Incipient stem cell niche conversion
To study the cytokinin-induced conversion of LRP/LRMs to shoots we first generated synchronous LRP initiation by exposing 3- or 4-day-old seedlings to 10 μm 1-naphthaleneacetic acid (1-NAA), a synthetic auxin used in transcriptomic studies of LRP (Himanen et al., 2004). Seedlings have few LRP prior to NAA treatment, which initiates LRP at every available position along the primary root xylem-pole pericycle. Transfer to cytokinin-enriched media induces the synchronous conversion of induced LRP into SMs, and emerging shoots become visible within 5 days (Figure 1a–c). This simple methodology appears robust, and we have used it to rapidly induce shoots from roots of other brassicas and poplar (Figure 1d).
Figure 1. Shoot induction via conversion of lateral root promordia (LRP) to shoot meristems (SMs). (a, b) High-throughput method: Nitex sheets were used to transfer tens to hundreds of seedlings between treatments. Seedlings were transferred from germination media 3–4 days after imbition to media supplemented with 10 μm 1-naphthaleneacetic acid (1-NAA) for 24 h for rapid LRP induction, and then to 4.4 μm 2iP (a cytokinin) promoting LRP → SM conversion (b). After 6 days of treatment with 2iP, dense shoots were visible. (c, d) Close-ups of Arabidopsis shoot induction (5 days 2iP; c) and poplar (14 days 2iP; d). Scale bars: 2 cm (a, b); 2 mm (c, d).
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A high-throughput methodology, adapted from Birnbaum et al. (2005), permitted the transfer of hundreds of plants between hormone treatments and rapid sampling (Figure 1a,b). In agreement with previous studies, 24 h after auxin treatment the LRP induced by the treatment reached between three and five cell layers, a stage that precedes commitment to self-sustaining LRMs (Sussex et al., 1995).
WUSCHEL reporters are expressed in cLRP within 30 h
In our experiments we used well-characterized transcriptional reporters for WUS and CLV3, with expression patterns reflecting mRNA in situ hybridization experiments (Reddy and Meyerowitz, 2005; Gordon et al., 2007; Yadav et al., 2009). Within the treated roots, WUS reporters were first visible 19 h after transfer from NAA to isopentenyladenine (2iP), and were weakly and sporadically expressed outside the LRP (Figure 2a). WUS reporter expression was first seen within cLRP 24–36 h after transfer to 2iP (Figure 2b), but rarely in the outermost layer of cells (2C–E). Expression of this transcriptional reporter for the organizing center of the SAM within former LRP shows that an important change in cell identity, towards that associated with SMs, is already underway just a day after exposure to cytokinin.
Figure 2. Confocal images of WUS and CLV3 transcriptional reporter expression during conversion of lateral root primordia (LRP) to shoot meristems (SMs). (a) After 19–24 h of 2iP treatment, sporadic and weak pWUS::DsRED-NLS (red nuclei) marks cells peripheral to LRP and scattered cells within the vascular cylinder. (b) After 30 h of 2iP treatment, pWUS::DsRED-NLS is upregulated within cLRP. (c, d) After 48 h of 2iP, pCLV3::GFP-ER (green cells) is seen in small isodiametric cells near the apices of 10–25% of cLRP. The pCLV3::GFP-ER domain was found above populations of small cells expressing pWUS::DsRED-NLS. (e) pWUS::GFP–ER (green) expression after 30 h of 2iP treatment is also upregulated within the cLRP. Scale bars: 65 μm.
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A pCLV3::GFP-ER reporter appeared 32–48 h after transfer to 2iP marking between two and four cell layers at the apex of cLRP, above and overlapping with populations of small cells expressing pWUS::DsRED-N7 (Figure 2c,d), and was never seen in the absence of the WUS reporter, consistent with previous studies that have found CLV3 expression to be dependent upon the WUS gene product (Laux et al., 1996; Brand et al., 2002). Thus, transcriptional reporter expression of these key regulators of the SAM stem cell population assumed a spatial relationship within cLRP, reflecting that in SAMs. Expression of these two components of a regulatory network responsible for maintaining a shoot meristematic stem cell population constitutes another significant step in the establishment of a new SM.
Transcriptome analysis of LRP–SM conversion reveals changes related to hormone signaling, meristem identity, cell cycle and photosynthesis
By enriching samples with synchronously developing LRP, we hoped to focus on gene expression pertaining to LRP–SM conversion. For transcriptome analysis, key time points in LRP → SM conversion were selected based on the aforementioned reporter analysis. Tissues were sampled after 24 h of exposure to NAA (0 h 2iP), and subsequently after 19, 30 and 48 h of 2iP treatment. The first time point corresponds to saturated LRP development within the primary roots, and 19 h of 2iP treatment corresponds with the initial expression of pWUS::DsRED-NLS. After 30 h of treatment with 2iP there is consistent expression of this marker within cLRP prior to the detection of pCLV3::mGFP-ER. Treatment with 2iP for 19 h precedes the detectable expression of SAM markers within cLRP, perhaps corresponding to an intermediate state between LRP and initiating SMs. We reasoned that a 30 h time point would reveal gene expression events associated with early SM initiation. After 48 h of treatment with 2iP the CLV3 marker is expressed within 10–25% of cLRP, reflecting gene expression representing the initial establishment of organized SMs.
Exogenous auxin and cytokinin were expected to generate marked changes in gene expression. Because expression of key developmental regulators could be relatively small when ranked against this background, lenient criteria were used for the initial selection of differentially expressed genes (DEGs). Using MAS5 processed data, each time point was compared pairwise with every other time point, applying a fold-change threshold of 1.75, a P-value cut-off of <0.05 and the rejection of mean expression values <50 units.
Figure 3 provides an overview of DEGs identified in each comparison (also Table S1). As expected, large numbers of DEGs (2399–3472) were identified in comparisons between 0 h and 2iP time points. There was also a marked overlap in the DEGs from each of these comparisons, with 1700 DEGs identified in all three (Figure 3). For comparisons between cytokinin treatment periods, 340 and 642 DEGs were identified between 19 and 30 h, and between 19 and 48 h, respectively. Interestingly, although the 30 and 48 h time points of 2iP treatment separate important changes in reporter gene expression, no DEGs were found in this comparison.
Figure 3. Distribution of differentially expressed genes (DEGs) during conversion of lateral root promordia (LRP) to shoot meristems (SMs) in a pairwise comparison of three different durations of cytokinin treatment (4.4 μm 2iP). ‘0 h’ corresponds to 24 h of LRP-induction using 10 μm 1-naphthaleneacetic acid (1-NAA). Overlap in DEGs between time points is high, suggesting substantial common transcriptional responses to auxin → cytokinin transfer.
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The expression of positive cell-cycle regulators and RM-associated genes is decreased by cytokinin, whereas the expression of photosynthetic and SAM-associated genes is increased
Consistent with the presumed changes in identity occurring as 2iP promotes LRP → SM conversion, reduced expression of many RM/LRP-associated genes and increased expression of SM-associated genes were observed (Table S2). However, several key SAM-associated genes, including CLV3, did not pass the expression value cut-off. Misexpression of CLV3 has been shown to precipitate consumption of the RAM (Fiers et al., 2005), but CLV3 reporter expression, and low expression values of CLV3, suggest it does not play a key role in the initial loss of LRM identity.
Unsurprisingly, transferring seedlings from high-auxin to high-cytokinin media is reflected in the increased expression of many cytokinin-responsive signaling genes, and in the reduced expression of auxin-induced signaling and metabolic genes (Tables S3 and S4, and over-represented gene ontology, GO, categories in Table S1), suggestive of an involvement in maintaining hormone signaling or metabolic homeostasis.
Cytokinins and certain cytokinin-signaling components promote differentiation of chloroplasts and expression of photosynthetic genes (Schmulling et al., 1997; Argyros et al., 2008), and all photosynthesis-related plant-encoded DEGs were found to be increased in steady-state transcript levels by exogenous 2iP in our study (over-represented GO categories in Table S1).
The reduced expression of type-A and -B cyclins and cyclin-dependent kinases, and the increased expression of three cyclin-dependent kinase inhibitors (Table S5), suggests a reduction in cell division/numbers of dividing cells on 2iP. Reduced expression of other positive regulators of cell division, such as EF2a, and decreased expression of many histones (e.g. S-phase marker HIS4; Table S5) support this interpretation. These observations are consistent with studies showing that cytokinins inhibit cell division in RMs and LRP founder cells (Werner et al., 2003; Li et al., 2006; Dello Ioio et al., 2007).
Gene ontology (GO) enrichment analysis revealed transcription factors were over-represented amongst DEGs in most pairwise comparisons (Table S1). This category contained 61 differentially expressed homeobox genes, some of which have known roles in meristem and organ initiation. For example, WOX13 is dynamically expressed during RAM/LRP initiation, and showed decreased expression on 2iP. Conversely, ARABIDOPSIS THALIANA HOMEOBOX 1 (ATH1) and PENNYWISE (PNY) interact with SHOOT MERISTEMLESS 1 (STM1) in the SAM, and their expression was increased.
Comparison with callus-based regeneration reveals an overlap with DEGs identified in LRP→SM conversion
To identify potential key regulators of shoot regeneration common to different in vitro systems, we compared targets identified in studies of shoot organogenesis from callus with LRP → SM conversion. Che et al. (2006) analysed transcriptome changes during root or shoot organogenesis from callus, and described the ‘top-20’ DEGs with increased/decreased expression during callus induction, or subsequent shoot or root induction. Of the top-20 DEGs identified during presumed commitment to shoot organogenesis, 11 and 12 of the genes with increased and decreased expression, respectively, also appeared amongst the DEGs identified in our study, with similar patterns of expression (Table S6).
We then surveyed genes previously identified as affecting shoot organogenesis for differential expression during LRP → SM conversion (Table S7). One of these, HLS1/COP3, is a negative regulator of callus-based shoot regeneration (Chatfield and Raizada, 2008), and exhibits >14-fold decrease in expression levels on 2iP in our study (Table S7). Interestingly, RSM1 overexpression phenocopies loss of HLS1 function (Hamaguchi et al., 2008), and RSM1 expression was increased more than eightfold on 2iP.
Transcriptome analysis of wus mutants identifies WUS-responsive DEGs
To further examine the role of WUS in LRP → SM conversion, we compared transcriptomes of loss-of-function wus mutants with the wild type (WT). We first compared a reported wus null allele, SAIL_150_G06 (McElver et al., 2001; Sonoda et al., 2007), with WT using the aforementioned 2iP treatment periods. Three biological replicates were recorded with Affymetrix ATH1 microarrays, and mas5 processed data analysed using the limma (linear models for microarrays) package (Smyth, 2005). A significance cut-off of P < 0.05, a minimum fold change >1.5 and a minimum expression value of 50 yielded 543 DEGs in total.
Figure 5 shows the distribution and overlap of the DEGs identified, and a summary of over-represented GO categories (details Tables S8 and S9). Using our lenient selection criteria, between 63 and 121 genes were found to have increased or decreased steady-state transcript levels in the mutant at each time point. So, although the developmental consequences of wus loss of function are dramatic, the perturbation of gene expression was small compared with that associated with the hormone treatments used to induce LRP and conversion. Additionally, unlike our WT time course, very little overlap was found between time points in terms of DEGs identified, suggesting that examining downstream consequences of wus loss of function successfully focused on transcriptome changes relevant to discrete stages in WUS-dependent development.
Figure 5. Distribution of differentially expressed genes (DEGs) in a wus loss-of-function mutant during cytokinin-induced conversion of lateral root promordia (LRP) to shoot meristems (SMs) and select gene ontology (GO) terms enriched at each time point. Compared with the transcriptome changes associated with the treatments driving LRP → SM conversion (Figure 3), those associated with loss of WUS function during this process were small, and the overlap in DEGs between time points is relatively low. This suggests we have identified discrete patterns of WUS-dependent gene expression associated with each developmental stage.
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After 30–48 h of 2iP treatment, the WUS reporter was consistently expressed in WT cLRP and the most relevant transcriptome differences were anticipated at these times. Amongst the DEGs identified in these comparisons, the most over-represented GO categories included the biological processes of post-embryonic development, apotosis, post-translational protein modification, glucoside biosynthesis, phenylpropanoid biosynthesis and responses to light. This output resembles the groups of GO categories described by Busch et al. (2010) in a genomic study identifying WUS-responsive genes: namely, the regulation of development (including meristem and cell death), metabolism (including glucosinolate) and response to stimuli. Furthermore, the distribution of WUS-repressed and -induced genes described by Busch et al. (2010) amongst the DEGs we identified (Figure 6) suggests that our approach has been successful in identifying a subset of WUS-responsive DEGs during LRP → SM conversion. After 19 h of 2iP treatment, WUS reporter expression is weak and sporadic in tissues around cLRP, and the ratio of WUS-repressed and -induced genes amongst DEGs suggests WUS loss of function has not yet directly affected the transcription of targets (Figure 6). Conversely, after 30 h of 2iP treatment the proportion of WUS-repressed genes amongst the DEGs with higher expression in wus increases to more than fivefold that of WUS-induced genes, suggesting that the loss of WUS has permitted elevated expression of these genes. Conversely, although the number of WUS-repressed genes has decreased amongst DEGs with reduced expression in wus at this time, they outnumber WUS-induced genes. However, after a further 18 h, WUS expression within WT cLRP indicates that >11% of the DEGs with reduced expression in wus belong to the WUS-induced group, and none to the WUS-repressed group (Figure 6). Furthermore, after 48 h of 2iP treatment the proportion of WUS-repressed genes amongst DEGs with increased expression in wus remained higher than WUS-induced genes. In addition, we surveyed promoter and intron sequences of DEGs for cis-element sequences bound by WUS (Lohmann et al., 2001; Busch et al., 2010), and found them to be over-represented amongst DEGs after 48 h of 2iP. Two or more instances of the 6-bp sequence CACGTG (Busch et al., 2010) were found within 500 bp upstream of 5.4% (1.7% expected, P = 0.003) of the DEGs with increased expression in wus, and two or more instances of the sequence TTAATSS (Lohmann et al., 2001) were found within the introns of 7.94% (3.45% expected) of DEGs with decreased expression in wus, although the latter observation was not deemed significant (P = 0.055).
Figure 6. Proportions of WUS-repressed and WUS-induced genes (Busch et al., 2010) amongst differentially expressed genes (DEGs) identified in transcriptome analysis of wus loss-of-function mutants during conversion of lateral root promordia (LRP) to shoot meristems (SMs) are consistent with rapid transcriptional responses to WUS expression in cLRP. (a) A comparison of a wus loss-of-function mutant (SAIL_150_G06) with the wild type (WT). WUS reporter expression was confined to scattered cells outside cLRP after 19 h of treatment with 2iP, and ratios of WUS-induced and WUS-repressed genes amongst the DEGs suggests WUS-responsive gene expression has not been perturbed. After 30 h of treatment with 2iP, WUS reporter expression was found within cLRP, and WUS-repressed genes now represent the majority of DEGs with increased expression in wus mutants, and made a reduced contribution to DEGs with lower expression in the mutant. After 48 h of treatment with 2iP, no WUS-repressed genes were found amongst DEGs with lower expression in wus. (b) In an additional comparison of two wus loss-of-function mutants with WT after 30 h of treatment with 2iP, WUS-repressed genes again outnumbered WUS-induced genes amongst DEGs with increased expression in wus, and WUS-induced genes outnumber WUS-repressed genes amongst DEGs with lower expression in wus.
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Thus, for a subset of genes the effect of WUS upregulation within cLRP seems broadly consistent with published findings on the regulation of gene expression by WUS. In addition, we have identified many targets not previously identified as WUS responsive that are differentially regulated in the loss-of-function mutant under the specific conditions associated with LRP → SM conversion (Table S8, and GO analysis in Table S9).
As might be expected, DEGs included those associated with developmental processes in SMs (Table S8), including CUP-SHAPED COTYLEDONS 1 and 3 (CUC1 and CUC3) and BLADE ON PETIOLE 2 (BOP2). However, many meristem-associated genes were expressed at low levels, and despite enrichment of samples with cLRP, SAM-associated genes positively regulated by WUS (e.g. CLV3; Brand et al., 2002; Yadav et al., 2011), or repressed by WUS (e.g. CLV1; Busch et al., 2010), were not identified as DEGs. It may be that the relevant cell types still represent an insubstantial fraction of samples, or that our sampling precedes the significant upregulation of many meristem-associated genes.
An important role of WUS in meristem function is believed to be the regulation of cytokinin-inducible ARRs. Leibfried et al. (2005) used inducible misexpression to isolate WUS-responsive genes, and identified four type-A ARR genes (ARR5, ARR6, ARR7 and ARR15) as WUS-repressed. In contrast, no ARR genes were amongst DEGs with higher expression in wus, and few cytokinin-related targets were identified as differentially expressed after 30–48 h of treatment with 2iP (Table S8), from which it is difficult to infer an outcome upon cytokinin-signaling output. This discrepancy may reflect different tissues sampled, and cytokinin treatments masking the impact of WUS on ARR expression in our study.
To identify associations and putative functional relationships between DEGs, cluster analysis was performed and each cluster analyzed for over-represented GO terms. The DEGs were grouped into 18 clusters by k-means clustering, seven of which yielded statistically over-represented GO terms (P < 0.05 Hochberg false discovery rate; Figure S1; Table S10). Although many GO categories overlapped with those identified in our time-point comparison (Table S9), several interesting new groups were highlighted. These included: lipase activity (cluster 5), nuclear protein import (cluster 6) and, endo-1,4-β-xylanase activity (cluster 7). Endo-1,4-β-xylanases are associated with cell expansion and shape changes, and inclusion of three (of five on the ATH1 microarray) within cluster 7 suggests WUS, or dependent processes, reduce these activities.
After 30 h of treatment with 2iP, we hoped to identify early events in WUS-dependent LRP → SM conversion. To provide greater resolution of WUS-related DEGs at this time point, we examined the transcriptome of another wus mutant allele (GABI_870H12). GABI-KAT constructs were designed for activation tagging, but in this line an intragenic insertion appears to drive elevated expression of a truncated non-functional product (Methods S1). Heterozygotes yield loss-of-function wus phenotypes in approximately 25% of progeny, and these homozygous mutants are unable to undergo LRP → SM conversion. For comparison of the two alleles with the WT, processed data were filtered to remove genes absent in one wus mutant allele, but not the other. Using the limma package of BioConductor, a P-value threshold of <0.05 and a minimum fold change of 1.5 in one genotype, 144 DEGs similarly regulated in both wus mutant alleles were identified (Table S11). Of these initial DEGs, 21% overlapped with those identified in our wus SAIL/WT comparison. Applying the fold change cut-off to both alleles increased overlap to 37%, comprising 25 and 48% of DEGs up- or downregulated in a wus mutant background, respectively. The observed differences in overlap between genes with increased or decreased levels of expression could reflect differences in the function of the mutant gene products, but as both mutant alleles seem functionally similar in terms of LRP → SM conversion, the subset of mutual DEGs appears to offer stronger candidates for genes mediating WUS-dependent LRP → SM development. Consistent with this view, the proportion of WUS-induced to WUS-repressed genes amongst DEGs with reduced expression in wus increased in this two-allele comparison (Figure 6).
Insertional knock-outs in WUS-responsive candidates affect LRP → SM conversion
To explore the roles of putative WUS-responsive targets identified by transcriptome analysis, we refined selection criteria to test insertional knock-outs of promising targets. To enrich for potential direct targets of WUS, we surveyed <1 kb upstream of the wus mutant DEGs for two or more instances of sequences corresponding to putative WUS-binding cis-elements: CACGTG and TTAATSS. As functional TTAATSS sequences were originally identified within an intron, we included DEGs with two or more instances within introns. These candidates were then surveyed with the Arabidopsis eFP Browser (Winter et al., 2007) for genes displaying differential expression in the SAM (Yadav et al., 2009) or embryo (Casson et al., 2005). Initially, 32 wus mutant DEGs were selected, of which 28 possessed corresponding T-DNA insert lines. Homozygous lines were assayed for LRP → SM conversion by scoring the numbers of shoots initiated on seedling roots treated with 4.4 or 2.2 μm 2iP for 5–7 days. The lower cytokinin concentration was included to screen for enhanced shoot-induction rates. A total of 39 homozygous insertion lines, corresponding to 27 genes, were tested in at least two replicates (Figure 7; Table S12). Four of these lines, corresponding to four different DEGs with reduced expression in wus during LRP → SM conversion, showed a consistent reduction in shoot initiation rates (Figure 7). All mutants appear phenotypically normal, apart from their deficit in shoot initiation. However, further work will be required to determine the relevance of the mutations to WUS-dependent processes and LRP → SM conversion.
Figure 7. T-DNA insertion lines corresponding to four candidate WUS-responsive targets identified from transcriptome analysis of wus loss-of-function alleles show reduced conversion of lateral root promordia (LRP) to shoot meristems (SMs) compared with the wild type (WT), as scored by shoot induction rates. (a) Homozygous T-DNA insertional mutants were also chosen based on the presence of potential WUS-binding cis-elements, and differential expression in SMs or embryonic domains. (b) Shoot induction from seedling primary roots after 24 h of treatment with 10 μm 1-naphthaleneacetic acid (1-NAA), followed by 6 days of 4.4 μm 2iP. Shoots were scored after 5–7 days of cytokinin treatment.
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Cell-specific profiling of the WUSCHEL domain of cLRP
Cell-specific expression profiling has been used to explore gene expression within specific domains of RMs (Birnbaum et al., 2003; Brady et al., 2007; Gifford et al., 2008) and SMs (Yadav et al., 2009), with improved sensitivity and resolution in relating transcriptome changes to development and responses to stimuli. Although our samples were enriched with cLRP, the relative contribution by key cell types/domains may be insufficient to resolve key genes, illustrated by low WUS expression values. We therefore isolated and profiled pWUS::mGFP5-ER cells to compare expression in the WUS domain of cLRP with the WUS domain of established SMs.
Tissues harvested after 30 h of treatment with 2iP were protoplasted rapidly (1 h) and cells expressing pWUS::mGFP5-ER were isolated with a fluorescence-activated cell sorter (FACS). Isolated RNA underwent two cycles of amplification and transcriptomes were recorded with Affymetrix ATH1 arrays. Previous studies, using RM and SM cells, have identified genes that respond to protoplasting with changes in expression (Birnbaum et al., 2003; Yadav et al., 2009), and these targets were removed from comparisons.
The mean expression value of WUS in pWUS::mGFP5-ER cells was about sevenfold higher than the whole root, suggesting successful enrichment for WUS-expressing cells, but was lower than that obtained for the WUSp domain of SAMs (Yadav et al., 2009). This latter observation was expected because the expression of transcriptional reporters for WUS only began in cLRP at this time.
Pearson correlation coefficients of 0.97–0.99 for comparisons indicate a high level of reproducibility between our pWUS::mGFP5-ER replicates (Table S13). Lower correlation coefficients (0.513–0.596) were found between pWUS::mGFP5-ER cells in our experiments and those isolated by Yadav et al. (2009). In addition to the likely differences in expression arising from harvesting WUS-expressing cells from different organs, low correlation values probably reflect differing culture conditions, particularly the high concentrations of hormones used in our experiments.
Next we identified genes differentially expressed within pWUS::mGFP5-ER cells from cLRP compared with whole root samples, and examined how these genes were distributed amongst those assigned to SAM domains (Yadav et al., 2009). Figure 8 shows that of the genes previously assigned to each SAM domain, the highest proportion of overlap with the DEGs from pWUS::mGFP5-ER cells is the WUSp domain of the SAM at 60.3%, compared with 35.7 and 45.5% for CLV3p and FILAMENTOUS FLOWER (FILp) domains, respectively. Moreover, a higher proportion of the overlapping genes in the WUS SAM domain are DEGs, with higher expression in pWUS::mGFP5-ER cells from cLRP: 42.0%, compared with 14.8 and 14.1% in CLV3 and FIL domains, respectively. This suggests that within hours of initiating WUS reporter expression in cLRP, the transcriptome of these cells began to resemble theWUSp domain of an SAM.
Figure 8. (a) Distribution of genes assigned to shoot meristem (SM) domains (Yadav et al., 2009) amongst differentially expressed genes (DEGs) from pWUS::mGFP5-ER sorted cells from converting lateral root primordia (cLRP; 30 h 2iP). The greatest overlap is with the WUSp domain of the SM (60.3%), compared with CLV3p (35.7%) and FILp (45.5%) domains. (b) Occurrence of potential WUS-binding cis-element-associated sequences, TTAATSS (Lohmann et al., 2001) and CACGTG (Busch et al., 2010), within promoters and introns of DEGs indentified in sorted pWUS::mGFP-ER cells from cLRP. Significant enrichment for CACGTG sequences was found within 1 kb upstream of DEGs.
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Examining the distribution of sequences associated with WUS cis-elements we found the 6-bp sequence CACGTG was significantly over-represented 500–1000 bp upstream of DEGs from pWUS::mGFP5-ER cells in cLRP (Figure 8). Two or more TAATTSS sequences within introns were also observed at a higher frequency than expected, but the cut-off of P < 0.05 was not met (P = 0.056). Enrichment of DEGs from this domain with elements mediating transcriptional responses to WUS is consistent with rapid transcriptional responses to WUS expression within cLRP.
We then examined the distribution WUS-responsive genes identified in our transcriptome analysis of wus mutants, and those identified by Busch et al. (2010), amongst DEGs from pWUS::mGFP5-ER cells in cLRP. Interestingly, genes found to have increased expression in pWUS::mGFP5-ER cells were enriched amongst those found to have increased expression in wus (Figure 9a). Conversely, those with decreased expression in pWUS::mGFP5-ER cells were enriched amongst those with decreased expression in wus (Figure 9a). Similarly, WUS-induced and WUS-repressed genes (Busch et al., 2010) were found to be enriched amongst those with decreased and increased expression, respectively, in pWUS::mGFP5-ER cells (Figure 9b). These findings mirror those of Busch et al. (2010), who found WUS-repressed genes were enriched within the WUS domain of the SAM, whereas WUS-induced genes were enriched among transcripts expressed in the combined CLV3 and WUS domains, and combined CLV3 and FIL domains. It was suggested that this finding reflected a contribution by both direct and indirect targets amongst WUS-responsive genes, and linked the prevalence of transcripts with reduced expression in the WUS domain to evidence that WUS acts primarily as a transcriptional repressor (Leibfried et al., 2005; Ikeda et al., 2009), modulating target gene expression. Our findings are in line with these hypotheses, and may also reflect the duration of WUS expression in cLRP and WUS non-cell autonomous activity (Mayer et al., 1998; Gallois et al., 2004; Yadav et al., 2011).
Figure 9. WUS-responsive genes amongst differentially expressed genes (DEGs) identified in pWUS::mGFP5-ER cells from converting lateral root primordia (cLRP). (a) Genes with decreased expression in pWUS::mGFP5-ER cells were enriched amongst those with decreased transcript levels in wus mutants. Conversely, genes found to have increased expression in pWUS::mGFP5-ER cells were enriched amongst those found to have increased transcript levels in the wus mutants. (b) Distribution of DEGs from pWUS::mGFP5-ER cells in cLRP amongst WUS-repressed and WUS-induced genes (Busch et al., 2010). WUS-repressed genes were enriched amongst DEGs with increased expression in pWUS::mGFP5-ER cells, whereas WUS-induced genes were enriched amongst DEGs with decreased expression in pWUS::mGFP5-ER cells. Fold-change categories are not cumulative.
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To characterize transcriptome differences between WUS domains within established SAMs and within cLRP we compared pWUS::mGFP5-ER cells in our experiment with WUSp SAM cells (Yadav et al., 2009). pWUS::mGFP5-ER-associated DEGs with more than twofold difference in expression comprised 1224 DEGs with elevated expression in cLRP, compared with WUSp SAM, and 1452 DEGs with lower expression (Table S14). GO analysis (Tables 1 and S15) revealed the enrichment of various ‘response to stimuli’ terms, including the responses to hormones (ABA, ethylene, cytokinin and auxin), which may reflect the exogenous hormones applied in our study. Differential over-representation of root versus shoot developmental terms (Table 1) amongst DEGs from cLRP is consistent with continuing conversion of the treated root tissues. Over-representation of the GO categories for meristem initiation, and meristem structural organization amongst DEGs with lower expression in the WUS domain of cLRP, may also reflect the transitional state of these organs.
Table 1. Selected enriched gene ontology (GO) terms amongst differentially expressed genes (DEGs) identified in the transcriptome of fluorescence-activated cell sorted (FACS) cells expressing pWUS::mGFP-ER from lateral root primordia (LRP) undergoing conversion to shoot meristems (SMs; after 30 h of treatment with 2iP), compared with the WUSp expression domain of apetala1/cauliflower double mutant SMs (Yadav et al., 2009). GO analysis was performed using agriGO (http://bioinfo.cau.edu.cn/agriGO)
|GO acc||Term type||Term||Query item||Query total||bg item||bg total||P value||Yekutieli FDR|
|Selected enriched GO terms. DEG >2 fold UP in pWUS::GFP 30 h 2iP versus SAM WUSp (Yadav et al., 2009)|
|GO:0042221||Biological process||Response to chemical stimulus||188||1224||1684||22 479||1.90E-18||7.40E-15|
|GO:0006950||Biological process||Response to stress||175||1224||1766||22 479||6.60E-13||6.50E-10|
|GO:0009407||Biological process||Toxin catabolic process||17||1224||44||22 479||5.90E-11||3.30E-08|
|GO:0010876||Biological process||Lipid localization||10||1224||15||22 479||5.60E-10||2.40E-07|
|GO:0009725||Biological process||Response to hormone stimulus||73||1224||687||22 479||1.10E-07||2.50E-05|
|GO:0016137||Biological process||Glycoside metabolic process||15||1224||101||22 479||4.00E-04||4.70E-02|
|GO:0000302||Biological process||Response to reactive oxygen species||10||1224||52||22 479||4.50E-04||5.20E-02|
|GO:0009791||Biological process||Post-embryonic morphogenesis||5||1224||26||22 479||1.20E-02||5.40E-01|
|GO:0007568||Biological process||Aging||9||1224||71||22 479||1.50E-02||6.20E-01|
|GO:0048364||Biological process||Root development||15||1224||170||22 479||4.60E-02||1.00E+00|
|Selected enriched GO terms. DEG >2 fold DOWN in pWUS::GFP 30 h 2iP versus SAM WUSp (Yadav et al., 2009)|
|GO:0009791||Biological process||Post-embryonic development||83||1452||501||22 479||1.20E-14||5.90E-11|
|GO:0001510||Biological process||RNA methylation||5||1452||6||22 479||6.40E-06||5.30E-03|
|GO:0048367||Biological process||Shoot development||34||1452||240||22 479||1.70E-05||8.90E-03|
|GO:0034660||Biological process||ncRNA metabolic process||23||1452||136||22 479||2.30E-05||9.40E-03|
|GO:0051641||Biological process||Cellular localization||52||1452||450||22 479||5.20E-05||1.60E-02|
|GO:0048366||Biological process||Leaf development||24||1452||156||22 479||7.50E-05||1.80E-02|
|GO:0010014||Biological process||Meristem initiation||5||1452||9||22 479||1.10E-04||2.20E-02|
|GO:0060918||Biological process||Auxin transport||10||1452||44||22 479||4.20E-04||5.00E-02|
|GO:0009933||Biological process||Meristem structural organization||9||1452||40||22 479||8.70E-04||8.20E-02|
|GO:0034470||Biological process||ncRNA processing||14||1452||83||22 479||8.70E-04||8.20E-02|
|GO:0009908||Biological process||Flower development||30||1452||257||22 479||1.40E-03||1.20E-01|
|GO:0016570||Biological process||Histone modification||9||1452||46||22 479||2.50E-03||1.70E-01|
|GO:0007049||Biological process||Cell cycle||25||1452||275||22 479||3.30E-03||2.10E-01|
|GO:0009765||Biological process||Photosynthesis, light harvesting||5||1452||18||22 479||4.80E-03||2.60E-01|
|GO:0010228||Biological process||Veg. to reprod. phase transition of meristem||11||1452||71||22 479||5.80E-03||2.90E-01|
|GO:0009790||Biological process||Embryonic development||37||1452||376||22 479||8.20E-03||3.60E-01|
Meristem initiation/organization includes targets interacting with WUS, or transcriptionally modulated by it. TOPLESS (TPL) and TOPLESS-RELATED 4 (TPR4) encode for transcriptional co-repressors that interact with WUS (Kieffer et al., 2006). Expression of TPL is enhanced by WUS (Busch et al., 2010), and directly targets PLETHORA 1 and 2 (PLT1 and PLT2) genes (Smith and Long, 2010), which are master regulators promoting basal/root fate (Aida et al., 2004; Galinha et al., 2007). Two other WUS-induced meristematic genes, STM1 and a CYCLIN-DEPENDENT KINASE B2;1 (CDKB2;1) were also found to have lower expression in the newly initiated WUS-reporter domain. CLV1 is a direct target of WUS, which represses CLV1 expression (Busch et al., 2010). If factors that positively regulate the expression of CLV1 within the SAM are absent in cLRP this might account for the lower expression of CLV1 in the WUS domain of these organs. As a shoot stem cell population, marked by CLV3 reporter expression, has not developed at this time, it is unsurprising that expression of other elements of the WUS–CLV pathway have yet to be established, including CLV1 and POLTERGEIST (POL) (Song et al., 2006).
Other interesting enriched biological processes amongst DEGs with lower expression in cLRP included: non-coding RNA processing, RNA methylation and histone modification. The establishment of a functional shoot stem cell niche can be expected to involve coordinated regulation of transcription, and the stability and functional output of numerous interacting targets. The categories histone modification, RNA methylation and ncRNA categories may include genes mediating these processes in initiating or established SAMs that have yet to be upregulated in LRP.