PerspectiveDevelopmental Biology

Intercellular Peptide Signals Regulate Plant Meristematic Cell Fate Decisions

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Sci. Signal.  09 Dec 2008:
Vol. 1, Issue 49, pp. pe53
DOI: 10.1126/scisignal.149pe53

Abstract

Plant stem cells secrete peptides that, after processing to release the active form, prevent neighboring cells from adopting a stem cell fate by activating a leucine-rich repeat (LRR) receptor–mediated pathway. Other plant meristematic cell fate decisions, such as those made during the patterning of veins and stomata, also appear to be controlled by similar LRR receptor pathways that are activated by secreted peptide signals. It is therefore probable that peptide ligands regulate meristematic activity in many plant developmental processes.

Stem cells, found in the surface layers at the center of the plant shoot apical meristem (SAM), divide to produce daughter cells with two possible fates: They can either remain in the stem cell niche and retain stem cell identity, or exit to become progenitor cells for organogenesis. The fate of these cells is determined by positional cues rather than by cell lineage. The population of SAM pluripotent stem cells is specified and restricted in number by mechanisms that are among the best-characterized plant developmental signaling pathways, largely because of studies of the Arabidopsis clavata mutants clv1, clv2, and clv3, which have enlarged shoot apical and floral meristems and excessive numbers of stem cells (13). CLV3 is a small secretory peptide present in the cells of the stem cell niche (4); however, stem cell identity is determined by signals from a group of underlying cells, called the organizing center (OC). OC cells express the homeodomain transcription factor encoded by WUSCHEL (WUS) that acts non–cell-autonomously to promote stem cell fate in the overlying cells, which in turn express CLV3 (5). CLV1, a leucine-rich repeat (LRR) domain receptor–like kinase (LRR-RLK), and CLV2, a related LRR receptor–like protein lacking a kinase domain, are both present in the cells underlying the stem cell niche, including the OC.

The CLV1 ectoplasmic domain of the proposed heterodimeric transmembrane CLV1-CLV2 receptor complex binds the extracellular CLV3 ligand (6), which signals to prevent unrestricted stem cell proliferation by repressing WUS transcription (Fig. 1). This feedback loop between the stem cells and the OC is mediated by opposing WUS and CLV3 signals and limits the size of the meristem while allowing the number of cells in the stem cell domain to remain stable (5). A Rho guanosine triphosphatase (GTPase)–related protein acts downstream of CLV1-CLV2 receptor activation (7), which, by analogy to other LRR receptor–mediated pathways, is thought to activate a mitogen-activated protein kinase (MAPK) cascade (8). The protein phosphatases POLTERGEIST (POL) and PLL1 act downstream of CLV components to promote WUS expression (9), but specific MAPK cascade components are yet to be identified.

Fig. 1

The CLV signaling pathway that restricts stem cell proliferation at the shoot apical meristem (A) has similarities to the mechanism that regulates meristemoid identity during stomatal patterning in Arabidopsis (B). Both potentially involve the secretion of a small peptide signal (red) from the meristematic cell (pale blue) and its activation by proteolytic processing (scissor symbols). The activated peptide signal is believed to be perceived in neighboring cells (yellow) in both situations by a LRR receptor complex, comprising an LRR-RLK (yellow) and a LRR receptor–like protein (pink), linked to downstream phosphorylation and dephosphorylation events. Arrowheads indicate activation; T-bars indicate inhibitory interactions.

CLV3 is a member of a family of small secretory peptides (more than 30 in Arabidopsis) that are exclusive to plants and share a conserved CLE (CLAVATA/ESR) motif close to their C terminus (10). Many of the CLE peptides are able to regulate the size of the shoot or the root apical meristem, or both, when misexpressed (1113). CLV3 is processed in vivo to release a 12–amino acid fragment called MCLV3 that encompasses most of the CLE motif and contains two hydroxylated proline residues, although activity is not dependent on hydroxylation (11). It is not known how CLE proteins are processed to release the active fragment, although a carboxypeptidase A has been implicated in the activation of CLE19 (14).

CLE peptides have roles beyond the stem cell niche and have emerged as regulators of plant vascular development (12). The vasculature consists of specialized conducting cells of the phloem and xylem, which includes tracheary elements (TEs), and the meristematic procambium, which provides the stem cell pool. Tracheary element differentiation inhibitory factor (TDIF), which inhibits TE differentiation from procambial cells while promoting cell division, was purified from an extracellular fraction of Zinnia elegans cultures. Like MCLV3, TDIF is a dodecapeptide with two hydroxyproline residues; it is predicted to be processed from a larger peptide precursor and is identical to the C-terminal dodecapeptides predicted for Arabidopsis CLE41 and CLE44 (12). All the predicted Arabidopsis CLE dodecapeptides were tested for their ability to affect TE differentiation or root growth. Only CLE41, CLE44, and CLE42 mimicked the effects of TDIF and, unlike most CLE peptides, did not inhibit root growth; these findings suggest that they have specific roles in TE differentiation. CLV3 was found to promote TE differentiation, which suggests that CLE signaling may have a dual function, with some CLE peptides promoting and others inhibiting stem cell differentiation.

No LRR-RLKs have been shown to act as receptors for CLE peptides in vascular tissue, but candidates with the expected expression pattern have been identified. In Arabidopsis, the putative LRR-RLKs VASCULAR HIGHWAY1 (VH1) and PHLOEM INTERCALATED WITH XYLEM (PXY) are required for differentiation events in vascular development (15) and to regulate the orientation of meristematic divisions to produce correct xylem and phloem positioning (16). CLE peptides are not the only secreted peptides implicated in vascular differentiation. Phytosulfokine-α, a disulfated pentapeptide, also induces differentiation of TEs (17, 18) in a pathway believed to be mediated by a LRR-RLK (19).

A putative secreted peptide signal, unrelated to the CLE or phytosulfokine peptides, regulates the patterning of stomata, pores in the leaf epidermis that are surrounded by two guard cells (20). Stomata are formed early in leaf development from meristemoids, which are stem cell–like precursor cells but are dispersed and have only transient stem cell activity, unlike SAM stem cells. Like stem cells at the meristem, the progeny of Arabidopsis meristemoids have two possible fates. They may retain meristematic activity and subsequently give rise to guard cells, or they may differentiate into epidermal pavement cells. As stomata are normally inhibited from developing adjacent to one another, meristemoid fate must be inhibited in the neighboring cells of stomatal precursors in a way that is perhaps analogous to the inhibition of stem cell identity in cells bordering the SAM stem cell niche. In Arabidopsis, at least, this is primarily achieved by the orientation of subsequent cell divisions or developmental arrest of adjacent meristemoids (21). A stomatal spacing mechanism by lateral inhibition was first suggested 60 years ago (22), but an inhibitory (and division-orienting) signal was not reported until 2007. The recently discovered inhibitory signal, which prevents Arabidopsis stomata from forming adjacent to one another, is the putative secreted peptide EPIDERMAL PATTERNING FACTOR 1 (EPF1) (20).

In combination with previously identified factors, the identification of EPF1 as a regulator of meristemoid fate indicates that the mechanisms regulating stomatal development share a striking similarity to the CLE signaling pathways. Among the first stomatal development mutants to be reported were stomatal density and distribution1 (sdd1) and too many mouths (tmm), which, like epf1 mutants, are characterized by pairs or clusters of stomata in the leaf epidermis (20, 23, 24). SDD1 and EPF1 are predominantly expressed in stomatal precursor cells, but TMM is expressed in both stomatal precursors and their neighboring daughter cells (20, 25, 26). TMM is an LRR receptor–like protein that lacks a kinase domain and, like CLV2, is likely to form a heterodimeric complex with a receptor kinase. The most likely LRR-RLK partners for TMM are the ERECTA family of proteins (ER, ERL1, and ERL2) that mediate a range of Arabidopsis developmental events, including stomatal patterning. Genetic analysis suggests that the ERECTA family proteins act in the same pathway as TMM and EPF1 (20, 27). Thus, EPF1 secreted from meristemoids may be a ligand for the TMM-ERECTA receptor complex, orienting cell division and inhibiting meristemoid identity in neighboring cells (Fig. 1), but ligand binding has yet to be demonstrated. SDD1 is a putative subtilisin-like protease that also acts in the TMM receptor–mediated pathway (23, 25). Subtilisins process peptide hormones, and it might be expected that SDD1 cleaves and activates EPF1. However, although SDD1 and EPF1 both act in the TMM pathway, they do so independently (20).

Therefore, other peptide regulators of stomatal patterning may exist that are processed by SDD1. Downstream of TMM receptor activation is a MAPK cascade consisting of YODA, putative MAPK kinase kinase, MAPK kinases, and MAPKs (28, 29). Basic helix-loop-helix (bHLH) transcription factors such as SPEECHLESS (SPCH) also act in the TMM pathway, but as promoters rather than inhibitors of meristemoid fate (30). SPCH heterodimerizes with the bHLH proteins ICE1 and SCREAM2, and these interactions are believed to contribute to its function in meristemoids (31). Whether these transcription factors act upstream or downstream of the EPF1 signal, or as part of a feedback loop, is not yet clear.

In addition to the CLE, EPF1, and phytosulfokine (PSK) peptides, other small peptides act as secreted signals in plants (32). These include rapid alkalinization factors (RALFs), which inhibit root growth and development through a MAPK pathway (33). AtPSK4 is a target for subtilisin processing, and AtRALF1 is cleaved by an unidentified protease (34, 35). EPF1 also has a putative subtilisin dibasic residue processing site, but a proteolytically cleaved fragment has not yet been identified.

The role of secreted peptide hormones is well established in animal systems, and plant genome sequences suggest that they may be at least as abundant in plants (32). Although multicellularity evolved separately in plants and animals, it appears that families of plant peptides have been recruited to fulfill similar functions, such as those of the WNT family of secreted signaling proteins that in animals regulate a diverse range of developmental processes, including stem cell maintenance and proliferation (36). It appears, however, that in plants, peptide signals are more likely to be perceived by LRR receptor complexes with serine/threonine kinase activity than by G protein–coupled receptors or receptor tyrosine kinases. Currently, we have insight into how only a handful of the hundreds of predicted secreted peptide ligands, their activating proteases, and LRR-RLK complexes interact to control plant cell fate and development. Over the next few years, as more plant receptor-ligand interactions are characterized, we shall gain a clearer picture of the complexity of plant peptide cell-to-cell signals.

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