the gift of pMA12 and of maize vp2 seeds, Dr G.Burkard (CNRS Strasbourg, France) for the gift of p23.2 (pMAH9), Dr Lebrun Poulenc, (Rh6ne Lyon, France) for the gift of pCSl 1 and Pr. R.Cerff (Brunschweig University, Germany) for the gift of maize GAPDH polyclonal antibody. We thank Dr De Rose (Rh6ne Poulenc, Lyon, France) for comments and critical reading of this manuscript. This work was supported by the Centre National de la Recherche Scientifique (URA 1178) and by the Ministere de la Recherche et de la Technologie (grant 89 C 0869) to J.F.B. References Andrews,S.C., Arosio,P., Bottke,W., Briat,J.F., von Darl,M., Harrison,P.M., Laulhere,J.P., Levi,S., Lobreaux,S. and Yewdall,S.J. (1992) J. Biochem., 47, Inorg. 161-174. Bagg,A. and Neilands,J.B. (1987) 26, 5471-5477. Biochemistry, Bostock,R.M. and Quatrano,R.S. (1992) Plant Physiol., 98, 1356-1363. Chu,C.C., Wang,C.C., Sun,C.S., Hsu,C., Yin,K.C., Chu,C.Y. and Bi,F.Y. (1975) Sci. Sin., 659-668. 18, Crichton,R.R. (1971) Biochim. Acta, 229, 75-82. Biophys. Crichton,R.R. (1990) Adv. Protein Chem., 40, 281-353. Degani,N. and Itai,C. (1978) Exp. Bot., 18, 113-115. Environ. Didierjean,L., Frendo,P. and Burkard,G. (1992) Plant Mol. Biol., 18, -849. Drysdale,J.W. and Shafritz,D.A. (1975) Biochem. Acta, 383, Biophys. 97-105. Hu,S., Hackett,R. and Hamer,D. (1988) Fiirst,P., Cell, 55, 705-717. Gomez,J., Sanchez-Martinez,D., Stiefel,V., Rigau,J., Puigdomnech,P. and Pages,M. (1988) Nature, 334, 262-264. Halliwell,B. (1987) FASEB J., 1, 358-364. Harrison,P.M., Artymiuk,P.J., Ford,G.C., Lawson,D.M., Smith,J.M.A., Treffry,A. and White,J.L. (1989) In Mann,S., Webb,J. and Williams,R.J.P. (eds), Biomineralization: Chemical and Biomedical Perspectives. VCH, Weinheim, pp. 257-294. Imlay,J.A. and Linn,S. (1988) Science, 240, 1302-1309. Klausner,R.D. and Harford,J.B. (1989) Science, 246, 870-872. Lebrun,M. and Freyssinet,G. (1991) Plant Mol. Biol., 17, 265-268. Lescure,A.M., Proudhon,D., Pesey,H., Ragland,M., Theil,E.C. and Briat,J.F. (1991) Proc. Natl. Acad. Sci. USA, 88, 8222-8226. Lobredaux,S. and Briat,J.F. (1991) Biochem. J., 274, 601-606. Lobreaux,S., Massenet,O. and Briat,J.F. (1992) Plant Mol. Biol., 19, 563 -575. Mundy,J. and Chua,N.H. (1988) EMBO J., 7, 2279-2286. Pena-Cortez,H., Sanchez-Serrano,J.J., Mertens,R., Willmitzer,L. and Prat,S. (1989) Proc. Natl. Acad. Sci. USA, 86, 9851-9855. Pena-Cortez,H., Willmitzer,L. and Sanchez-Serrano,J.J. (1991) Plant Cell, 3, 963-972. Pla,M., Goday,A., Vilardell,J., Gomez,J. and Pages,M. (1989) Plant Mol. Biol., 13, 385-394. Proudhon,D., Briat,J.F. and Lescure,A.M. (1989) Plant Phvsiol., 90, 586-590. Ragland,M., Briat,J.F., Laulhere,J.P., Massenet,O. and Gagnon,J., Theil,E.C. (1990) J. Biol. Chem., 265, 18339-18344. Seckbach,S. (1982) J. Plant. Nutr., 5, 369-394. and Mundy,J. (1990) Plant Cell, 2, 503-512. Skriver,K. Spence,M.J., Henzl,M.T. and Lammers,P.J. (1991) Plant Mol. Biol., 17, 499-504. Theil,E.C. (1987) Annu. Rev. Biochem., 56, 289-315. Theil,E.C. (1990) J. Biol. Chem., 265, 4771-4774. Thompson,M.D., Jacks,C.M., Lenvik,T.R. and Gantt,J.S. (1992) Plant Mol. Biol., 18, 931-944. Van der Mark,F., Bienfait,H.F. and Van der (1983a) Biochem. Ende,H. Biophys. Res. Commun., 115, 463-469.

Journal

The EMBO Journal – Springer Journals

Published: Feb 1, 1993

There are no references for this article.