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In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots

In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots Background: Tonoplast intrinsic proteins (TIPs) are widely used as markers for vacuolar compartments in higher plants. Ten TIP isoforms are encoded by the Arabidopsis genome. For several isoforms, the tissue and cell specific pattern of expression are not known. Results: We generated fluorescent protein fusions to the genomic sequences of all members of the Arabidopsis TIP family whose expression is predicted to occur in root tissues (TIP1;1 and 1;2; TIP2;1, 2;2 and 2;3; TIP4;1) and expressed these fusions, both individually and in selected pairwise combinations, in transgenic Arabidopsis. Analysis by confocal microscopy revealed that TIP distribution varied between different cell layers within the root axis, with extensive co-expression of some TIPs and more restricted expression patterns for other isoforms. TIP isoforms whose expression overlapped appeared to localise to the tonoplast of the central vacuole, vacuolar bulbs and smaller, uncharacterised structures. Conclusion: We have produced a comprehensive atlas of TIP expression in Arabidopsis roots, which reveals novel expression patterns for not previously studied TIPs. Background three δ-TIP (TIP2), the seed-specific α- and β-TIP (TIP3;1 Tonoplast intrinsic proteins (TIPs) are a subfamily of and TIP3;2), one ε -TIP (TIP4;1) and one ζ-TIP (TIP5;1). aquaporins, small integral membrane proteins belonging to the major intrinsic protein (MIPs) family. Aquaporins Several TIP isoforms have been studied in detail as regards form channels that facilitate the movement of water, their expression [3,5,6] and function [7,8]. TIPs have also small uncharged solutes (glycerol, urea, boric acid, silicic been widely employed as intracellular markers for vacu- acid, hydrogen peroxide) and gases (ammonia, carbon olar biogenesis and identity. Immunofluorescence experi- dioxide) across biological membranes. (For recent reviews ments in root tips and mature embryos of different plant see [1,2]). TIPs have been either detected, or predicted to species led to the identification of separate vacuolar com- localise, to the tonoplast [3]. partments within the same cell [9-13]. These experiments indicated an association of γ -TIP (TIP1;1) with vegetative, The Arabidopsis genome encodes 10 TIP isoforms [4], fur- lytic-type vacuoles and of α-TIP (TIP3;1) and δ-TIP ther classified into five subgroups: three γ -TIP (TIP1), (TIP2;1) with protein storage vacuoles. The detection of Page 1 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 different TIP isoforms on separate tonoplasts provided tion of TIP1;2, no YFP-tagged TIP isoforms yielded a evidence for multiple, functionally different vacuolar detectable signal in the root cap or meristem (Fig. 1, pan- compartments within plant cells (reviewed in Frigerio et els A to F). In general, TIP-YFP expression initiates at the al, 2008). Recently we compared expression of TIP3;1 and elongation zone. While TIP1;1-YFP and TIP4;1-YFP are TIP1;1 in Arabidopsis and found minimal overlap in the detectable from the base of the elongation zone (panels A timing of their expression, with TIP3;1 being abundant in and F), TIP2;2-YFP and TIP2;3-YFP expression is first embryos of mature seeds and sharply declining during observed at the zone of transition with the differentiation seed germination, to be replaced by TIP1;1 [14]. The latter zone (panels D and E). The onset of fluorescence occurs in was not present in root tips, thus raising some doubt as to different cell types depending on the isoforms. TIP1;1-YFP the applicability of these particular isoforms as vacuolar is initially visible in endodermal cells, before extending to markers in Arabidopsis [5,14]. As the investigation was every cell type in the differentiation zone (Fig. 1, compare limited to the three TIP isoforms against which peptide panels A and G). This pattern is faithfully replicated in lat- antibodies were raised for the immunofluorescence stud- eral roots (Fig. 1M). TIP1;1 expression is strongest at the ies [10], the possibility remained that other TIP family differentiation zone (Fig. 1G). TIP2;2-YFP becomes first members with similar immunoreactivity may be present detectable in the cortex and epidermis, but its expression in different vacuoles within Arabidopsis root tissues. extends to the pericycle as the root matures (Fig. 1, com- Indeed, the tissue-specificity of expression of some TIP pare panels D and J). TIP2;3-YFP has a similarly wide- family members has not yet been investigated in detail. spread distribution in more mature root axes but its expression initiates in the pericycle, then extends to cortex In this report we have mapped the expression of every Ara- and epidermal cells (Fig. 1, panels E and K). Again, the ini- bidopsis TIP isoform that is predicted to be present in root tial expression patterns of TIP2;2 and TIP2;3 are mirrored tissues by transcriptomic analysis [15]. This excludes in the lateral roots (Fig 1, panels P and Q). In contrast to TIP3;1 and TIP3;2 (α and β-TIP), which have seed-specific the previous isoforms, TIP4;1-YFP is only expressed in epi- expression patterns [14,16]; Gattolin and Frigerio, unpub- dermal and (less strongly) in cortical cells of the differen- lished), and both TIP1;3 (γ -TIP3) and TIP5;1 (ζ-TIP), tiation zone (Fig. 1, panels F and L), with fluorescence which are predicted by bioinformatic analysis to be decreasing in more mature parts of the root where lateral expressed solely in floral organs and pollen [15-17]. roots emerge (Fig. 1R). Our results indicate that expression of some TIP isoforms under their native promoters is remarkably tissue and cell- specific. In general, when multiple isoforms are co- expressed in the same cell, they appear to localise mainly to the tonoplast of the central vacuole. Our identification of the sites of expression of every TIP isoform paves the way to understanding TIP specialisation and function in Arabidopsis root tissues. Results In addition to the fluorescent TIP reporters we generated previously for TIP1;1 (γ -TIP1; At2g36830) and TIP2;1 (δ- TIP1; At3g16240) [14], we cloned the genomic sequences of not previously studied isoforms: TIP1:2 (γ -TIP2; At3g26520), TIP2;2, TIP2;3 (δ-TIP2 and δ-TIP3; Expression p Figure 1 atterns of TIP isoforms in Arabidopsis roots At4g17340 and At5g47450) and TIP4;1 (ε -TIP; Expression patterns of TIP isoforms in Arabidopsis At2g25810). We produced chimeric constructs in which roots. 8-day old roots from the indicated transgenic lines either YFP or monomeric RFP were fused in frame to the were excised, stained with propidium iodide for 2 min and C-terminus of each TIP genomic sequence (including their visualised by CLSM. The images show representative results promoter regions, 5' UTR and introns), and generated for each construct. The signals from YFP (green) and propid- transgenic plants which were analysed for TIP-XFP expres- ium iodide fluorescence (red) are merged. Top panels: single sion patterns by confocal laser scanning microscopy optical sections of the root tips, Middle panels: single optical (CLSM). We first observed TIP-YFP expression at low mag- sections of root differentiation zones. Bottom panels: maxi- nification. 8-day old roots from seedlings expressing indi- mal projection of 16 optical z sections (4 μm step-size) vidual YFP-tagged TIPs were stained with propidium through mature root axes and young lateral roots. Scale bars: 100 μm. iodide and analysed by CLSM. With the possible excep- Page 2 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 In the case of TIP1;2, expression seems to be exclusively limited to the root cap and the columella (Fig. 1B). A very limited YFP signal can also be detected in the same region of the young lateral root (Fig 1N, arrowhead) and in older lateral roots (Additional file 1A). Perhaps the most remarkable expression pattern observed is that of TIP2;1, which in 8-day old roots is only detecta- ble in a small region at the base of the lateral roots (Fig. 1O, arrowhead). Having identified the general patterns of expression of the different isoforms at low magnification, we then studied the cell-specificity of TIP-YFP expression in more detail. We analysed propidium iodide -stained roots by CLSM by performing optical z-sections through differentiation zones at 63× magnification (Fig. 2). TIP1;1-YFP is clearly expressed in epidermis and cortex, but its expression is particularly strong in the endodermis and pericycle (Fig. 2A). Here TIP1;1-YFP highlights numerous bright circular structures in the lumen of the central vacuole. We hypothesise these are vacuolar 'bulbs', which have previously been described as tonoplast invagi- nations, which occur independently of the ectopic expres- sion of XFP-tagged membrane proteins, and where TIP1;1-GFP is concentrated [18,19]. It is however difficult in some cases to observe a continuity between these struc- tures and the central vacuole tonoplast. At higher magnification, the overlapping patterns of expression of TIP2;2-YFP and TIP2;3-YFP are confirmed. Both are present in pericycle cells, particularly in the rows of pericycle cells that form the xylem poles [20]. Both TIP- YFPs tend to be absent from the endodermis (Fig. 2, pan- els B and D), although we could detect discontinuous Ce Figure 2 ll-type specificity of TIP-YFP expression in the root axis endodermal expression at various positions along most Cell-type specificity of TIP-YFP expression in the root axis. 8-day old roots from the indicated transgenic root axes (Fig. 2, panels C and E; and highlighted in blue lines were excised, stained with propidium iodide for 2 min in panels G and H). and visualised by CLSM. Stacks of 80 optical z sections (1 μm step-size) were collected from root axes at the differentia- In contrast to the previous isoforms labelling inner root tion zone. The images show representative results for each cell layers, TIP4;1 expression is clearly restricted to the construct. A to F: for each panel, the top section shows a sin- root epidermis and cortex, with no signal detectable in the gle xy optical section, and the bottom section shows the xz inner layers (Fig. 2F). projection of the whole image stack, revealing the cross sec- tion of the root axis. The signals from YFP fluorescence Localisation of TIP1;1 and TIP1;2 (green) and propidium iodide fluorescence (red) are merged. Having analysed the TIPs with the broadest expression G and H: the YFP fluorescence trace from representative patterns, we focussed on the two TIPs which seem to have image stacks for the indicated transgenic lines was recon- structed, segmented and rendered in 3D with Mimics 12.1. a more limited expression range in roots. TIP1;2-YFP pre- The different tissues are colour-coded as follows: brown, sented a patchy distribution in cells of the root cap (Fig epidermis; red, cortex; blue, endodermis; green, pericycle. 1B). To ascertain that this was not an artefact due to Ep, epidermis; c, cortex; e, endodermis; p, pericycle; x, expression of our chimeric gene, we also generated a con- xylem. Scale bar: 20 μm. struct (YFP-TIP1;2) where YFP was fused downstream of the promoter and 5'UTR and in frame with the 5' of the Page 3 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 TIP coding sequence. In transgenic plants, YFP-TIP1;2 the labelled cells are in the process of detaching from the presents a similar expression pattern to TIP1;2-YFP, thus root (Fig. 3, panels C and F, arrowheads), suggesting that ruling out YFP fusion artefacts (compare Fig. 3A with Fig. they may correspond to 'border-like' cells [22]. The distri- 1B). Expression is confined to the columella and the lat- bution of YFP-TIP1;2 is therefore radically different to that eral root cap [21], with the labelled cells disappearing at observed for its paralogue, TIP1;1-YFP, which has the wid- the boundary with the elongation zone (Fig. 3A). Some of est pattern of expression but is excluded from the root tip, including the root cap (Fig. 1, panels A to M). At the subcellular level, YFP-TIP1;2 localises to the endo- plasmic reticulum (ER) of young root cap cells (Fig 3e: note the characteristic reticular pattern and the nuclear envelope; see also Additional file 1B). The chimeric pro- tein is mostly found on the tonoplast of elongated lateral root cap cells (Fig. 3D). This is likely to reflect different stages of TIP1;2 trafficking in cells of different ages, rather than impaired capacity to reach the tonoplast. This is fur- ther demonstrated by the fact that in the epidermis of cot- yledonary cells, where TIP1;2 is uniformly expressed, the fusion protein appears to localise to the tonoplast (Addi- tional file 1C-D). TIP2;1 is localised in lateral root primordia We have previously shown that TIP2;1 expression becomes detectable in old root regions nearing the hypocotyl, and is then widespread in hypocotyl and coty- ledonary leaves [14]. We did not initially notice expres- sion in young roots, but closer analysis revealed that in 8- day old roots TIP2;1-YFP has a very specialised expression pattern (Fig. 4). The YFP signal is detected in a ring-like cluster at the base of emerging lateral roots (Fig. 4, panels A-D). In very early lateral root primordia (LRP), TIP2;1 expression is detectable in 2-4 cells at the LRP. As the LRP grows further, the number of cells expressing TIP2;1-YFP increases but remains confined to a cluster underlying the YFP Figure 3 -TIP1;2 is expressed in the root cap base the lateral root (Fig. 4, panels F-I). In rare cases, when YFP-TIP1;2 is expressed in the root cap. 8-day old the lateral root is fully emerged, the expression of TIP2;1- roots from the indicated transgenic lines were excised, YFP can extend to some cells within the lateral root axis stained with propidium iodide for 2 min and visualised by (Fig 4I). Co-labelling with propidium iodide shows that CLSM. Stacks of 80 optical z sections (1 μm step-size) were the TIP2;1 expressing cells are located in close proximity collected from root tips. The images show a representative to the xylem (fig. 4E), suggesting a pericycle localisation. result for this construct. The signals from YFP fluorescence Co-expression with TIP2;3-RFP, which we found to be (green) and propidium iodide fluorescence (red) are merged. enriched in the pericycle (Fig. 2, panels D, E, H) confirms A: maximal 3D projection of the root tip at the base of the that TIP2;1-YFP expression originates from pericycle cells elongation zone. The image shows two adjacent z-stacks of (Fig. 4, panels F-I). This indicates that the initial expres- the same root, separated by a black line. B and C: xz projec- sion of TIP2;1-YFP is likely to occur in the LRP founder tions of the image stack in panel a, revealing two cross-sec- tions of the root axis, taken in the regions of the root cells. Remarkably, the expression of TIP2;1-YFP and indicated by the arrowheads in A. D and E: the regions indi- TIP2;3-RFP appears to be mutually exclusive, with a clear cated by dotted boxes in A were observed at high magnifica- boundary between cells expressing one or the other iso- tion. Single optical sections are shown. Note YFP-TIP1;2 in form (Fig. 4H, inset; see Additional file 2 for individual the ER of young root cap cells and in the tonoplast of root channels). cap cells closer to the elongation zone. F: The fluorescent traces from YFP (green) and propidium iodide (red) from the Overlapping TIP isoforms are mostly detectable at the image stack in panels A were reconstructed, segmented and central vacuole tonoplast rendered in 3D with Mimics 12.1. Scale bars: (a), 20 μm; (d) We have shown that the various TIP isoforms under study and (e), 10 μm. present diverse tissue specificity within roots. Several iso- Page 4 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 with different spectral variants of fluorescent proteins and co-expressed in transgenic Arabidopsis (Fig. 5). The individual TIP expression patterns in double trans- genic lines mirrored those observed in the lines expressing individual isoforms (Additional file 3). The widespread !" !" !" TIP 2;1-YFP expression in Figure 4 lateral root primordia TIP 2;1-YFP expression in lateral root primordia. A-E: 8-day old roots from TIP2;1-YFP transgenic seedlings were excised, stained with propidium iodide and visualised by !" CLSM. Stacks of 80 optical z sections (1 μm step-size) were collected from mature root axes. The images show repre- sentative results for this construct. Maximal projections of the z-stacks are shown, with the individual signals for YFP (A), propidium iodide (B) or the merged signals (C and D). E: The fluorescent traces from YFP (green) and propidium iodide (red) from the image stack in panels (A-C) was recon- structed, segmented and rendered in 3D with Mimics 12.1. Note that the TIP2;1-YFP-expressing cells are in close prox- imity to the xylem (labelled with x). F-I: Roots from 8-day old transgenic seedlings expressing TIP2;1-YFP (green) and TIP2;3-RFP (red) were imaged. Sequential stages of lateral Overlapping TIP plast of Figure 5 the central vacuole isoforms are mainly detected at the tono- root development are shown. Inset in H: note the boundary Overlapping TIP isoforms are mainly detected at the between pericycle cells expressing TIP2;3-RFP (top) and tonoplast of the central vacuole. Transgenic seedlings TIP2;1-YFP (bottom). Scale bars: 20 μm. co-expressing the indicated TIP-YFP and TIP-RFP constructs were grown for 8 days on MS medium--agar plates. Roots were excised and visualised by CLSM. (A, D, G, J): YFP fluo- forms, however, are co-expressed in certain tissues, rescence (green); (B, E, H, K): RFP fluorescence (red); (C, F, namely TIP1;1, TIP2;2, TIP2;3, and TIP4;1. In order to I, L): merged images. Arrowheads in panel I indicate struc- ascertain whether these isoforms were specific to distinct tures labelled by TIP2;3-RFP but not TIP2;2-YFP. Scale bars:10 μm. vacuolar compartments, we focused on the subcellular localisation of selected pairs of the above isoforms, tagged Page 5 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 TIP1;1-YFP and TIP2;3-RFP are co-expressed in epidermis, is intriguing. Transcripts of the maize aquaporin ZmTIP1 cortex and pericycle cells (Fig. 2). In these tissues, both were also localised in the lateral root by in situ hybridisa- proteins are detected on the tonoplast of the central vacu- tion, but every cell in the LRP seemed to contain the tran- ole (Fig. 5A-C). Both the tonoplast and the smaller, bulb- script [25]. TIP2;1 can therefore be considered an like vacuolar structures [18] are labelled. Likewise, TIP2;2- additional marker for the Arabidopsis LRP margins, YFP and TIP1;1-RFP mostly label the same tonoplast in alongside the auxin efflux carriers Pin4 and Pin6 [26] and the cell layers where they are co-expressed (Fig. 5D-F). the transcription factor CUC3 [27], which have a similar TIP2;2-YFP and TIP2;3-RFP, which almost overlap in root localisation. It will be interesting to study the specific role tissues (Fig. 2), are also co-localised on tonoplast and of TIP2;1 in these cells and determine why this stage of lat- 'bulbs' (Fig. 5G-I). Occasionally, TIP2;3-RFP highlighted eral root development demands such a precise TIP iso- smaller vesicular structures that did not appear to contain form activation. TIP2;2-YFP (Fig 5I, arrowheads). The nature of these struc- tures was not investigated further. Finally, TIP4;1-YFP, As a general observation, we could not detect expression which is restricted to epidermis and cortex (Fig. 2F), co- of any of the TIP-XFP fusions under study in the root tip localises with TIP2;3 in those tissues (Fig. 5J-L). Note that meristem. This lack of expression was previously reported the relative abundance of these two isoforms mirrors the for TIP1;1 both by histochemical detection of GUS pattern observed in single isoform localisation, with fusions [5] and YFP tagging [14,19]. It is of course possi- TIP4;1 expression being strongest in the epidermis and ble that expression levels of our fusions are too low in this weaker in the cortex (Fig. 2F), and TIP2;3 expression being region to be detected by confocal microscopy. However, stronger in cortex but weaker in epidermis (Fig. 2, panels the fact that the more sensitive histochemical GUS stain- D-E). ing also fails to detect expression of TIP1;1, which micro- array data indicate is the most abundantly transcribed Taken together, these co-expression results indicate that isoform in roots [16], strongly suggest that the protein is each TIP isoform-fluorescent protein fusion we analysed not expressed in the root meristem. This is in contrast with is predominantly found at the central vacuole tonoplast in data from other species such as pea and barley, where TIPs Arabidopsis root tissues. have been located in isolated root tip cells [10,12] and in root tip sections by immunohistochemical methods [28]. Analysis in the Olbrich et al. study was performed on 3- Discussion We have produced a complete expression map for all day old seedlings. At the same age in Arabidopsis seed- members of the TIP family that are present in Arabidopsis lings we could already detect all the TIP isoforms root tissues, including isoforms not previously studied. described in this study, with the exception of TIP2;1. The use of XFP fusions to TIP genomic sequences allowed However their expression pattern was already the same as us to investigate both the tissue specificity and the subcel- observed at 8 days (data not shown). We therefore lular localisation of these proteins. resolved to present results at 8 days, when the complete set of root TIPs is detectable. In general, our fluorescent reporter - TIP localisation data correlate well with the relative TIP transcript levels, as This lack of observable expression in root tips makes it dif- observed by microarray analysis [15,23,24], with the ficult to perform meaningful comparisons between the exception of TIP1;2. TIP1;2 is indeed the isoform with the vacuolar complement of Arabidopsis root tip cells and highest level of mRNA expression in the root cap [23], that of other plant species. which matches our observations (Fig. 3). However, tran- script levels for TIP1;2 have also been shown to be almost TIP-YFP expression was also not detected in the root vas- as high as TIP1;1 throughout the root axis [15,23,24]. We culature, regardless of the developmental stage. This mir- can only speculate at this stage that post-transcriptional rors observations in barley and pea root sections, where control processes prevent TIP1;2-YFP protein from being the stele was not labelled by TIP antisera [28]. While it is detectable in these tissues. easy to rationalise the absence of a vacuole in the xylem cells, which underwent autolysis, and in mature sieve ele- TIP1;1 is the most widely expressed isoform along the ments, which lack true vacuoles (reviewed in [29]) it was root axis. TIP2;2 and 2;3 have very similar expression pat- somewhat surprising not to find TIPs in the companion terns, with their expression being low in the endodermis, and parenchima cells. We think it unlikely that this lack of but high in the xylem pole pericycle. It appears that TIP2;1 detection is caused by a loss of sensitivity by the confocal becomes strongly expressed in the pericycle when this microscope detectors in the inner layers of the roots, undergoes differentiation to form the lateral root primor- because both propidium iodide staining and YFP signal dium (Fig. 4). This narrow range of localisation of TIP2;1 are easily detected in the xylem and xylem pole pericycle, Page 6 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 respectively (Fig. 2 and Additional file 3). In addition, we the promoter (if longer than 1.5 Kb), plus 5' UTR and could easily detect 35S::TIP2;1-YFP in the vascular tissue introns, was amplified from total genomic DNA from Ara- using the same settings (Additional file 4). Accordingly, bidopsis thaliana Columbia ecotype. Primers included Boursiac et al [24] recently showed that constitutively restriction sites KpnI at the 5' and XhoI at the 3' of the tar- expressed TIP1;1-GFP and TIP1;2-GFP clearly label the get sequences. Amplified fragments were cloned into the vascular tissue [24]. KpnI and XhoI sites of pGREEN0029, upstream of a XhoI/ SacI fragment containing the YFP coding sequence and Recently it has been shown that Arabidopsis knockout the OCS 3' terminator fragment. A similar strategy was mutants lacking TIP1;1, TIP1;2, or both isoforms, do not adopted to fuse TIP sequences to RFP, but in this case the have any major defects [7,19]. This is in contrast with forward primers included both KpnI and SacI restriction drastic defects observed in Arabidopsis upon downregula- sites, generating a TIP-RFP cassette that could be mobi- tion of TIP1;1 by RNAi [30]. A possible explanation for lized with SacI. To obtain pairwise TIP-YFP/TIP-RFP com- the latter result is off-target silencing in the RNAi lines [7]. binations, selected TIP-RFP cassettes were excised with Our data provide a rationale for the lack of a macroscopic SacI and ligated into TIP-YFP vectors linearised with SacI, phenotype in the double TIP1 knockouts observed by giving rise to constructs harbouring both reporter genes in Schussler et al. We have shown that, in roots, expression a tandem. All the chimeric constructs were introduced of TIP1;1 and TIP1;2 does not appear to overlap, with into strain C58 of Agrobacterium tumefaciens harbouring TIP1;1 being expressed in epidermis, cortex, endodermis the pSoup vector [31]. Arabidopsis plants were then trans- and perycycle starting from the elongation zone, and formed using the floral dip method [32]. TIP1;2 being restricted to the root cap. As TIP1;1 and 1;2 show different tissue specificities, it seems unlikely that Confocal analysis and image processing they are reciprocally redundant. In addition, we have About 30 seeds from at least 4 independent transgenic shown that other TIP isoforms, namely TIP2;2, TIP2;3 and lines per construct were germinated onto agar plates con- TIP4;1 would still be present in the tissues lacking TIP1;1 taining half-strength Murashige and Skoog (MS) Basal (Fig. 2). It is therefore possible that these remaining iso- Medium (Sigma-Aldrich) and grown for 8 days at 22°C, forms compensate for the lack of TIP1;1 in the knockout. in a 16:8 light:dark regime. Roots were excised, mounted On the other hand, the effect of the absence of TIP1;2 in half-strength liquid MS medium and immediately from the root cap may be subtle and may have gone unde- observed with a Leica TSC SP5 confocal laser scanning tected under the experimental conditions adopted for the microscope, using either a 10× (NA 0.3) air or a 63× (NA whole-plant analysis of the double mutants. A lack of phe- 1.4) oil immersion objective. In some cases roots were notype in the aerial parts of the single knockout plants preincubated for 2 min in 10 μg/ml propidium iodide, may be explained by the fact that both TIP1;1 and TIP1;2 diluted in half-strength MS medium. YFP was excited at are expressed in leaves [14] and Additional file 2) and 514 nm and detected in the 525 to 550 nm range. RFP was may well be acting redundantly there. As for the double excited at 561 nm and detected in the 553 to 638 nm knockout, redundancy may be afforded by TIP2;1 [14] range. Propidium iodide was excited at 561 nm and and TIP2;2 [16], which are also expressed in leaves. detected in the 650 to 720 nm range. Simultaneous detec- tion of YFP and RFP or YFP and propidium iodide was Conclusion performed by combining the settings indicated above in We have identified novel patterns of expression of TIP iso- the sequential scanning facility of the microscope, as forms in Arabidopsis roots. This information may provide instructed by the manufacturer. a useful starting point for a more targeted approach to dis- sect the function of individual TIP isoforms in root tissues. 3D reconstruction of z-stacks of optical sections was per- It also provides the foundation for further analysis of the formed with the Leica LAS-AF Lite free software (Leica intracellular targeting of different TIPs. Microsystems, Germany). Segmentation analysis and 3D rendering were performed with Mimics 12.1 (Materialise Methods N.V., Leuwen, Belgium). Recombinant DNA and generation of transgenic plants The constructs encoding native TIP1;1-YFP and native Abbreviations TIP2;1YFP have been described previously [14]. CLSM: confocal laser scanning microscopy; ER: endoplas- mic reticulum; PI: propidium iodide; TIP: tonoplast A full list of primers designed to amplify the genomic intrinsic protein. sequences of the root-expressed TIPs is shown in Addi- tional file 5. Each TIP genomic sequence, including either Authors' contributions the complete promoter region (up to the UTR of the gene SG generated the majority of the constructs and transgenic immediately upstream in the chromosome) or 1.5 Kb of plants and performed the bulk of the confocal analysis. Page 7 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 MS produced the TIP1;2-RFP and 35S:TIP2;1 constructs Additional file 5 and transgenic lines and performed confocal analysis. PH Primers used in this study. The diagram indicates the target sequences produced the native TIP1;1-YFP and TIP2;1 YFP constructs for the indicated primers in the final constructs. Restriction sites are and transgenic plants and performed confocal analysis. shown in bold. RK performed the 3D image analysis in MIMICS. LF Click here for file designed the experimental programme, gave technical [http://www.biomedcentral.com/content/supplementary/1471- and intellectual guidance and wrote the manuscript. All 2229-9-133-S5.PDF] authors read and approved the final manuscript. Additional material Acknowledgements We are grateful to Robert Spooner and Alessandro Vitale for critical read- Additional file 1 ing of the manuscript. This work was orted in part by the European Union Expression and subcellular localisation of TIP1;2. 8-day old seedlings (LSH-2002-1.2.5-2 "Recombinant Pharmaceuticals from Plant for Human expressing YFP-TIP1;2 were visualised by CLSM. A: 10× magnification Health -Pharma-Planta") and by the Leverhulme Trust (grant F/00215/AP). of a lateral root. The signals from YFP fluorescence (green) and propidium A grant from the Fondation 'Les Gueules Cassées' funded the acquisition of iodide fluorescence (red) are merged. B: single root cap cell with TIP1;2- the Mimics software. YFP showing typical ER labelling. C-D: epidermal cells in cotyledons where TIP1;2-YFP shows typical tonoplast labelling (green). Red: chloro- References phyll autofluorescence (excitation 514 nm, detection 600-650 nm). Scale 1. Kaldenhoff R, Fischer M: Functional aquaporin diversity in bars: A, 100 μm; B and D, 5 μm; C, 20 μm. plants. Biochim Biophys Acta 2006, 1758:1134-1141. Click here for file 2. Maurel C: Plant aquaporins: Novel functions and regulation [http://www.biomedcentral.com/content/supplementary/1471- properties. FEBS Letters 2007, 581:2227-2236. 3. Hofte H, Hubbard L, Reizer J, Ludevid D, Herman EM, Chrispeels MJ: 2229-9-133-S1.PDF] Vegetative and Seed-Specific Forms of Tonoplast Intrinsic Protein in the Vacuolar Membrane of Arabidopsis thaliana. Additional file 2 Plant Physiol 1992, 99:561-570. Mutually exclusive expression of TIP2;1 and TIP2;3 in lateral root pri- 4. Johanson U, Karlsson M, Johansson I, Gustavsson S, Sjovall S, Fraysse L, Weig AR, Kjellbom P: The complete set of genes encoding mordia. Roots from 8-day old transgenic seedlings expressing TIP2;1-YFP major intrinsic proteins in Arabidopsis provides a frame- (green) and TIP2;3-RFP (red) were visualised by CLSM. Scale bar, 20 work for a new nomenclature for major intrinsic proteins in μm. plants. Plant Physiol 2001, 126:1358-1369. Click here for file 5. Ludevid D, Hofte H, Himelblau E, Chrispeels MJ: The Expression [http://www.biomedcentral.com/content/supplementary/1471- Pattern of the Tonoplast Intrinsic Protein gamma-TIP in 2229-9-133-S2.PDF] Arabidopsis thaliana Is Correlated with Cell Enlargement. Plant Physiol 1992, 100:1633-1639. 6. Daniels MJ, Chaumont F, Mirkov TE, Chrispeels MJ: Characteriza- Additional file 3 tion of a new vacuolar membrane aquaporin sensitive to Co-expression of selected TIP-XFP pairs. Transgenic seedlings co- mercury at a unique site. Plant Cell 1996, 8:587-599. 7. Schussler MD, Alexandersson E, Bienert GP, Kichey T, Laursen KH, expressing the indicated TIP-YFP and TIP-RFP constructs were grown for Johanson U, Kjellbom P, Schjoerring JK, Jahn TP: The effects of the 8 days on MS medium-agar plates. Roots were excised and visualised by loss of TIP1;1 and TIP1;2 aquaporins in Arabidopsis thaliana. CLSM. Stacks of 80 optical z sections (1 μm step-size) were collected from Plant J 2008, 56:756-767. root axes at the differentiation zone. The images show representative 8. Loque D, Ludewig U, Yuan L, von Wiren N: Tonoplast Intrinsic results for each construct. Each panel shows the xz projection of the whole Proteins AtTIP2;1 and AtTIP2;3 Facilitate NH3 Transport image stack, revealing the cross section of the root axis. into the Vacuole. Plant Physiol 2005, 137:671-680. 9. Jauh GY, Fischer AM, Grimes HD, Ryan CA Jr, Rogers JC: delta- Click here for file Tonoplast intrinsic protein defines unique plant vacuole [http://www.biomedcentral.com/content/supplementary/1471- functions. Proc Natl Acad Sci USA 1998, 95:12995-12999. 2229-9-133-S3.PDF] 10. Jauh G-Y, Phillips TE, Rogers JC: Tonoplast intrinsic protein iso- forms as markers for vacuolar functions. Plant Cell 1999, Additional file 4 11:1867-1882. 11. Gillespie J, Rogers SW, Deery M, Dupree P, Rogers JC: A unique Constitutively expressed TIP2;1-YFP is detectable in every root tissue. family of proteins associated with internalized membranes Roots from 8-day old transgenic seedlings expressing 35S::TIP2;1-YFP in protein storage vacuoles of the Brassicaceae. Plant J 2005, (green) were excised, stained with propidium iodide (red) for 2 min and 41:429-441. visualised by CLSM. A: stacks of 80 optical z sections (1 μm step-size) 12. Paris N, Stanley CM, Jones RL, Rogers JC: Plant cells contain two were collected from root axes at the differentiation zone. The images show functionally distinct vacuolar compartments. Cell 1996, 85:563-572. representative results for this construct. The signals from YFP fluorescence 13. Poxleitner M, Rogers SW, Lacey Samuels A, Browse J, Rogers JC: A (green) and propidium iodide fluorescence (red) are merged. B-D: single role for caleosin in degradation of oil-body storage lipid dur- optical section through the vascular tissue, indicating that constitutive ing seed germination. Plant J 2006, 47:917-933. expression of TIP2;1 is easily detectable in these cell types. B: YFP, C, pro- 14. Hunter PR, Craddock CP, Di Benedetto S, Roberts LM, Frigerio L: pidium iodide, D, merged images. Scale bar, 10 μm. Fluorescent Reporter Proteins for the Tonoplast and the Click here for file Vacuolar Lumen Identify a Single Vacuolar Compartment in Arabidopsis Cells. Plant Physiol 2007, 145:1371-1382. [http://www.biomedcentral.com/content/supplementary/1471- 15. Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, 2229-9-133-S4.PDF] Scholkopf B, Weigel D, Lohmann JU: A gene expression map of Page 8 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 Arabidopsis thaliana development. Nat Genet 2005, 37:501-506. 16. 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Molecular and cellular features of aquaporin expres- sion. Plant Physiol 2005, 139:790-805. 25. Chaumont F, Barrieu F, Herman EM, Chrispeels MJ: Characteriza- tion of a maize tonoplast aquaporin expressed in zones of cell division and elongation. Plant Physiol 1998, 117:1143-1152. 26. Benkova E, Michniewicz M, Sauer M, Teichmann T, Seifertova D, Jur- gens G, Friml J: Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 2003, 115:591-602. 27. Vroemen CW, Mordhorst AP, Albrecht C, Kwaaitaal MA, de Vries SC: The CUP-SHAPED COTYLEDON3 gene is required for boundary and shoot meristem formation in Arabidopsis. Plant Cell 2003, 15:1563-1577. 28. Olbrich A, Hillmer S, Hinz G, Oliviusson P, Robinson DG: Newly formed vacuoles in root meristems of barley and pea seed- lings have characteristics of both protein storage and lytic vacuoles. Plant Physiol 2007, 145:1383-1394. 29. De D: Plant Cell Vacuoles Collingwood, Australia: CSIRO Publishing; 30. Ma S, Quist TM, Ulanov A, Joly R, Bohnert HJ: Loss of TIP1;1 aquaporin in Arabidopsis leads to cell and plant death. Plant J 2004, 40:845-859. 31. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM: pGreen: a versatile and flexible binary Ti vector for Agrobac- terium-mediated plant transformation. Plant Mol Biol 2000, 42:819-832. 32. Clough SJ, Bent AF: Floral dip: a simplified method for Agro- bacterium-mediated transformation of Arabidopsis thal- Publish with Bio Med Central and every iana. Plant J 1998, 16:735-743. scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots

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Copyright © 2009 by Gattolin et al; licensee BioMed Central Ltd.
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Life Sciences; Plant Sciences; Agriculture; Tree Biology
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Abstract

Background: Tonoplast intrinsic proteins (TIPs) are widely used as markers for vacuolar compartments in higher plants. Ten TIP isoforms are encoded by the Arabidopsis genome. For several isoforms, the tissue and cell specific pattern of expression are not known. Results: We generated fluorescent protein fusions to the genomic sequences of all members of the Arabidopsis TIP family whose expression is predicted to occur in root tissues (TIP1;1 and 1;2; TIP2;1, 2;2 and 2;3; TIP4;1) and expressed these fusions, both individually and in selected pairwise combinations, in transgenic Arabidopsis. Analysis by confocal microscopy revealed that TIP distribution varied between different cell layers within the root axis, with extensive co-expression of some TIPs and more restricted expression patterns for other isoforms. TIP isoforms whose expression overlapped appeared to localise to the tonoplast of the central vacuole, vacuolar bulbs and smaller, uncharacterised structures. Conclusion: We have produced a comprehensive atlas of TIP expression in Arabidopsis roots, which reveals novel expression patterns for not previously studied TIPs. Background three δ-TIP (TIP2), the seed-specific α- and β-TIP (TIP3;1 Tonoplast intrinsic proteins (TIPs) are a subfamily of and TIP3;2), one ε -TIP (TIP4;1) and one ζ-TIP (TIP5;1). aquaporins, small integral membrane proteins belonging to the major intrinsic protein (MIPs) family. Aquaporins Several TIP isoforms have been studied in detail as regards form channels that facilitate the movement of water, their expression [3,5,6] and function [7,8]. TIPs have also small uncharged solutes (glycerol, urea, boric acid, silicic been widely employed as intracellular markers for vacu- acid, hydrogen peroxide) and gases (ammonia, carbon olar biogenesis and identity. Immunofluorescence experi- dioxide) across biological membranes. (For recent reviews ments in root tips and mature embryos of different plant see [1,2]). TIPs have been either detected, or predicted to species led to the identification of separate vacuolar com- localise, to the tonoplast [3]. partments within the same cell [9-13]. These experiments indicated an association of γ -TIP (TIP1;1) with vegetative, The Arabidopsis genome encodes 10 TIP isoforms [4], fur- lytic-type vacuoles and of α-TIP (TIP3;1) and δ-TIP ther classified into five subgroups: three γ -TIP (TIP1), (TIP2;1) with protein storage vacuoles. The detection of Page 1 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 different TIP isoforms on separate tonoplasts provided tion of TIP1;2, no YFP-tagged TIP isoforms yielded a evidence for multiple, functionally different vacuolar detectable signal in the root cap or meristem (Fig. 1, pan- compartments within plant cells (reviewed in Frigerio et els A to F). In general, TIP-YFP expression initiates at the al, 2008). Recently we compared expression of TIP3;1 and elongation zone. While TIP1;1-YFP and TIP4;1-YFP are TIP1;1 in Arabidopsis and found minimal overlap in the detectable from the base of the elongation zone (panels A timing of their expression, with TIP3;1 being abundant in and F), TIP2;2-YFP and TIP2;3-YFP expression is first embryos of mature seeds and sharply declining during observed at the zone of transition with the differentiation seed germination, to be replaced by TIP1;1 [14]. The latter zone (panels D and E). The onset of fluorescence occurs in was not present in root tips, thus raising some doubt as to different cell types depending on the isoforms. TIP1;1-YFP the applicability of these particular isoforms as vacuolar is initially visible in endodermal cells, before extending to markers in Arabidopsis [5,14]. As the investigation was every cell type in the differentiation zone (Fig. 1, compare limited to the three TIP isoforms against which peptide panels A and G). This pattern is faithfully replicated in lat- antibodies were raised for the immunofluorescence stud- eral roots (Fig. 1M). TIP1;1 expression is strongest at the ies [10], the possibility remained that other TIP family differentiation zone (Fig. 1G). TIP2;2-YFP becomes first members with similar immunoreactivity may be present detectable in the cortex and epidermis, but its expression in different vacuoles within Arabidopsis root tissues. extends to the pericycle as the root matures (Fig. 1, com- Indeed, the tissue-specificity of expression of some TIP pare panels D and J). TIP2;3-YFP has a similarly wide- family members has not yet been investigated in detail. spread distribution in more mature root axes but its expression initiates in the pericycle, then extends to cortex In this report we have mapped the expression of every Ara- and epidermal cells (Fig. 1, panels E and K). Again, the ini- bidopsis TIP isoform that is predicted to be present in root tial expression patterns of TIP2;2 and TIP2;3 are mirrored tissues by transcriptomic analysis [15]. This excludes in the lateral roots (Fig 1, panels P and Q). In contrast to TIP3;1 and TIP3;2 (α and β-TIP), which have seed-specific the previous isoforms, TIP4;1-YFP is only expressed in epi- expression patterns [14,16]; Gattolin and Frigerio, unpub- dermal and (less strongly) in cortical cells of the differen- lished), and both TIP1;3 (γ -TIP3) and TIP5;1 (ζ-TIP), tiation zone (Fig. 1, panels F and L), with fluorescence which are predicted by bioinformatic analysis to be decreasing in more mature parts of the root where lateral expressed solely in floral organs and pollen [15-17]. roots emerge (Fig. 1R). Our results indicate that expression of some TIP isoforms under their native promoters is remarkably tissue and cell- specific. In general, when multiple isoforms are co- expressed in the same cell, they appear to localise mainly to the tonoplast of the central vacuole. Our identification of the sites of expression of every TIP isoform paves the way to understanding TIP specialisation and function in Arabidopsis root tissues. Results In addition to the fluorescent TIP reporters we generated previously for TIP1;1 (γ -TIP1; At2g36830) and TIP2;1 (δ- TIP1; At3g16240) [14], we cloned the genomic sequences of not previously studied isoforms: TIP1:2 (γ -TIP2; At3g26520), TIP2;2, TIP2;3 (δ-TIP2 and δ-TIP3; Expression p Figure 1 atterns of TIP isoforms in Arabidopsis roots At4g17340 and At5g47450) and TIP4;1 (ε -TIP; Expression patterns of TIP isoforms in Arabidopsis At2g25810). We produced chimeric constructs in which roots. 8-day old roots from the indicated transgenic lines either YFP or monomeric RFP were fused in frame to the were excised, stained with propidium iodide for 2 min and C-terminus of each TIP genomic sequence (including their visualised by CLSM. The images show representative results promoter regions, 5' UTR and introns), and generated for each construct. The signals from YFP (green) and propid- transgenic plants which were analysed for TIP-XFP expres- ium iodide fluorescence (red) are merged. Top panels: single sion patterns by confocal laser scanning microscopy optical sections of the root tips, Middle panels: single optical (CLSM). We first observed TIP-YFP expression at low mag- sections of root differentiation zones. Bottom panels: maxi- nification. 8-day old roots from seedlings expressing indi- mal projection of 16 optical z sections (4 μm step-size) vidual YFP-tagged TIPs were stained with propidium through mature root axes and young lateral roots. Scale bars: 100 μm. iodide and analysed by CLSM. With the possible excep- Page 2 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 In the case of TIP1;2, expression seems to be exclusively limited to the root cap and the columella (Fig. 1B). A very limited YFP signal can also be detected in the same region of the young lateral root (Fig 1N, arrowhead) and in older lateral roots (Additional file 1A). Perhaps the most remarkable expression pattern observed is that of TIP2;1, which in 8-day old roots is only detecta- ble in a small region at the base of the lateral roots (Fig. 1O, arrowhead). Having identified the general patterns of expression of the different isoforms at low magnification, we then studied the cell-specificity of TIP-YFP expression in more detail. We analysed propidium iodide -stained roots by CLSM by performing optical z-sections through differentiation zones at 63× magnification (Fig. 2). TIP1;1-YFP is clearly expressed in epidermis and cortex, but its expression is particularly strong in the endodermis and pericycle (Fig. 2A). Here TIP1;1-YFP highlights numerous bright circular structures in the lumen of the central vacuole. We hypothesise these are vacuolar 'bulbs', which have previously been described as tonoplast invagi- nations, which occur independently of the ectopic expres- sion of XFP-tagged membrane proteins, and where TIP1;1-GFP is concentrated [18,19]. It is however difficult in some cases to observe a continuity between these struc- tures and the central vacuole tonoplast. At higher magnification, the overlapping patterns of expression of TIP2;2-YFP and TIP2;3-YFP are confirmed. Both are present in pericycle cells, particularly in the rows of pericycle cells that form the xylem poles [20]. Both TIP- YFPs tend to be absent from the endodermis (Fig. 2, pan- els B and D), although we could detect discontinuous Ce Figure 2 ll-type specificity of TIP-YFP expression in the root axis endodermal expression at various positions along most Cell-type specificity of TIP-YFP expression in the root axis. 8-day old roots from the indicated transgenic root axes (Fig. 2, panels C and E; and highlighted in blue lines were excised, stained with propidium iodide for 2 min in panels G and H). and visualised by CLSM. Stacks of 80 optical z sections (1 μm step-size) were collected from root axes at the differentia- In contrast to the previous isoforms labelling inner root tion zone. The images show representative results for each cell layers, TIP4;1 expression is clearly restricted to the construct. A to F: for each panel, the top section shows a sin- root epidermis and cortex, with no signal detectable in the gle xy optical section, and the bottom section shows the xz inner layers (Fig. 2F). projection of the whole image stack, revealing the cross sec- tion of the root axis. The signals from YFP fluorescence Localisation of TIP1;1 and TIP1;2 (green) and propidium iodide fluorescence (red) are merged. Having analysed the TIPs with the broadest expression G and H: the YFP fluorescence trace from representative patterns, we focussed on the two TIPs which seem to have image stacks for the indicated transgenic lines was recon- structed, segmented and rendered in 3D with Mimics 12.1. a more limited expression range in roots. TIP1;2-YFP pre- The different tissues are colour-coded as follows: brown, sented a patchy distribution in cells of the root cap (Fig epidermis; red, cortex; blue, endodermis; green, pericycle. 1B). To ascertain that this was not an artefact due to Ep, epidermis; c, cortex; e, endodermis; p, pericycle; x, expression of our chimeric gene, we also generated a con- xylem. Scale bar: 20 μm. struct (YFP-TIP1;2) where YFP was fused downstream of the promoter and 5'UTR and in frame with the 5' of the Page 3 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 TIP coding sequence. In transgenic plants, YFP-TIP1;2 the labelled cells are in the process of detaching from the presents a similar expression pattern to TIP1;2-YFP, thus root (Fig. 3, panels C and F, arrowheads), suggesting that ruling out YFP fusion artefacts (compare Fig. 3A with Fig. they may correspond to 'border-like' cells [22]. The distri- 1B). Expression is confined to the columella and the lat- bution of YFP-TIP1;2 is therefore radically different to that eral root cap [21], with the labelled cells disappearing at observed for its paralogue, TIP1;1-YFP, which has the wid- the boundary with the elongation zone (Fig. 3A). Some of est pattern of expression but is excluded from the root tip, including the root cap (Fig. 1, panels A to M). At the subcellular level, YFP-TIP1;2 localises to the endo- plasmic reticulum (ER) of young root cap cells (Fig 3e: note the characteristic reticular pattern and the nuclear envelope; see also Additional file 1B). The chimeric pro- tein is mostly found on the tonoplast of elongated lateral root cap cells (Fig. 3D). This is likely to reflect different stages of TIP1;2 trafficking in cells of different ages, rather than impaired capacity to reach the tonoplast. This is fur- ther demonstrated by the fact that in the epidermis of cot- yledonary cells, where TIP1;2 is uniformly expressed, the fusion protein appears to localise to the tonoplast (Addi- tional file 1C-D). TIP2;1 is localised in lateral root primordia We have previously shown that TIP2;1 expression becomes detectable in old root regions nearing the hypocotyl, and is then widespread in hypocotyl and coty- ledonary leaves [14]. We did not initially notice expres- sion in young roots, but closer analysis revealed that in 8- day old roots TIP2;1-YFP has a very specialised expression pattern (Fig. 4). The YFP signal is detected in a ring-like cluster at the base of emerging lateral roots (Fig. 4, panels A-D). In very early lateral root primordia (LRP), TIP2;1 expression is detectable in 2-4 cells at the LRP. As the LRP grows further, the number of cells expressing TIP2;1-YFP increases but remains confined to a cluster underlying the YFP Figure 3 -TIP1;2 is expressed in the root cap base the lateral root (Fig. 4, panels F-I). In rare cases, when YFP-TIP1;2 is expressed in the root cap. 8-day old the lateral root is fully emerged, the expression of TIP2;1- roots from the indicated transgenic lines were excised, YFP can extend to some cells within the lateral root axis stained with propidium iodide for 2 min and visualised by (Fig 4I). Co-labelling with propidium iodide shows that CLSM. Stacks of 80 optical z sections (1 μm step-size) were the TIP2;1 expressing cells are located in close proximity collected from root tips. The images show a representative to the xylem (fig. 4E), suggesting a pericycle localisation. result for this construct. The signals from YFP fluorescence Co-expression with TIP2;3-RFP, which we found to be (green) and propidium iodide fluorescence (red) are merged. enriched in the pericycle (Fig. 2, panels D, E, H) confirms A: maximal 3D projection of the root tip at the base of the that TIP2;1-YFP expression originates from pericycle cells elongation zone. The image shows two adjacent z-stacks of (Fig. 4, panels F-I). This indicates that the initial expres- the same root, separated by a black line. B and C: xz projec- sion of TIP2;1-YFP is likely to occur in the LRP founder tions of the image stack in panel a, revealing two cross-sec- tions of the root axis, taken in the regions of the root cells. Remarkably, the expression of TIP2;1-YFP and indicated by the arrowheads in A. D and E: the regions indi- TIP2;3-RFP appears to be mutually exclusive, with a clear cated by dotted boxes in A were observed at high magnifica- boundary between cells expressing one or the other iso- tion. Single optical sections are shown. Note YFP-TIP1;2 in form (Fig. 4H, inset; see Additional file 2 for individual the ER of young root cap cells and in the tonoplast of root channels). cap cells closer to the elongation zone. F: The fluorescent traces from YFP (green) and propidium iodide (red) from the Overlapping TIP isoforms are mostly detectable at the image stack in panels A were reconstructed, segmented and central vacuole tonoplast rendered in 3D with Mimics 12.1. Scale bars: (a), 20 μm; (d) We have shown that the various TIP isoforms under study and (e), 10 μm. present diverse tissue specificity within roots. Several iso- Page 4 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 with different spectral variants of fluorescent proteins and co-expressed in transgenic Arabidopsis (Fig. 5). The individual TIP expression patterns in double trans- genic lines mirrored those observed in the lines expressing individual isoforms (Additional file 3). The widespread !" !" !" TIP 2;1-YFP expression in Figure 4 lateral root primordia TIP 2;1-YFP expression in lateral root primordia. A-E: 8-day old roots from TIP2;1-YFP transgenic seedlings were excised, stained with propidium iodide and visualised by !" CLSM. Stacks of 80 optical z sections (1 μm step-size) were collected from mature root axes. The images show repre- sentative results for this construct. Maximal projections of the z-stacks are shown, with the individual signals for YFP (A), propidium iodide (B) or the merged signals (C and D). E: The fluorescent traces from YFP (green) and propidium iodide (red) from the image stack in panels (A-C) was recon- structed, segmented and rendered in 3D with Mimics 12.1. Note that the TIP2;1-YFP-expressing cells are in close prox- imity to the xylem (labelled with x). F-I: Roots from 8-day old transgenic seedlings expressing TIP2;1-YFP (green) and TIP2;3-RFP (red) were imaged. Sequential stages of lateral Overlapping TIP plast of Figure 5 the central vacuole isoforms are mainly detected at the tono- root development are shown. Inset in H: note the boundary Overlapping TIP isoforms are mainly detected at the between pericycle cells expressing TIP2;3-RFP (top) and tonoplast of the central vacuole. Transgenic seedlings TIP2;1-YFP (bottom). Scale bars: 20 μm. co-expressing the indicated TIP-YFP and TIP-RFP constructs were grown for 8 days on MS medium--agar plates. Roots were excised and visualised by CLSM. (A, D, G, J): YFP fluo- forms, however, are co-expressed in certain tissues, rescence (green); (B, E, H, K): RFP fluorescence (red); (C, F, namely TIP1;1, TIP2;2, TIP2;3, and TIP4;1. In order to I, L): merged images. Arrowheads in panel I indicate struc- ascertain whether these isoforms were specific to distinct tures labelled by TIP2;3-RFP but not TIP2;2-YFP. Scale bars:10 μm. vacuolar compartments, we focused on the subcellular localisation of selected pairs of the above isoforms, tagged Page 5 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 TIP1;1-YFP and TIP2;3-RFP are co-expressed in epidermis, is intriguing. Transcripts of the maize aquaporin ZmTIP1 cortex and pericycle cells (Fig. 2). In these tissues, both were also localised in the lateral root by in situ hybridisa- proteins are detected on the tonoplast of the central vacu- tion, but every cell in the LRP seemed to contain the tran- ole (Fig. 5A-C). Both the tonoplast and the smaller, bulb- script [25]. TIP2;1 can therefore be considered an like vacuolar structures [18] are labelled. Likewise, TIP2;2- additional marker for the Arabidopsis LRP margins, YFP and TIP1;1-RFP mostly label the same tonoplast in alongside the auxin efflux carriers Pin4 and Pin6 [26] and the cell layers where they are co-expressed (Fig. 5D-F). the transcription factor CUC3 [27], which have a similar TIP2;2-YFP and TIP2;3-RFP, which almost overlap in root localisation. It will be interesting to study the specific role tissues (Fig. 2), are also co-localised on tonoplast and of TIP2;1 in these cells and determine why this stage of lat- 'bulbs' (Fig. 5G-I). Occasionally, TIP2;3-RFP highlighted eral root development demands such a precise TIP iso- smaller vesicular structures that did not appear to contain form activation. TIP2;2-YFP (Fig 5I, arrowheads). The nature of these struc- tures was not investigated further. Finally, TIP4;1-YFP, As a general observation, we could not detect expression which is restricted to epidermis and cortex (Fig. 2F), co- of any of the TIP-XFP fusions under study in the root tip localises with TIP2;3 in those tissues (Fig. 5J-L). Note that meristem. This lack of expression was previously reported the relative abundance of these two isoforms mirrors the for TIP1;1 both by histochemical detection of GUS pattern observed in single isoform localisation, with fusions [5] and YFP tagging [14,19]. It is of course possi- TIP4;1 expression being strongest in the epidermis and ble that expression levels of our fusions are too low in this weaker in the cortex (Fig. 2F), and TIP2;3 expression being region to be detected by confocal microscopy. However, stronger in cortex but weaker in epidermis (Fig. 2, panels the fact that the more sensitive histochemical GUS stain- D-E). ing also fails to detect expression of TIP1;1, which micro- array data indicate is the most abundantly transcribed Taken together, these co-expression results indicate that isoform in roots [16], strongly suggest that the protein is each TIP isoform-fluorescent protein fusion we analysed not expressed in the root meristem. This is in contrast with is predominantly found at the central vacuole tonoplast in data from other species such as pea and barley, where TIPs Arabidopsis root tissues. have been located in isolated root tip cells [10,12] and in root tip sections by immunohistochemical methods [28]. Analysis in the Olbrich et al. study was performed on 3- Discussion We have produced a complete expression map for all day old seedlings. At the same age in Arabidopsis seed- members of the TIP family that are present in Arabidopsis lings we could already detect all the TIP isoforms root tissues, including isoforms not previously studied. described in this study, with the exception of TIP2;1. The use of XFP fusions to TIP genomic sequences allowed However their expression pattern was already the same as us to investigate both the tissue specificity and the subcel- observed at 8 days (data not shown). We therefore lular localisation of these proteins. resolved to present results at 8 days, when the complete set of root TIPs is detectable. In general, our fluorescent reporter - TIP localisation data correlate well with the relative TIP transcript levels, as This lack of observable expression in root tips makes it dif- observed by microarray analysis [15,23,24], with the ficult to perform meaningful comparisons between the exception of TIP1;2. TIP1;2 is indeed the isoform with the vacuolar complement of Arabidopsis root tip cells and highest level of mRNA expression in the root cap [23], that of other plant species. which matches our observations (Fig. 3). However, tran- script levels for TIP1;2 have also been shown to be almost TIP-YFP expression was also not detected in the root vas- as high as TIP1;1 throughout the root axis [15,23,24]. We culature, regardless of the developmental stage. This mir- can only speculate at this stage that post-transcriptional rors observations in barley and pea root sections, where control processes prevent TIP1;2-YFP protein from being the stele was not labelled by TIP antisera [28]. While it is detectable in these tissues. easy to rationalise the absence of a vacuole in the xylem cells, which underwent autolysis, and in mature sieve ele- TIP1;1 is the most widely expressed isoform along the ments, which lack true vacuoles (reviewed in [29]) it was root axis. TIP2;2 and 2;3 have very similar expression pat- somewhat surprising not to find TIPs in the companion terns, with their expression being low in the endodermis, and parenchima cells. We think it unlikely that this lack of but high in the xylem pole pericycle. It appears that TIP2;1 detection is caused by a loss of sensitivity by the confocal becomes strongly expressed in the pericycle when this microscope detectors in the inner layers of the roots, undergoes differentiation to form the lateral root primor- because both propidium iodide staining and YFP signal dium (Fig. 4). This narrow range of localisation of TIP2;1 are easily detected in the xylem and xylem pole pericycle, Page 6 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 respectively (Fig. 2 and Additional file 3). In addition, we the promoter (if longer than 1.5 Kb), plus 5' UTR and could easily detect 35S::TIP2;1-YFP in the vascular tissue introns, was amplified from total genomic DNA from Ara- using the same settings (Additional file 4). Accordingly, bidopsis thaliana Columbia ecotype. Primers included Boursiac et al [24] recently showed that constitutively restriction sites KpnI at the 5' and XhoI at the 3' of the tar- expressed TIP1;1-GFP and TIP1;2-GFP clearly label the get sequences. Amplified fragments were cloned into the vascular tissue [24]. KpnI and XhoI sites of pGREEN0029, upstream of a XhoI/ SacI fragment containing the YFP coding sequence and Recently it has been shown that Arabidopsis knockout the OCS 3' terminator fragment. A similar strategy was mutants lacking TIP1;1, TIP1;2, or both isoforms, do not adopted to fuse TIP sequences to RFP, but in this case the have any major defects [7,19]. This is in contrast with forward primers included both KpnI and SacI restriction drastic defects observed in Arabidopsis upon downregula- sites, generating a TIP-RFP cassette that could be mobi- tion of TIP1;1 by RNAi [30]. A possible explanation for lized with SacI. To obtain pairwise TIP-YFP/TIP-RFP com- the latter result is off-target silencing in the RNAi lines [7]. binations, selected TIP-RFP cassettes were excised with Our data provide a rationale for the lack of a macroscopic SacI and ligated into TIP-YFP vectors linearised with SacI, phenotype in the double TIP1 knockouts observed by giving rise to constructs harbouring both reporter genes in Schussler et al. We have shown that, in roots, expression a tandem. All the chimeric constructs were introduced of TIP1;1 and TIP1;2 does not appear to overlap, with into strain C58 of Agrobacterium tumefaciens harbouring TIP1;1 being expressed in epidermis, cortex, endodermis the pSoup vector [31]. Arabidopsis plants were then trans- and perycycle starting from the elongation zone, and formed using the floral dip method [32]. TIP1;2 being restricted to the root cap. As TIP1;1 and 1;2 show different tissue specificities, it seems unlikely that Confocal analysis and image processing they are reciprocally redundant. In addition, we have About 30 seeds from at least 4 independent transgenic shown that other TIP isoforms, namely TIP2;2, TIP2;3 and lines per construct were germinated onto agar plates con- TIP4;1 would still be present in the tissues lacking TIP1;1 taining half-strength Murashige and Skoog (MS) Basal (Fig. 2). It is therefore possible that these remaining iso- Medium (Sigma-Aldrich) and grown for 8 days at 22°C, forms compensate for the lack of TIP1;1 in the knockout. in a 16:8 light:dark regime. Roots were excised, mounted On the other hand, the effect of the absence of TIP1;2 in half-strength liquid MS medium and immediately from the root cap may be subtle and may have gone unde- observed with a Leica TSC SP5 confocal laser scanning tected under the experimental conditions adopted for the microscope, using either a 10× (NA 0.3) air or a 63× (NA whole-plant analysis of the double mutants. A lack of phe- 1.4) oil immersion objective. In some cases roots were notype in the aerial parts of the single knockout plants preincubated for 2 min in 10 μg/ml propidium iodide, may be explained by the fact that both TIP1;1 and TIP1;2 diluted in half-strength MS medium. YFP was excited at are expressed in leaves [14] and Additional file 2) and 514 nm and detected in the 525 to 550 nm range. RFP was may well be acting redundantly there. As for the double excited at 561 nm and detected in the 553 to 638 nm knockout, redundancy may be afforded by TIP2;1 [14] range. Propidium iodide was excited at 561 nm and and TIP2;2 [16], which are also expressed in leaves. detected in the 650 to 720 nm range. Simultaneous detec- tion of YFP and RFP or YFP and propidium iodide was Conclusion performed by combining the settings indicated above in We have identified novel patterns of expression of TIP iso- the sequential scanning facility of the microscope, as forms in Arabidopsis roots. This information may provide instructed by the manufacturer. a useful starting point for a more targeted approach to dis- sect the function of individual TIP isoforms in root tissues. 3D reconstruction of z-stacks of optical sections was per- It also provides the foundation for further analysis of the formed with the Leica LAS-AF Lite free software (Leica intracellular targeting of different TIPs. Microsystems, Germany). Segmentation analysis and 3D rendering were performed with Mimics 12.1 (Materialise Methods N.V., Leuwen, Belgium). Recombinant DNA and generation of transgenic plants The constructs encoding native TIP1;1-YFP and native Abbreviations TIP2;1YFP have been described previously [14]. CLSM: confocal laser scanning microscopy; ER: endoplas- mic reticulum; PI: propidium iodide; TIP: tonoplast A full list of primers designed to amplify the genomic intrinsic protein. sequences of the root-expressed TIPs is shown in Addi- tional file 5. Each TIP genomic sequence, including either Authors' contributions the complete promoter region (up to the UTR of the gene SG generated the majority of the constructs and transgenic immediately upstream in the chromosome) or 1.5 Kb of plants and performed the bulk of the confocal analysis. Page 7 of 9 (page number not for citation purposes) BMC Plant Biology 2009, 9:133 http://www.biomedcentral.com/1471-2229/9/133 MS produced the TIP1;2-RFP and 35S:TIP2;1 constructs Additional file 5 and transgenic lines and performed confocal analysis. PH Primers used in this study. The diagram indicates the target sequences produced the native TIP1;1-YFP and TIP2;1 YFP constructs for the indicated primers in the final constructs. Restriction sites are and transgenic plants and performed confocal analysis. shown in bold. RK performed the 3D image analysis in MIMICS. LF Click here for file designed the experimental programme, gave technical [http://www.biomedcentral.com/content/supplementary/1471- and intellectual guidance and wrote the manuscript. All 2229-9-133-S5.PDF] authors read and approved the final manuscript. Additional material Acknowledgements We are grateful to Robert Spooner and Alessandro Vitale for critical read- Additional file 1 ing of the manuscript. This work was orted in part by the European Union Expression and subcellular localisation of TIP1;2. 8-day old seedlings (LSH-2002-1.2.5-2 "Recombinant Pharmaceuticals from Plant for Human expressing YFP-TIP1;2 were visualised by CLSM. A: 10× magnification Health -Pharma-Planta") and by the Leverhulme Trust (grant F/00215/AP). of a lateral root. The signals from YFP fluorescence (green) and propidium A grant from the Fondation 'Les Gueules Cassées' funded the acquisition of iodide fluorescence (red) are merged. B: single root cap cell with TIP1;2- the Mimics software. YFP showing typical ER labelling. C-D: epidermal cells in cotyledons where TIP1;2-YFP shows typical tonoplast labelling (green). Red: chloro- References phyll autofluorescence (excitation 514 nm, detection 600-650 nm). Scale 1. Kaldenhoff R, Fischer M: Functional aquaporin diversity in bars: A, 100 μm; B and D, 5 μm; C, 20 μm. plants. Biochim Biophys Acta 2006, 1758:1134-1141. Click here for file 2. Maurel C: Plant aquaporins: Novel functions and regulation [http://www.biomedcentral.com/content/supplementary/1471- properties. FEBS Letters 2007, 581:2227-2236. 3. 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