The post-embryonic development of above-ground tissues in plants is dependent upon the maintenance and differentiation of stem cells at the shoot meristem. The Arabidopsis WUSCHEL (WUS) transcription factor establishes an organizing center within the shoot meristem that is essential for specification of stem-cell identity in overlying cells. The CLAVATA (CLV)signaling pathway, including the CLV1 receptor-kinase, promotes the differentiation of stem cells by limiting the WUS expression domain,yet the mechanism of CLV signaling is largely unknown. Previously, we have shown that mutations in two protein phosphatases, POLTERGEIST (POL) and PLL1,partially suppress clv mutant phenotypes. Here, we demonstrate that POL and PLL1 are integral components of the CLV1 signaling pathway. POL and PLL1 are essential for stem-cell specification, and can also block stem-cell differentiation when overexpressed. We provide extensive evidence that POL and PLL1 act downstream of CLV signaling to maintain WUS expression and that they regulate WUS at a transcriptional level. Our findings suggest that POL and PLL1 are central players in regulating the balance between stem-cell maintenance and differentiation, and are the closest known factors to WUS regulation in the shoot meristem.

The adult plant body is generated from the continuous and re-iterative organogenesis at two stem-cell containing structures: the shoot and root meristems. The plant shoot meristem is established at the apical end of the embryo, and is responsible for generating all of the organs and lateral meristems found above ground. A functional shoot meristem is maintained through a tightly controlled balance between the proliferation of a group of stem cells residing in the center, and the differentiation of their peripheral and basal progeny cells for the formation of organ primordia and other differentiated tissues. Flower meristems initiated during inflorescence development function similarly to shoot meristems, except for the transient nature of their stem-cell population.

The homeodomain-containing transcription factor WUSCHEL (WUS) is necessary and sufficient within the meristem to specify stem-cell identity. wusmutations lead to the loss of shoot meristem stem cells, and WUSoverexpression gives rise to ectopic stem cells within the meristem(Brand et al., 2002; Gallois et al., 2002; Laux et al., 1996; Lenhard et al., 2002; Mayer et al., 1998; Schoof et al., 2000). WUS is expressed immediately basal to the stem cells in what is termed as an `organizing center' (Mayer et al., 1998).

The CLAVATA (CLV) signal transduction components CLV1, CLV2 and CLV3 act to restrict the domain of WUS expression(Brand et al., 2000; Schoof et al., 2000). CLV1 encodes a receptor-like kinase containing leucine-rich repeat(LRR) motifs, CLV2 a LRR receptor-like protein and CLV3 a small secreted polypeptide (Clark et al.,1997; Fletcher et al.,1999; Jeong et al.,1999; Rojo et al.,2002; Ni and Clark,2006). Recent findings have shown that transient inactivation of the CLV pathway leads to rapid alterations in the expression of meristem regulators, consistent with earlier studies investigating the effect of clv mutations on WUS expression(Reddy and Meyerowitz, 2005). Despite our improving understanding of the crucial biological role of this signaling pathway in regulating stem-cell specification and differentiation,no downstream signaling intermediates have been definitively identified.

The best candidate for a CLV signaling intermediate is the protein phosphatase kinase-associated protein kinase (KAPP)(Stone et al., 1994; Stone et al., 1998; Williams et al., 1997). KAPP binds, among many receptor-kinases, to CLV1, and both overexpression and cosuppression studies have suggested that KAPP plays a role in repressing CLV1 function (Stone et al., 1994; Stone et al., 1998; Williams et al., 1997). However, neither a definitive genetic study nor a clear mechanism for KAPP function has been reported.

Another potential source of signaling components are modifier mutants. Previous studies have identified mutations in many genes that enhance or suppress the phenotype of clv mutants and are potential candidates for signaling intermediates, including SHOOTMERISTEMLESS, ULTRAPETALA,REVOLUTA, PHABULOSA, PHAVOLUTA, CORONA, PERIANTHIA, and WIGGUM/ENHANCED RESPONSE TO ABA 1(Clark et al., 1996; Fletcher, 2001; Green et al., 2005; Otsuga et al., 2001; Prigge et al., 2005; Running et al., 1998; Running and Meyerowitz, 1996). However, detailed analyses of all of these genes suggest that each acts independently of the CLV signaling pathway.

Two additional genes that modify the clv phenotype when mutated are POLTERGEIST (POL) and PLL1, which encode related protein phosphatases. Mutations in either gene provide partial,additive suppression of the stem-cell accumulation of clv mutants,raising the possibility that these two genes act redundantly to promote stem-cell identity (Song and Clark,2005; Yu et al.,2003; Yu et al.,2000). However, the pol pll1 double mutant is seedling lethal, complicating previous efforts to analyze these genes and their potential role in CLV signaling.

In this study, we report a detailed analysis of POL, PLL1, WUS and CLV genetics. We overcome seedling lethality through grafting, and show interactions between mutations in these genes and their overexpression. All of our findings are consistent with a model in which POL and PLL1 act downstream of the CLV proteins, CLV signaling represses POL/PLL1 and POL/PLL1 are required for WUS expression.

Plant materials and growth condition

The pol-1, pol-6 and pll1-1 mutants were obtained as described previously (Song and Clark,2005; Yu et al.,2003; Yu et al.,2000). pol-6 pll1-1+ plants were isolated from the F2 progeny obtained from the cross between pol-6 and pll1-1, and, subsequently, pol-6 pll1-1 double mutants were identified among the progeny of pol-6 pll1-1+ plants based on polymerase chain reaction (PCR) genotyping, as described previously(Song and Clark, 2005). Plants were grown as described previously (Song and Clark, 2005). When plants were grown in sterile conditions,seeds were germinated on the half strength of MS media (Sigma) containing 1%sucrose and 0.02% MES solidified with 0.6% agarose after imbibition at 4°C for 2 days following sterilization.

pol pll1 seedlings 4-6 days after germination grown in sterile condition were micro-grafted as described previously(Turnbull et al., 2002). A cotyledon shoulder region of a pol pll1 seedling was dissected with a razor blade under a dissecting microscope and transferred on top of a wild-type stock that was prepared by being cut at the hypocotyl region. Whether a scion and a stock were positioned correctly along the axis was examined under a dissecting microscope. Grafted plants were moved into soil approximately 5 days after grafting.

Complementation of pol pll1 with PLL1 expression and antisense expression of PLL1

For the complementation of the seedling-lethal phenotype of pol-1 pll1-1 double mutants, the PLL1 cis-elements including 3.0 kb promoter and 0.5 kb terminator were used for the expression of PLL1cDNA. PLL1 cDNA fragment (Song and Clark, 2005) digested with SmaI/SpeI was first inserted into a pUC19 vector (digested with SmaI/XbaI)and then the PLL1 promoter that was digested with EcoRI/SmaI was inserted in front of the PLL1 cDNA. Subsequently, the fused fragment was digested with EcoRI/SalI and introduced into a pOCA28 vector(Olszewski et al., 1988)containing a PLL1 0.5 kb terminator (SalI/XhoI)digested with EcoRI/SalI. pol-1 pll1-1/+ plants were transformed with this construct as described previously(Clough and Bent, 1998). T1 plants displaying kanamycin resistance were screened and PCR genotyped with PLL1c1/SynLB3 and PLL1c1/PLL1c2 primers to identify plants containing both the pll1-1 T-DNA insertion and the transgene for complementation (Song and Clark,2005). Subsequently, their T2 progeny were PCR genotyped to screen transgenic plants homozygous for pll1-1.

For the anti-sense expression of PLL1, a full-length cDNA fragment, PCR amplified with PLL1-NSpe/PLL1-C primers (PLL1-NSpe,5′-AACTAGTATGGGAAGTGGATTCTCCTCCT-3′); PLL1-C,5′-CGCACTAGTTCAAAGATACTTTCCTGATGAC-3′), was introduced in reverse orientation into a pCB302-3 binary vector containing cauliflower mosaic virus(CaMV) 35S cis regulatory elements(Xiang et al., 1999). This transgene was introduced into pol-6 mutants.

Genetic interaction of POL/PLL1 with CLV, WUS and the P35S:CLV3 transgene

clv3-2 pol-6 pll1-1 triple mutants were screened among the progeny of clv3-2 pol-6 pll1/+ that were identified from PCR genotyping among the F2 progeny obtained from the cross between clv3-2 pol-6 and clv3-2 pll1 (Song and Clark, 2005). The seedlings of the triple mutants were grown in sterile conditions and grafted as described above, and their floral organ numbers were counted. PER:PLL1 and P35S:PLL1 transgenes(Song and Clark, 2005) were introduced into the clv1-1, clv1-11, clv2-1 and clv3-2mutant backgrounds by crosses. The individual F2 progeny seed pools segregating both clv and the transgene were selected, and their phenotypes were examined. Scanning electron microscopy (SEM) analysis was performed as described previously(Diévart et al., 2003)using plants at 10 days after germination.

The P35S:PLL1 transgene was introduced into the wus-1 mutant background by crosses. The individual F2progeny segregating both wus-1 and P35S:PLL1 was examined to see whether P35S:PLL1 alters the floral phenotype of wus-1.

F1 plants were obtained from crosses between a transgenic plant containing P35S:PLL1 and a transgenic plant containing P35S:CLV3 (Brand et al., 2000). Plants possessing both P35S:PLL1and P35S:CLV3 were screened among the F1 plants and their phenotypes were examined to see whether they displayed the Wus- phenotype. To determine the presence of the P35S:PLL1 and P35S:CLV3 transgenes,PCR genotyping was performed using PLL1c1/PLL1c2 primers (PLL1c1,5′-GTGTTTACTCGAAGAAGAGACGGA-3′; PLL1c2,5′-GTGCTCGTTTTTTATTCTTGTTACTTC-3′) and 35Sp1/CLV3r primers (35Sp1,5′-GATGACGCACAATCCCACTA-3′; CLV3r,5′-TCAAGGGAGCTGAAAGTTGTT-3′), respectively.

Expression of these genes was assessed using primers for PLL1(PLL1c1/PLL1c2), CLV3 (CLV3f,5′-ATGGATTCTAAAAGCTTTGTGCT-3′; CLV3r,5′-TCAAGGGAGCTGAAAGTTGTT-3′) and TUB (TUBf,5′-AGAGGTTGACGAGCAGATGA-3′; TUBr,5′-CCTCTTCTTCCTCCTCGTAC-3′).

Expression pattern analyses of reporter genes

PWUS:GUS and PCLV3:GUS reporter genes (Gross-Hardt et al.,2002; Lenhard et al.,2002) were introduced into mutant backgrounds by crosses. GUS activity was examined in the plants, which were grown in sterile conditions,at 10 days after germination, or in the inflorescence of grafted plants as described previously (Sessions et al.,1999). Tissues were incubated in the staining solution overnight.

Complementation of pol pll1 by the ectopic expression of WUS

For ectopic WUS expression, the pOpL two-component expression system was used (Moore et al.,1998). APETALA1 (AP1)(Hempel et al., 1998) and ERECTA (ER) promoters(Diévart et al., 2003)were used to drive the expression of LhG4 (kindly provided by Michael Prigge, University of Indiana, Indiana, USA). Several independent transgenic lines were screened and crossed with transgenic plants expressing pOp:WUS (Schoof et al.,2000) (kindly provided by Thomas Laux) to examine the strength of the promoter based on the phenotypes of F1 plants. F1progeny obtained from the crosses between the selected AP1:LhG4 line(or ER:LhG4 line) and pol-6 pll1-1/+ were crossed to F1 progeny obtained from the crosses between pOp6:WUS and pol-6 pll1-1/+. Among the resulting new progeny, pol pll1plants were isolated, grafted and PCR genotyped using genespecific primers(LhG4f/LhG4r for AP1:LhG4 and ER:LhG4; WUS-N/WUS-C for pOp6:WUS) to test for the presence of both transgenes (LhG4f,5′-TAACGTTATACGATGTCGCAGAG-3′; LhG4r,5′-CCAATGCGACCAGATGCT-3′; WUS-N,5′-CCCGGGGATGGAGCCGCCACAGCATCAG-3′; WUS-C,5′-GGATCCCTAGTTCAGACGTAGCTCAAG-3′). AP1:LhG4 and pOp6:WUS transgenes were introduced into the wus-1 mutant background in a similar manner.

Grafted pol pll1 tissue phenocopies wusmutants

A major obstacle in analyzing POL/PLL1 function in stem-cell specification was the embryo/seedling lethality of the pol pll1 double mutants(Song and Clark, 2005). We determined that pol pll1 lethality was largely the result of major defects in basal embryo patterning, and that we could grow pol pll1double-mutant tissue by grafting the apical portion of a pol-6 pll1-1seedling onto the hypocotyl/root of a wild-type seedling(Fig. 1A-E). Both pol-6 and pll1-1 are T-DNA insertion alleles that are putative nulls (Song and Clark,2005).

The pol pll1 tissues (Fig. 1F,G) growing in such grafted plants closely phenocopied wus mutants (Fig. 1I),with re-iterative termination of shoot apices during vegetative development. Eventually transitioning to flowering as wus mutants do, the pol pll1 grafted tissue (Fig. 1K,L) gave rise to inflorescence phenotypes similar to wus mutants (Fig. 1N),including flowers with reduced numbers of floral organs, presumably as a result of the loss of flower-meristem stem cells (compare Fig. 1P,Q with Fig. 1S). pol pll1 flowers lacked central carpels and developed reduced numbers of stamens, although the phenotype was slightly less severe than wusmutants (Fig. 2). The meristem-termination phenotypes were also observed, albeit less frequently, in pol/pol pll1/+ plants, in pol/pol plants with antisense expression of PLL1 and in pol/pol pll1/pll1 plants with incomplete complementation by PLL1 (Fig. 1U-Y). This indicates that these phenotypes are not related to the grafting technique used to generate pol pll1 tissue, but are a consequence of reduced POL/PLL1 activity.

pol pll1 is epistatic to clv3

The severe loss of meristem activity in pol pll1 double-mutant tissue allowed us to address whether POL/PLL1 act upstream or downstream of the CLV signaling pathway by generating clv pol-6 pll1-1 triple mutants. A similar genetic approach was previously used to establish that WUS acts downstream of CLV signaling, a finding bourn out by subsequent detailed studies (Laux et al.,1996; Mayer et al.,1998; Schoof et al.,2000). The seedling lethality of pol pll1 mutants was unaffected by the introduction of the putative null clv3-2 mutation. The ratio between the viable plants and seedling-lethal plants in the progeny of clv3-2 pol pll1/+ plants (79:31) and the progeny of clv3-2 pol/+ pll1 plants (54:19) did not vary significantly from 3:1 based on χ2 analysis. clv2-1 pol pll1, clv1-7 pol pll1and clv1-1 pol pll1 triple mutants also exhibited seedling-lethal phenotypes.

To examine the post-embryonic phenotypes, clv3-2 pol pll1 mutants were grafted onto the wild-type hypocotyls. clv3-2 pol pll1 tissue developed in an identical fashion to pol pll1 double-mutant tissue during vegetative and inflorescence development(Fig. 1H,M). The mean number of organs developing in clv3-2 pol pll1 flowers was statistically indistinguishable from that of pol pll1 flowers(Fig. 1R, Fig. 2). These results indicate that pol pll1 is fully epistatic to clv3-2, indicating that POL/PLL1 act downstream of the CLV signaling pathway.

Alteration of stem-cell marker gene expression in pol pll1mutants

To determine the nature of meristem defects in pol pll1 mutants,we crossed pol pll1 to well-characterized transgenes in which the CLV3 and WUS cis regulatory elements driveβ-glucuronidase (GUS) expression (PCLV3:GUS and PWUS:GUS) and monitored the expression of these key meristem regulatory factors (Gross-Hardt et al., 2002; Lenhard et al.,2002). For PWUS:GUS, we observed a clear spot of GUS activity at the shoot meristem in wild-type seedlings, but no signal at all was observed in pol pll1 seedlings (data not shown). Around the transition to flowering, when WUS becomes expressed in the many wild-type flower meristems, we observed occasional punctate spots of WUS expression in pol pll1 plants(Fig. 3A-F). These spots corresponded to expression in internal cell layers of what morphologically appeared to be nascent meristems (Fig. 3C,F). When compared to PWUS:GUS expression in wild-type plants, the spots in pol pll1 mutants appeared to correspond to transient apices forming in leaf axils. These results suggest that POL/PLL1 are required for the maintenance, but not the initiation, of WUS expression. Because data indicate that CLV signaling is also important for maintenance, but not initiation, of WUS expression,these results are consistent with the hypothesis that POL/PLL1 functions within the CLV pathway. If CLV signaling achieves repression of WUSthrough the inhibition of POL/PLL1 activity, one would expect constitutive inhibition of WUS in pol pll1 mutants after initiation.

PCLV3:GUS activity was detected in the initiating shoot apical meristem of pol pll1 embryos; however, the activity was weaker, compared with wild type, and was restricted to the epidermal layer(see Fig. S2 in the supplementary material). Post-embryonically, PCLV3:GUS behaved similarly to PWUS:GUS in wild-type plants and in pol pll1mutants, with punctate spots of PCLV3:GUS activity in pol pll1 mutants in apparent transient shoots(Fig. 3G-O). Similar to wild-type meristems, the PCLV3:GUS signal was largely within apical cells layers within these apparently transient meristems(Fig. 3L,O). Both reporter-gene expression patterns and morphology suggest that meristems are initiated but immediately lost in pol pll1 plants, consistent with hyper-repression of WUS after meristem initiation.

Ectopic WUS expression bypasses the requirement of POL/PLL1 for stem cells

To definitively test whether the loss of meristem activity in pol pll1 mutants was the consequence of the loss of WUS expression maintenance, we designed a transgene arrangement to determine if ectopic WUS expression could bypass the requirement for POL/PLL1. We set expression of WUS under the control of the flower-specific APETALA1 (AP1) cis regulatory elements in a transactivation system, in which AP1-driven WUS expression would only occur in the progeny of plants carrying both the PAP1:LHG4driver and the POP6:WUS responder(Hempel et al., 1998; Moore et al., 1998; Schoof et al., 2000). In wild-type plants with PAP1:LHG4/POP6:WUS (hereafter referred to as PAP1:WUS), flowers underwent extensive meristematic proliferation and eventual carpeloid organ formation(Fig. 4A). wus plants with PAP1:WUS exhibited defective vegetative development typical of wus mutants; however, upon flowering, these plants developed vigorous meristem activity in each flower, giving rise to meristem proliferation and carpeloid organ formation(Fig. 4D). An identical restoration of floral-meristem activity was observed when PAP1:WUS was introduced into pol pll1 grafted tissue, including extensive meristem proliferation and organogenesis(Fig. 4B,C). PAP1:WUS in pol pll1 tissue drove activation of PCLV3, indicating the meristem-like nature of the proliferations (Fig. 4E,F). PAP1:WUS did not drive activation of PWUS, suggesting that WUS is not under autoregulatory control (Fig. 4G,H). A similar restoration of meristem activity in pol pll1 mutants was observed when WUS expression was driven by the cis regulatory elements for the receptor-kinase ERECTA (ER)(Diévart et al., 2003; Yokoyama et al., 1998). Using the same transactivation transgene arrangement followed by grafting to generate PER:WUS pol pll1 tissue, we observed restoration of meristem proliferation that was less extensive than PAP1:WUS and gave rise to more normal floral organs(Fig. 4I,J). Thus, the loss of pol pll1 meristem activity is directly attributable to the loss of WUS expression maintenance, indicating that POL/PLL1 act through WUS to promote stem-cell identity.

Ectopic PLL1 expression blocks differentiation in clv mutants

If POL/PLL1 are indeed targeted for negative regulation by CLV signaling,one would predict that overexpressing POL/PLL1 would enhance clvmutants, providing de-repression of excess POL/PLL1. Given the extensive gene families for CLV1, CLV2 and CLV3(Botella et al., 1997; Sharma et al., 2003; Shiu and Bleecker, 2001), and evidence that clv1 and clv2 null alleles exhibit rather weak phenotypes (Diévart et al.,2003; Kayes and Clark,1998), it is unclear whether any clv single mutant represents a complete loss of signaling. We have previously shown that PLL1 overexpression in an otherwise wild-type background gives rise to weak Clv- phenotypes (Song and Clark, 2005). Here, we drove PLL1 expression under the control of both ER (PER) and the cauliflower mosaic virus (CaMV) 35S (P35S) cis regulatory elements in the clv2-1 and clv3-2 mutant backgrounds, and observed dramatic enhancement of the Clv- phenotype(Fig. 5). In many cases, the transgenic plants simply produced meristem tissue at the shoot apex, with a complete absence of organ formation over a long developmental window. Some plants senesced and died without producing organs, whereas others developed`escape' tissue, presumably as a result of transgene repression, that went on to produce a small number of organs after a sustained period without organogenesis (see Fig. S1 in the supplementary material). To determine whether the overproliferating tissue was stem cell-like in nature, we introduced PCLV3:GUS and PWUS:GUS(Gross-Hardt et al., 2002; Lenhard et al., 2002) into the P35S:PLL1 clv3-2 background(Fig. 5O,P). In both cases, we observed GUS activity throughout the apex, with the strongest PWUS:GUS signal in internal cell layers and the strongest PCLV3:GUS signal in the overlying cells. The presence and pattern of both meristem markers, as well as the morphology of these structures, indicate that these accumulated cells are equivalent to the central zone of a wild-type meristem, including stem cells in the top three cell layers. This data also indicate that clv mutants have a partial loss of differentiation that is further antagonized by the overexpression of PLL1. This is consistent with both a detailed analysis of clv mutants, as well as a recent study showing that inducible downregulation of CLV3 leads to the rapid expansion of the CLV3 expression domain (Reddy and Meyerowitz, 2005). Indeed, it would be of interest to repeat this sort of analysis in a PLL1 overexpression background, as P35S:PLL1 clv3 results in a more complete loss of differentiation than a clv3 single mutant.

Ectopic stem cells induced by PLL1 overexpression require WUS and suppress CLV3 overexpression effects

To determine whether the effect of PLL1 overexpression was entirely dependent upon WUS, we assessed the effect of the P35S:PLL1 transgene in the wus-1 mutant background. P35S:PLL1 wus plants were readily distinguishable from wus single mutants during vegetative development because PLL overexpression results in altered leaf morphology, namely smaller, rounder leaves (Song and Clark,2005). Beyond this difference in leaf development, P35S:PLL1 had no effect that we could detect on the terminated vegetative or inflorescence meristems observed in wusmutants (see Fig. S1 in the supplementary material). Similarly, within the flowers, P35S:PLL1 had no statistically measurable effect on the number of organs formed in wus flowers. Most importantly, no increase in the number of stamens or carpels per flower as a result of PLL1 overexpression was observed(Fig. 6). wus is thus fully epistatic to P35S:PLL1 within the meristem,consistent with the hypothesis that PLL1 acts within the meristem by regulating WUS.

Overexpression of CLV3 leads to shoot- and flower-meristem termination, resulting in plants phenotypically similar to wusmutants, albeit less severe (Brand et al.,2000). If CLV signaling acts to repress POL/PLL1 activity, then overexpression of PLL1 in the P35S:CLV3background would be predicted to suppress, at least partially, the meristem termination phenotype. Progeny from a cross between plants carrying the P35S:CLV3 transgene and plants carrying the P35S:PLL1 transgene were assayed for the shoot-meristem termination phenotype and genotyped for the presence of the two transgenes. Whereas every plant carrying P35S:CLV3 alone developed terminated shoot meristems, 80% of the plants carrying both transgenes developed normal shoot meristems (Table 1). Transgene expression analysis indicated that this was not the result of suppression of the P35S:CLV3 transgene (see Fig. S3 in the supplementary material).

Taken together, our analyses strongly suggest that POL/PLL1 are intermediates downstream of the CLV factors in the regulation WUSexpression and stem-cell differentiation. POL/PLL1 are required for stem-cell maintenance through their regulation of WUSexpression. POL/PLL1 overexpression blocks differentiation and drives stem-cell accumulation, especially in a clv mutant background. This would suggest that CLV signaling inhibits POL/PLL1 posttranscriptionally and that a combination of excess PLL1transcription and loss of CLV-mediated inhibition is sufficient to block stem-cell differentiation.

POL/PLL1 regulation of WUS appears to be on the level of transcription, as evidenced by the loss of WUS transcription in pol pll1 mutants, as well as by the ability to bypass POL/PLL1 by expressing WUS under a different set of cis regulatory elements. The initiation of WUS expression in transient shoots in pol pll1double mutants, as well as the slightly weaker phenotype of pol pll1double-mutant tissue in comparison with wus null alleles, reflects that POL/PLL1 regulation of WUS expression acts only after meristem initiation. Thus, POL/PLL1 are primarily regulators of the maintenance, not the initiation, of WUS expression. This is fully consistent with data on the function of the CLV pathway in regulating WUS expression.

It is formally possible that POL/PLL1 act independently on WUSrather than acting as intermediates of CLV signaling, but this alternative hypothesis is not supported by the data. First, pol pll1 mutants do express WUS, albeit only in transient shoots. If POL/PLL1 and CLV were acting separately, one would expect the removal of CLV signaling to expand WUS expression, and hence alter phenotypes, in pol pll1 mutants. Therefore, this alternative hypothesis would predict that clv should suppress, to some extent, pol pll1 double-mutant phenotypes. This is especially true in the flower meristem, where we see transient WUS and CLV3 expression in pol pll1, and these mutants exhibit flower-meristem defects that are slightly weaker than wus. Even here, there is no effect of removing CLV signaling, as evidence by the full epistasis of pol pll1 to clv mutants. In addition, there is an incredible level of dosage sensitivity between CLV, POL/PLL1 and WUS in both loss- and gain-of-function situations, suggesting a common pathway.

CLV repression of POL/PLL1 is likely to be post-transcriptional, based on both the broad expression of POL and PLL1 throughout the plant and within the meristem (Yu et al., 2003; Song and Clark, 2005), and on the interaction of clv mutants with PLL1 overexpression. This would be quite typical for receptor signaling intermediates.

Little is known about downstream targets of other receptor kinases. POL/PLL1 share similarity in general pathway function with the BIN2 kinase in BRI1 receptor kinase-mediated brassinosteroid signaling(Li and Nam, 2002). POL/PLL1 and BIN2 appear to be negatively regulated by the corresponding receptor kinase, and said repression allows for signaling to occur. In the case of BIN2 repression, brassinosteroid signaling occurs as the result of the loss of BIN2-mediated phosphorylation and subsequent degradation of a set of transcription factors (Wang et al.,2002; Yin et al.,2002; Zhao et al.,2002). As with BIN2, the exact link between the POL/PLL1 and the upstream receptor kinase remains unclear. Whether CLV1 directly modulates POL/PLL1 activity and what are the targets of POL/PLL1 phosphatase activity are crucial issues to pursue.

pol pll1 mutants have pleiotropic phenotypes, with evidence for POL/PLL1 regulation of basal embryo development, of pedicel development and of leaf vascular patterning (Song and Clark,2005). These activities may represent the function of POL/PLL1 downstream of other receptor kinases. The redundant nature of many receptor kinases may explain why the corresponding receptors have not yet been identified (Cano-Delgado et al.,2004; DeYoung et al.,2006; Shpak et al.,2004; Zhou et al.,2004).

It is of interest to consider that the CLV1-related BAM receptors in Arabidopsis may also utilize POL and related proteins as signaling intermediates. bam mutant combinations exhibit many developmental defects, including effects on leaf vascular patterning similar to those that result from the overexpression of PLL1(DeYoung et al., 2006; Song and Clark, 2005), raising the possibility that BAM receptors may regulate leaf development by signaling through POL/PLL1 or related phosphatases. The observation that CLV1 can replace BAM function during leaf, stem and floral-organ development suggest a common signaling pathway in each of these different tissues(DeYoung et al., 2006). Given the large receptor-kinase gene family in Arabidopsis, it will be crucial to test the association of specific receptors with potential downstream intermediates.

This work was supported by a grant from the National Institute of Health(R01GM62962) to S.E.C.; and, in part, by a grant from the Plant Signaling Network Research Center (R11-2003-008-03004-0) to M.M.L.

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