Plants maintain pools of pluripotent stem cells which allow them to constantly produce new tissues and organs. Stem cell homeostasis in shoot and root tips depends on negative regulation by ligand–receptor pairs of the CLE peptide and leucine-rich repeat receptor-like kinase (LRR-RLK) families. However, regulation of the cambium, the stem cell niche required for lateral growth of shoots and roots, is poorly characterized. Here we show that the LRR-RLK MOL1 is necessary for cambium homeostasis in Arabidopsis thaliana. By employing promoter reporter lines, we reveal that MOL1 is active in a domain that is distinct from the domain of the positively acting CLE41/PXY signaling module. In particular, we show that MOL1 acts in an opposing manner to the CLE41/PXY module and that changing the domain or level of MOL1 expression both result in disturbed cambium organization. Underlining discrete roles of MOL1 and PXY, both LRR-RLKs are not able to replace each other when their expression domains are interchanged. Furthermore, MOL1 but not PXY is able to rescue CLV1 deficiency in the shoot apical meristem. By identifying genes mis-expressed in mol1 mutants, we demonstrate that MOL1 represses genes associated with stress-related ethylene and jasmonic acid hormone signaling pathways which have known roles in coordinating lateral growth of the Arabidopsis stem. Our findings provide evidence that common regulatory mechanisms in different plant stem cell niches are adapted to specific niche anatomies and emphasize the importance of a complex spatial organization of intercellular signaling cascades for a strictly bidirectional tissue production.
The coordinated determination of cell fate during the transformation of pluripotent stem cells to specialized body cells is crucial for the development of higher organisms. Intercellular communication between stem cells and their immediate derivatives is a common motif in this context (Seuntjens et al., 2009; Doma et al., 2013). However, how stem cell properties are controlled without strict determination by cell lineage and how directionality of tissue production is achieved in these cases is unknown. Plants are extraordinary in this regard, because cell fate is constantly readjusted allowing indeterminate increase in body size by the constant formation of new tissues. Stem cells facilitating longitudinal growth of organs are embedded in protective niches located at shoot and root tips, called the shoot and root apical meristems (SAM and RAM) (Heidstra and Sabatini, 2014). Although the orientation of cell divisions is tightly controlled in cases such as the Arabidopsis RAM, divisions in apical meristems of higher plants are often random (Heimsch and Seago, 2008; Aichinger et al., 2012). In contrast, lateral growth of roots and shoots predominantly depends on strictly oriented cell divisions in the cambium, a bifacial meristem producing secondary vascular tissue (Brackmann and Greb, 2014).
Interestingly, although plant meristems are anatomically distinct, limited numbers of comparative studies have shown commonalities in their regulation (Sarkar et al., 2007; Stahl et al., 2013; Zhou et al. 2014), suggesting the existence of common motifs promoting plant stem cell attributes. One set of regulatory components found in all plant stem cell niches is composed of specific members of the LRR-RLK family, their ligands belonging to the CLAVATA3/ESR-RELATED peptide (CLEp) family and members of the WUSCHEL-RELATED HOMEOBOX (WOX) transcription factor family (Lenhard and Laux, 2003; DeYoung et al., 2006; Hirakawa et al., 2008, 2010; Ogawa et al., 2008; Guo et al., 2010; Depuydt et al., 2013). In the SAM, binding of the CLE peptide CLAVATA3 (CLV3p) to the LRR-RLK CLV1 inhibits transcription of the stem cell promoting WUSCHEL (WUS) transcription factor gene (Schoof et al., 2000; Ogawa et al., 2008). In turn, the WUS protein moves from the center of the meristem to the apical stem cells to activate CLV3 expression, thus completing a feedback loop that balances stem cell proliferation (Mayer et al., 1998; Yadav et al., 2011; Daum et al., 2014). In the RAM, CLV1 acts together with the receptor kinase ARABIDOPSIS CRINKLY4 (ACR4) to inhibit the WOX5 transcription factor gene maintaining root stem cells (Sarkar et al., 2007; De Smet et al., 2008; Stahl et al., 2009, 2013). In this case, CLE40p is most likely produced in differentiated root tip cells and binds to CLV1, which is expressed in the distal stem cells (Sarkar et al., 2007; Stahl et al., 2009, 2013; Guo et al., 2010).
While regulatory mechanisms underlying stem cell homeostasis in the two apical meristem types (SAM and RAM) have been studied intensively, it is not well understood how proliferation of the stem cells in lateral meristems, for instance the cambium, is controlled. Only a positive regulatory cascade has been found there: The CLE41/44p is generated in differentiated phloem cells, a cambium-derived vascular tissue produced toward the organ periphery (distally). From there it moves to the stem cells within the cambium where it binds to the LRR-RLK PHLOEM INTERCALATED WITH XYLEM (PXY, also known as TDR). Thereby, CLE41/44p inhibits differentiation of xylem, the vascular tissue produced toward the organ center (proximally) (Fisher and Turner, 2007; Hirakawa et al., 2008; Etchells and Turner, 2010; Kondo et al., 2014). In addition, the binding enhances expression of WOX4, another member of the WOX transcription factor family, which in turn promotes cambial cell divisions (Hirakawa et al., 2010; Suer et al., 2011; Etchells et al., 2013). The distinct directionality of the CLE41/PXY-dependent signaling seems to provide spatial information for instructing the almost strictly periclinal (parallel to the organ surface) orientation of cell divisions in the cambium (Etchells and Turner, 2010). However, considering the bifacial character of the cambium, a more complex system for providing spatial and directional information than the unidirectional CLE41/PXY system may be predicted to act in the cambium zone. Furthermore, in analogy to the apical meristems, a negative regulation for cambium homeostasis is to be expected. Although several LRR-RLKs have been detected to be expressed associated with the cambium in woody species (Wang et al., 2015), no such negative regulation has been characterized to date. Thus, it is unclear how strictly bidirectional tissue production is achieved and how the regulatory networks that are involved are spatially organized to produce such a tightly controlled system.
Here, we use fluorescent promoter reporters to show that the LRR-RLK MORE LATERAL GROWTH1 (MOL1) is expressed in the distal domain of the cambium. Histological and genetic analyses demonstrate that MOL1 contributes to stem cell homeostasis within the cambium and niche organization and that PXY and WOX4 act epistatically to MOL1. MOL1 is closely related to PXY but by swapping the promoters of these genes we found that PXY and MOL1 are not interchangeable. Interestingly, MOL1 is able to replace CLV1 in the SAM but this does not hold true for PXY. We also show that MOL1 represses stress-related hormonal signaling pathways that positively influence cambium activity. Altogether, we identify MOL1 as a member of a predicted intercellular communication cascade acting antagonistically to the CLE41/PXY/WOX4 cascade and connecting distinct niche areas. This signaling module is functionally relevant for the dynamics of the anatomically complex cambium and, thus, provides scenarios for the evolution of plant stem cell niches.
MOL1 activity defines the distal cambium domain
As MOL1 was an excellent candidate for representing a negative signaling cascade repressing cambium activity (Agustí et al., 2011b), we set out to analyze the role of the MOL1 gene in cambium regulation. To determine spatial organization of MOL1 activity in the cambium area, we generated Arabidopsis lines carrying a proMOL1:YELLOW FLUORESCENT PROTEIN (YFP) reporter harboring MOL1 promoter regions sufficient to fully complement the mol1 mutant phenotype when driving MOL1 expression (see below). The reporter was combined with cyan fluorescent protein (CFP)-based reporters driven by the promoters of PXY or APL, which are active in the predicted stem cells within the cambium or in the differentiated phloem, respectively (Agustí et al., 2011a). Analysis of laterally growing stems showed that the MOL1 promoter was mostly active distally to the area of proPXY:CFP activity (Figure 1a–f) and overlapped with proAPL:CFP activity (Figure 1g–l), but also included APL-negative developing phloem cells and phloem parenchyma (Figure 1g,h). A similar arrangement of activity domains was found in hypocotyls from seedlings and mature plants and in primary vascular bundles from inflorescence stems (Figure S1a–g). These expression patterns together with the observation that cambium activity is enhanced in mol1 mutants (Agustí et al., 2011b) suggested that MOL1 either represses stem cell divisions or, alternatively, regulates the transition of cambium stem cells to phloem cells delivered toward the organ periphery.
MOL1 represses stem cell activity within the cambium
To decide between both possibilities, we first examined the expansion of secondary vascular tissues in mature mol1-1 mutant (Agustí et al., 2011b) hypocotyls. The overall hypocotyl diameter was increased in mol1-1 compared with wild type, with an increase in both phloem and xylem production (Figure 2a,e,f). This alteration was similar to the increase in lateral growth in the inflorescence stem (Figure 2b,g,h). To verify that tissue extension is a good proxy for the number of cells within these tissues we prepared new samples and counted the number of cells along the line of measurement in the inflorescence stem. This analysis showed that mol1-1 not only displayed a significantly larger extension of the interfascicular cambium-derived tissues (Figure 2b,g,h), but that there are indeed more cells within these regions (Figure S1h).
In contrast with mol1-1 mutants, expression of MOL1 fused to YFP under the control of the strong and ubiquitous CaMV 35S promoter (Benfey and Chua, 1990) led to a reduction in the amount of phloem and xylem cells in the hypocotyl (Figure 2c,i,j) and a reduction in lateral growth at the base of the inflorescence stem (Figure 2d,k,l). In addition, the production of well aligned files of compact cells derived from cambial divisions was strongly reduced in the inflorescence stem (Figure 2k,l). Furthermore, cambium cells were poorly organized and often absent in mature hypocotyls (Figure 2i,j), indicating that enhanced and/or ectopic MOL1 activity alters the organization of the cambium zone and that MOL1 negatively regulates cambium attributes. Consistent with the latter conclusion, enhanced tissue production in mol1-1 mutants was not observed when these mutants also carried wox4-1, or pxy-4 mutations, which diminish cambium activity (Fisher and Turner, 2007; Suer et al., 2011) (Figure 2b,m–p) but do not alter MOL1 mRNA abundance on the organ level (Agustí et al., 2011b; Suer et al., 2011; Etchells et al., 2012) (Dataset S1). This favors the conclusion that WOX4 and PXY act epistatically to MOL1 and that MOL1 acts on a fully functional cambium. Collectively, these observations argue for a primary role of MOL1 in repressing the division rate of cambium stem cells.
In order to determine whether MOL1 has additional effects on plant growth outside the cambium we counted the primary rosette leaves to gauge floral transition and measured the fresh weight of the aerial parts at the same growth stage as was analyzed above. This analysis revealed a small but statistically significant delay in floral transition of mol1-1 mutants (Figure 3a). In addition, mol1-1 mutants were slightly heavier than wild type plants (Figure 3b). However, the time until plants reached 15 to 20 cm shoot height was almost identical (49.1 days for wild type and 50.8 days for mol1-1), indicating that these differences most likely were due to changes in growth rate, rather than the length of the growth period. In spite of these changes the overall morphology of mol1-1 mutants did not appear to be different to wild type plants (Figure 3c,d). These findings were in line with a role of MOL1 predominantly in cambium regulation and underlined the specificity of the observed effects. Supporting this conclusion, the anatomy of primary vascular bundles was not altered in mol1-1 mutants (Figure S1i,j) and a β-glucuronidase (GUS)-based transcriptional proMOL:GUS reporter demonstrated an activity of MOL1 exclusively in the mature vascular system harboring an established cambium-like stem cell niche (Figure S1k–o).
MOL1 does not influence PXY, CLE41 or WOX4 transcription
To see whether there is a direct interaction between MOL1 and the positively acting PXY/CLE41/WOX4 signaling cascade, we compared transcriptional profiles of mol1-1 and wild type from the bottom-most centimeter of inflorescence stems harboring a secondary tissue conformation (Sehr et al., 2010). Consistent with increased lateral growth in mol1-1 mutants, we identified genes linked to the stress-related ethylene and jasmonic acid signaling pathways, which are positively associated with lateral stem growth (Love et al., 2009; Sehr et al., 2010; Etchells et al., 2012), to be more expressed in mol1-1 (Figure S2a and Dataset S1). Particularly striking was the observation that 20% (38/190) of genes that are upregulated in mol1-1 are also upregulated in pxy mutants, including several ETHYLENE RESPONSE FACTOR (ERF) transcription factor genes which partly compensate the loss of PXY activity (Etchells et al., 2012), (Figure S2b and Dataset S1). In contrast, CLE41, WOX4 or PXY were not found to be differentially expressed. These findings were confirmed by qRT-PCR analysis (Figure 3e,f) and by analyzing proWOX4:YFP and proPXY:YFP promoter reporters which did not display an altered pattern of activity when comparing wild type and mol1-1 mutant backgrounds (Figure S2c). Analysis of transcriptomes from the second elongated internode, with primary tissue conformation, revealed just 16 genes, again not including PXY, CLE41 or WOX4, to be significantly (adjusted P-value <0.1) changed in mol1-1 (Dataset S1). In addition to supporting the idea of a role of MOL1 predominantly in the regulation of lateral growth, these observations imply that MOL1 does not directly repress PXY, CLE41 or WOX4 gene activities.
MOL1 and PXY functions are distinct
Importantly, although the extracellular domain of PXY is the most similar one to the extracellular domain of MOL1 within the Arabidopsis proteome (Figure 4a) both LRR-RLKs were not able to functionally replace each other when their respective promoters were swapped (Figure 4b–k). This was although all transgenes were actively expressed (Figure S3a–d). In contrast, a proMOL1:MOL1 transgene was able to fully complement the mol1-1 mutant phenotype and a proPXY:PXY transgene partly restored cambium dynamics in pxy-4 mutant backgrounds (Figure 4b–k). This finding suggests that both MOL1 and PXY act in distinct signaling cascades. To investigate whether MOL1 activity in the distal cambium domain is essential, we expressed MOL1 under the control of the PXY promoter in wild type and in mol1-1 mutants. Interestingly, we observed severe disorganization of the cambium in some cases (n = 1/7 for proPXY:MOL1 in wild type and 2/6 for proPXY:MOL1 in mol1-1) (Figure 4l–o) underlining the importance of a proper spatial organization of MOL1 for organized cambium activity. More generally, our observations underline the importance of a MOL1 and PXY-dependent regulation of distinct regulatory programs in discrete cambium domains.
To see whether MOL1 is able to act as a general repressor of stem cell activity, we expressed MOL1 under the control of the CLV1 promoter in clv1-20 mutants (Durbak and Tax, 2011) (Figure S4). We found that MOL1 rescued the petal number defect of clv1-20 mutants in a similar manner as CLV1 itself (Figure 5a–e). In contrast, when we expressed PXY under the control of the CLV1 promoter (Figure S4), we found that the primary apical meristem was prematurely terminated and numerous axillary inflorescence stems developed (Figure 5f). This showed that MOL1 and CLV1, two LRR-RLKs negatively influencing meristem activity, are able to replace each other in the SAM, while PXY behaves differently in this context.
Regulation of stem cell activity is a paradigm for cell fate determination in multicellular organisms (Sanchez Alvarado and Yamanaka, 2014). Due to the lack of cell motility and of fixed cell lineages in plants, the spatial arrangement of the signaling networks that are involved is essential for creating a cellular environment promoting stem cell properties. Here we identified MOL1 as a negative regulator of cambium activity acting antagonistically to the CLE41/PXY/WOX4 cascade. We revealed that the expression domain of MOL1 is strongly associated with the radial tissue organization within the cambium area suggesting that MOL1 represents an intercellular communication module that is important for stem cell homeostasis within the cambium.
In contrast with the SAM and RAM, for which negative regulatory feedback loops via LRR-RLK/CLE cascades are well characterized (Schoof et al., 2000; Stahl et al., 2009, 2013; Daum et al., 2014), only local regulators acting positively on cambium stem cell activity were previously known (Fisher and Turner, 2007; Hirakawa et al., 2008, 2010; Etchells and Turner, 2010). Based on its influence on both phloem and xylem production, the epistasis of WOX4 and PXY to MOL1 and its capacity to replace CLV1 in the SAM, we identified MOL1 as a negative regulator of cambium homeostasis. We also show that it is expressed in the distal cambium domain including differentiated phloem cells and mostly distinct from the PXY activity domain. Similarly to the RAM (Bennett and Scheres, 2010), the cambium delivers cells in opposite directions, in this case, by strongly favoring periclinal cell divisions along the longest cell axis. In light of these tightly controlled dynamics, reciprocal signaling cascades connecting the central stem cells with their progenitors and providing positional and directional information can be expected. CLE41p is produced in the phloem and travels to the central cambium domain where it promotes cambium activity via PXY and WOX4 (Hirakawa et al., 2008; Etchells et al., 2012). Although MOL1 and the positive cambium regulators CLE41 and CLE44 seem to be expressed in similar domains (Hirakawa et al., 2008; Etchells and Turner, 2010), neither CLE41 nor CLE44 transcript accumulation was detectably altered in mol1-1 mutants arguing against a MOL1-dependent non-cell autonomous regulation of cambium activity by influencing CLE41/44 transcription. This view is also supported by our finding that WOX4, whose activity is stimulated by CLE41, is not altered in mol1-1 mutants (Hirakawa et al., 2010). Of note, the finding that WOX4 activity is not altered in mol1-1 is in contrast to our previous analysis during which we detected higher WOX4 mRNA levels in stems from mol1 mutants (Agustí et al., 2011b). We propose that enhanced WOX4 activity was previously detected due to an enhanced acropetal progression of cambium initiation along the stem in mol1 mutants leading to an altered overall anatomy with more cambium-related cells in analyzed stem fragments, but not due to an enhanced WOX4 activity at a given position. In addition, because PXY and MOL1 are not able to replace each other when expressed in the respective expression domains, we suggest that two largely independent signaling cascades exist in the cambium acting antagonistically in functional terms and, potentially, in their spatial directionality of intercellular signaling. Although the three BARELY ANY MERISTEM (BAM1-3) LRR-RLKs have partly opposing functions to CLV1 in the SAM (DeYoung et al., 2006; Deyoung and Clark, 2008), counteracting LRR-RLKs/CLE cascades with a prominent spatial specificity have not been observed in apical plant stem cell niches in which tissue production is not strictly bidirectional. Thus, it is possible that the spatial contrast of the PXY/CLE41 cascades with MOL1 reflects the necessity for a radial communication between cambium-related tissues during lateral growth.
Interestingly, ERF transcription factor genes that are expressed in the central cambium domain and known to promote lateral stem growth downstream of WOX4 and PXY (Etchells et al., 2012) are more active in mol1-1 mutants. Because these stress-related signaling components are more active, even in primary mol1 stems in which tissue anatomy is not altered, it is possible that MOL1 primarily acts on cambium activity by repressing ethylene signaling independently of WOX4 and PXY. Cambium activity results in the expansion of peripheral organ tissues and mechano-sensitive stress signaling has been suggested to play a major role in the coordination of tissue dynamics (Ko et al., 2004; Sehr et al., 2010).
The current view is that lateral meristems, like the cambium, evolved after the apical meristems and allowed plants to adapt to a plethora of ecological niches on land (Rowe and Speck, 2005; Spicer and Groover, 2010; Hirakawa and Bowman, 2015). The diversification and adjustment of signaling networks involved in apical meristem regulation during the evolution of lateral plant growth, in particular CLE/LRR-RLK signaling modules seem to have been essential for this major breakthrough in the establishment of a broad range of different plant growth forms (Miwa et al., 2009). The identification of MOL1 as being important for cambium regulation and the spatial association with this particular stem cell niche suggests that they belong to a regulatory program diversified to regulate lateral plant growth. Its specific capacity to replace CLV1 in the SAM further demonstrates a functional similarity between the regulatory systems in both stem cell niches. Thus, the establishment of a complex spatial organization of signaling components might have been instrumental for developing a novel stem cell niche with a strict demand for directionality of tissue production.
Plant material and growth conditions
Arabidopsis thaliana (L.) Heynh. plants of the accession Columbia were used for all experiments and grown as described previously (Suer et al., 2011).
Protein sequence alignments
Protein sequences were aligned by CLC Main Workbench 6.9 (CLC bio, Denmark) using the default parameters (gap opening cost: 10; gap extension cost: 1.0; gap separation distance: 8) and visualized by the Create Tree tool of the same software. The phylogenetic tree was constructed by using the neighbor-joining (NJ) method and the Jukes Cantor method for estimating the number of amino acid substitutions between sequences. Bootstrap values were obtained by 1000 bootstrap replicates.
qRT-PCR was carried out using SensiMix™ SYBR® Green (Bioline Reagents Ltd, London, UK) mastermix and gene specific primers as detailed in Table S1, in a Roche Lightcycler480, with standard three-step amplification and detection protocols. Raw amplification data were exported and analyzed with LinRegPCR (Ruijter et al., 2009; Tuomi et al., 2010) software. Further analysis and statistical tests were done using Microsoft Excel®.
Confocal microscopy of inflorescence stems was based on the protocol of (Suer et al., 2011). Stem cross-sections were hand-cut with a razor blade (Classic Wilkinson, Wilkinson, Solingen, Germany) and mounted in water. For seedling hypocotyls, the plants were first mounted in 4% low melting point agar (Sigma-Aldrich, St. Louis, MO, USA) in water to facilitate handling during sectioning. Micrographs were taken using a Zeiss LSM780 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) with separate tracks for excitation and detection of each fluorophore. CFP was excited at 458 nm and imaged by collecting emissions between 463 and 508 nm. YFP was excited at 514 nm and emissions were collected between 517 and 543 nm. Propidium iodide (1:5000 v/v in water) was used to counter-stain secondary cell walls. Propidium iodide was excited by laser at 561 nm and emissions were collected between 561 and 608 nm. Transmitted light from the 561 nm laser was collected to make the bright field images.
Cloning and transgenic lines
All fluorescent reporter proteins were targeted to the endoplasmic reticulum (ER) by the appropriate target sequences (Haseloff et al., 1997). For MOL1 promoter constructs, initially two genomic fragments (1296 bp upstream of the start codon and 490 bp downstream of the stop codon) were inserted between NotI and ApaI sites of pGreen0229 or pGreen0029 (Hellens et al., 2000) resulting in pMS40 and pMS94, respectively. The open reading frames of YFP–ER or GUS were then inserted into restrictions sites introduced between the two genomic fragments in pMS40 (resulting in pNG1 and pMS92, respectively) and the MOL1 open reading frame was inserted into pMS94 (resulting in pMS95). For proMOL1:PXY (pKG49), the same strategy was used except that in this case pGreenII0179 served as a backbone. The proPXY:CFP, proAPL:CFP and proWOX4:YFP reporters were described previously (Agustí et al., 2011a; Suer et al., 2011). For proPXY:PXY (pMS85) and proPXY:MOL1 (pMS84) constructs the sequence of the CFP was replaced by the respective open reading frames. For the pro35S:MOL1–YFP construct (pMS90), a DNA fragment encoding the MOL1–YFP fusion was cloned into pGreen0229 containing the 35S promoter. All constructs were sequenced and, after plant transformation, single copy lines were identified by Southern blot analyses and representative lines were used for further investigations.
Histological analyses of the stem were performed as described previously (Agustí et al., 2011a). All samples compared histologically were grown in parallel. Measurements were made blind to sample identity.
RNA in situ hybridization
RNA in situ hybridization was carried out as described previously (Greb et al., 2003). For probe synthesis, PCR products (Table S1) generated using cDNA as a template were cloned into the pGEM-T vector (Promega, Madison, WI, USA) and used as a template for transcription from the T7 or SP6 promoter. MOL1 and PXY probes were described before (Agustí et al., 2011b).
Total RNA was extracted with TRIzol® (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's protocol. For RNA sequencing, total RNA was isolated from the bottom-most centimeter of the elongated inflorescence stem of 20 cm tall plants or from above the first node. Library preparation and Deep RNA sequencing comparing wild type and mol1-1 mutants with two replicates each in which RNA from two plants was pooled (single-end, 50 bp reads, at least 27.5 million aligned fragments for each sample) was performed at the Vienna Biocenter Core Facilities GmbH (VBCF) (Vienna, Austria). RNA sequencing of samples from above the first node comparing wild type, mol1 used three replicates per genotype and each replicate contained RNA pooled from two or three plants. Each replicate produced at least 37 million aligned reads (single read 50 bp). HiSeq machines (Illumina, San Diego, CA, USA) were used in all cases. All reads were aligned using CLC Genomics Work Bench 7 (CLC bio, Denmark). For statistical analysis the DESeq package from the R/Bioconductor software was used. Normalization and analysis of microarray data published previously was done by the Robust Multi-Array (RMA) method using the affy and Limma packages from the R/Bioconductor software (Gautier et al., 2004; Gentleman et al., 2004; Anders and Huber, 2010). An adjusted P-value of 0.1 was chosen as a threshold for selecting differentially expressed genes in Dataset S1.
Virtual plant software analysis
BioMaps analysis using the Virtual Plant software [version 1.3, www.virtualplant.org (Katari et al., 2010)], was done using the following parameters: gene ontology (GeneOntology_biological processes), method (Fisher), background (whole genome), cutoff (0.01).
We are grateful to Wolfgang Busch and Klaus Brackmann for helpful comments on the manuscript. Plant growth was performed with support of the Plant Sciences Facility at the Vienna Biocenter Core Facilities GmbH (VBCF), Austria. This work was supported by a Marie Curie PLANT FELLOWS (FP7, GA-2010-267243, no. 337) postdoctoral fellowship to N.R.G., by an EMBO long-term postdoctoral fellowship (ALTF 342-2012) to V.J. and by a Heisenberg Fellowship from the German Research Foundation (DFG, GR 2104/3-1) to T.G. Furthermore, support was provided by the DFG through grant GR 2104/4-1 and the SFB 873.