Is the Arabidopsis root niche protected by sequestration of the CLE40 signal by its putative receptor ACR4?

Abstract

A tight but also dynamic regulation is necessary to control the size of stem cell populations in response to internal and external cues. The stem cells of the Arabidopsis shoot and root meristems are governed by the niche cells of the organizing centre (OC) and the quiescent centre (QC), respectively. The well characterized CLV3/WUS negative feedback loop adjusts homeostasis of the stem cell population in the shoot. Here, the CLAVATA3 (CLV3) dodecapeptide, expressed by the stem cells, signals to repress WUSCHEL (WUS), which is expressed in the subjacent OC cells, and in turn activates CLV3 expression non-cell autonomously. However, a similar signaling module controlling the root stem cell population was as yet unknown. In the June issue of Current Biology we report on such a signaling module comprising CLE40 (a CLV3 homologue) that acts via the receptor kinase Arabidopsis Crinkly4 (ACR4) to repress the WUS homologue WOX5 which maintains distal root stem cells. Furthermore, we showed that CLE40 peptide (CLE40p) treatment upregulates ACR4 expression. In this Addendum, we are further elaborating our hypothesis in which the upregulation of ACR4 as a consequence of ectopic CLE40p builds a protective barrier for the QC niche cells.

Intercellular signaling processes mediated by small peptide ligands play important roles in plant development. The stem cells in the Arabidopsis shoot apical meristem express and secrete the CLV3 dodecapeptide and this signal is transmitted by the membrane localized leucine rich repeat (LRR) receptor kinase CLAVATA1 (CLV1), and independently by CORYNE (CRN) and CLAVATA2 (CLV2) to the subjacent niche cells of the OC. This signaling eventually leads to the downregulation of the homeodomain transcription factor WUS which is expressed in the OC. WUS in turn promotes stem cell fate, and thereby also CLV3 expression, in the overlying stem cells thus leading to a negative feedback loop adjusting stem cell homeostasis in the shoot.^1–4 In Arabidopsis, 31 members of the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) family, carrying a conserved 14 aa C-terminal CLE motif, are known.⁵ It was shown in previous studies that overexpression of CLE genes or addition of synthetic peptides can activate CLE-dependent signaling pathways and it was proposed that they could act through CLV-related LRR receptor-like kinases. Two classes of peptides are distinguishable by their effect on the root and shoot meristems. Class A peptides, e.g., CLV3p, CLE19p and CLE40p, promote cell differentiation resulting in reduced root meristem size and a short root phenotype.^6–9 Class B peptides, e.g., CLE41 (TDIF) do not reduce root size, but instead promote proliferation of vascular cells.⁹

The WUS homologue WUSCHEL-RELATED HOMEOBOX5 (WOX5) is expressed in the QC of the Arabidopsis root, which represents the equivalent to the OC of the shoot apical meristem harboring the niche cells of the root.¹⁰ The stem cells of the root, the so called initial cells, are surrounding the QC and give rise to all the different root tissues (labeled in Fig. 1). In wox5 mutants the columella stem cells (CSCs) differentiate which is visible by their accumulation of starch granules. WOX5 was shown to be necessary and sufficient for distal (towards the root tip) root stem cell maintenance. WUS and WOX5 can functionally replace each other if expressed in the appropriate domains, demonstrating their close homology.¹⁰

Figure 1.

Model of CLE40 sequestration in the distal root meristem by its receptor ACR4 allows WOX5 expression in the QC and thereby confers stem cell fate in the surrounding cells. Schematic representation of the Arabidopsis root meristem. Color codes indicate mRNA and protein abundance of WoX5 (red), ACR4 (green and light green) and CLE40 (blue). Arrows indicate CLE40p movement into surrounding cells. The stem cells surrounding the QC are outlined by a grey line. DM, distal meristem; PM, proximal meristem; QC, quiescent centre; CSC, columella stem cells; CC, columella cells; LRC, lateral root cap; Ep, epidermis; C, cortex; EN, endodermis.

Mutants in the receptor-like kinase ACR4 were recently shown to carry additional layers of columella stem cells (CSCs), arguing for a negative regulation of distal root stem cell fate by ACR4.¹¹

CLE40 is the closest homolog of CLV3 and was shown to be able to substitute CLV3 in the shoot; cle40 mutants were previously described to display a short root phenotype.¹²

In our recent publication,¹³ we describe two new mutant alleles of cle40 focusing on their distal meristem phenotypes. We show that CLE40 expression starts during embryogenesis and gets gradually confined to the future distal root meristem and the stele. cle40 mutants show, like acr4 mutants, additional CSCs. Addition of the synthetic CLE40p or the related CLV3p (but not TDIFp), can rescue this phenotype in cle40 mutants, but importantly not in acr4 mutants. Furthermore, in cle40 and acr4 mutant roots the WOX5 expression domains are equally enlarged laterally. Therefore CLE40 can negatively regulate WOX5 expression, resembling the CLV3/WUS feedback regulation in the shoot; however, the CLE40 signal in the root originates from differentiated instead of stem cells.

In light of these results we suggest ACR4 (a member of the CRINKLY4 receptor-like kinase family) as the receptor for the CLE40 signal. We propose a signaling module with CLE40 as a secreted peptide ligand originating from differentiated root cells to act via the ACR4 receptor to regulate WOX5 expression levels and it's expression domain in order to maintain distal root stem cells.

Addition of synthetic CLE40 or CLV3 peptides promotes differentiation of CSC cells towards CC cells (observable by starch accumulation in CSC cells). Intriguingly, the expression of WOX5 in the QC position is gradually lost and WOX5 expression emerges instead at more proximal positions.

In the root meristem ACR4 is expressed, as visualized by a pACR4:ACR4-GFP reporter line,¹⁴ mainly in the CSCs and at much lower levels in the CC cells, the root cap and QC in between the CLE40 and WOX5 expressing cells of the columella and QC cells, respectively. ACR4 expression levels are strongly upregulated after CLE40p or CLV3p treatment (but not after TDIFp treatment) and the ACR4 expression now systematically includes the QC position. This parallels the proximal displacement of the WOX5 expression domain upon CLE40p or CLV3p treatment. These observations indicate a dual role for ACR4 so that the receptor is not only involved in transmitting the CLE40 signal but is also positively regulated by its peptide ligand. Notably, the WOX5 expression is still proximally adjacent to the ACR4 expression now in QC position; therefore CLE40 is also able to control the proximo-distal axis of WOX5 expression.

The adjacent and partly overlapping expression domains of CLE40, ACR4 and WOX5 (see Fig. 1) and their changes upon CLE40p or CLV3p treatment lead us to the hypothesis that by upregulation of the receptor ACR4 the plant seeks to protect the WOX5 expression in the niche cells of the QC thereby maintaining the surrounding stem cells. CLE40 is expressed in the differentiated columella cells of the distal root meristem and overlaps there with weak ACR4 expression. As CLE40p is presumably secreted to the extracellular space it would be capable of spreading into the surrounding cells. The range of the intercellular movement of the CLE40 signal could be restricted via ligand sequestration by the ACR4 receptor as is also thought for CLV3 and CLV1 in the shoot. A protective barrier of ACR4 expressing CSCs could act to dynamically buffer alterations in ligand abundance during changes in requirement of differentiated cells in different developmental stages of the plant. ACR4 would not only relay the CLE40 signal into the receiving cells to restrict WOX5 expression but also sequester its own ligand to protect the QC from too much CLE40p; which in turn would allow WOX5 expression in the QC to maintain the surrounding stem cells (see Fig. 1).

Future analyses of ACR4 and CLE40 expression and localization dynamics might give further insight into this sequestration model.

Addendum to: Stahl Y, Wink RH, Ingram GC, Simon R. A signaling module controlling the stem cell niche in Arabidopsis root meristems. Curr Biol. 2009;19:909–914. doi: 10.1016/j.cub.2009.03.060.