Plant meristems: CLAVATA3/ESR-related signaling in the shoot apical ...

J Plant Res (2009) 122:31–39 DOI 10.1007/s10265-008-0207-3

CURRENT TOPICS IN PLANT RESEARCH

Plant meristems: CLAVATA3/ESR-related signaling in the shoot apical meristem and the root apical meristem Hiroki Miwa Æ Atsuko Kinoshita Æ Hiroo Fukuda Æ Shinichiro Sawa

Received: 6 November 2008 / Accepted: 27 November 2008 / Published online: 23 December 2008 Ó The Botanical Society of Japan and Springer 2008

Abstract The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species.

S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007. H. Miwa A. Kinoshita H. Fukuda S. Sawa (&) Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan e-mail: [email protected]

Keywords CLAVATA3/ESR (CLE) CLAVATA (CLV) Leucine-rich repeat-receptor like kinase (LRR-RLK) Meristem Root apical meristem (RAM) Shoot apical meristem (SAM)

Introduction The term meristem was first defined by Carl Wilhelm von Na¨geli in his book ‘‘Beitra¨ge zur Wissenschaftlichen Botanik’’ (von Na¨geli 1858). Meristem is a formative plant tissue made up of cells capable of dividing and giving rise to new cells (Scofield and Murray 2006). Because of their essential role in higher plants, meristems have received much attention from plant scientists for more than 150 years. The shoot apical meristem (SAM) and the root apical meristem (RAM) are known to be two important meristems that provide cells for postembryonic growth and development. The SAM is a collection of cells that continuously renew themselves by cell division and provide cells to new organs. Although continuous cell division and differentiation of their daughters are observed in meristematic regions, the meristem size and the number of stem cells are kept constant. These observations suggest that stem cell maintenance and new organ formation are well balanced. Recently, it has been shown that intercellular communication controls the organization and maintenance of the SAM, as well as cell-fate specification. Thus, stem cell maintenance requires the well-coordinated regulation of different signals for meristem homeostasis. SAM maintenance is regulated by two main signaling pathways: the WUSCHEL (WUS) pathway and the SHOOT MERISTEMLESS [STM; KN1-related homeobox (KNOX) protein] pathway (Lenhard et al. 2002). The WUS signaling

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Fig. 1 Models of structures and signaling cascade of SAM and c RAM. a Structure of SAM and the expression of regulatory genes. The SAM is divided into three zones, the peripheral zone (PZ), the central zone (CZ) and the rib zone (RZ), and three layers, L1, L2 and L3. CLV3 is expressed in the stem cell (yellow) whereas WUS is expressed in the organizing center (OC, red). The coordinated SAM structure is regulated by WUS–CLV3 negative feedback loop. b Molecular components involved in SAM maintenance. CLV3 precursor is maturated and secreted to the apoplastic space, then received by sets of receptors, CLV1, CLV2, and SOL2/CRN. The receptors transmit the signal to regulate WUS expression. Exogenously applied synthetic peptides presumably act through the same pathway. c Structure of the RAM. The quiescent center (QC, red) is surrounded by stem cells (yellow), and meristematic cells (blue) are located in the upper area. WUSCHEL-related homeobox 5 (WOX5) is expressed in the QC cells (inbox). The region of expression of CLE genes in the RAM remains to be found

pathway is mediated by the intercellular signal of the CLAVATA3/ESR (CLE) peptide and perception by leucine-rich repeat (LRR) receptor kinases. In this article we overview SAM organization and maintenance by focusing on CLE peptides, cognate LRR receptor-like kinases and the WUS-related molecules, together with the similar machinery operated in the RAM. We also review the conservation of the CLAVATA–WUSCHEL (CLV–WUS) signaling network in various plants.

CLV function in SAM The anatomy of the SAM is defined in terms of layers and radial zones (Kwiatkowska 2008). The SAM can be divided into three layers based on cell fate. The L1 layer cells divide parallel to the surface and give rise to the epidermis. The L2 layer gives rise to mesophyll cells, and the L3 layers give rise to the central tissues of the leaf and stem (Fig. 1a). The organization of the SAM is also divided into three areas: the peripheral zone (PZ), the central zone (CZ), and the rib zone (RZ) (Fig. 1a). The activity of cell division in the CZ is low, whereas cells in the PZ divide actively and provide cells that are needed for growth and differentiation of lateral organs. During the past two decades, genetic analyses of a variety of Arabidopsis thaliana (L.) Heynh. mutants have identified a number of molecular components involved in SAM maintenance. Among these, clv1, clv2, and clv3 are the loss-of-function mutants that exhibit a remarkable phenotype of shoot fasciation, enlarged floral meristems, and increased numbers of floral organs. Two of these mutants have mutations in membrane-associated receptorlike proteins that have LRRs in their predicted extracellular domain; CLV1 encodes an LRR-receptor like kinase (LRRRLK), and CLV2 encodes an LRR-receptor like protein lacking a kinase domain (Clark et al. 1997; Jeong et al.

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1999; Fig. 1b; Table 1). CLV3 encodes a 96-amino acid protein with a putative secretory signal peptide sequence in its N-terminal region (Fletcher et al. 1999).

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Table 1 Receptor-like kinases which function in meristem maintenance (FM floral meristem) Receptor-like kinase

Accession no.

Classification

Primary expression

Biological function

Reference

CLV1

At1g75820

Class XI LRR-RLK

SAM

Promotion of differentiation at SAM and FM

Clark et al. (1997)

CLV2

At1g65380

LRR-RLP

Whole tissue


Jeong et al. (1999)

SOL2/CRN

At5g13290

RLK

Whole tissue


Miwa et al. (2008) and Mu¨ller et al. (2008)

BAM1

At5g65700

Class XI LRR-RLK

SAM, root

Inhibition of stem cell differentiation at SAM

DeYoung et al. (2006)

BAM2

At3g49670

Class XI LRR-RLK

SAM, root



BAM3

At4g20270

Class XI LRR-RLK

Stele



ERECTA

At2g26330

Class XIII LRR-RLK

SAM

Shoot organ growth

Torii et al. (1996)

OsFON1

AB182389

Class XI LRR-RLK

SAM and FM

Promotion of differentiation at FM

Suzaki et al. (2004)

Class XI LRR-RLK

Vegetative seedling apex


Bommert et al. (2005)

ZmTD1 LjHAR1

AB092810

Class XI LRR-RLK

Whole tissue

Regulation of nodule numbers

Nishimura et al. (2002) and Krusell et al. (2002)

GmNARK

AY166655

Class XI LRR-RLK

Whole tissue


Searle et al. (2003)

PsSYM29

AJ495759

Class XI LRR-RLK


Krusell et al. (2002)

MtSUNN

AY769943

Class XI LRR-RLK

Whole tissue


Schnabel et al. (2005)

ZmFEA2

AY055124

LRR-RLP

Vegetative seedling apex


Taguchi-Shiobara et al. (2001)

Although it is believed that CLV3, as a small ligand, binds to the putative CLV1/CLV2 receptor complex to regulate stem cell identity in the SAM, the molecular characteristics of these proteins have long been unknown (Fig. 1b). Recently, biochemical studies provided key findings for the CLV signaling molecules (Kondo et al. 2006; Sawa et al. 2006; Ogawa et al. 2008). In situ matrixassisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS) analysis identified a mature form of CLV3 (MCLV3) as a 12-amino acid peptide with two hydroxy prolines (RTVPhSGPhDPLHH). The 12-amino acid sequence is located near the C-terminal end of CLV3, and an application of the chemically synthesized dodecapeptide results in the CLV3 overexpression-like phenotype, suggesting that this peptide regulates meristematic identity in both the SAM and the RAM (Kondo et al. 2006). Ogawa et al. (2008) detected biochemical interactions between the LRR domain of CLV1 and MCLV3 using membrane fraction of tobacco BY-2 cells expressing CLV1 that had an epitope tag instead of the kinase domain (CLV1-DKD-HT). This was the first molecular evidence that CLV3 and CLV1 function as a ligand-receptor pair in

the plant stem cell maintenance system. The interaction between CLV3 and CLV2 still remains to be assessed. CLV1 is a member of the large gene family, the LRRRLK family (Shiu and Bleecker 2001; Table 1). Since the CLV1 is categorized in class XI, in accordance with the distinctive LRRs in their putative extracellular domain, members of the CLV1 group might be the receptors for CLV3 homologs.

Functional CLE peptide in SAM and RAM Database searches using the CLV3 entire sequence yielded only a poor match with the maize (Zea mays) embryo-surrounding region (ESR) proteins in 14 amino acids. However, when this 14-amino acid sequence was used for database queries, a total of 31 related sequences were identified in the Arabidopsis genome (Cock and McCormick 2001; Sharma et al. 2003; Ito et al. 2006; Strabala et al. 2006). These genes were named CLE genes, and the conserved 14-amino acid sequence was designated as the CLE domain.

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The CLE family encodes small proteins characterized by the conserved CLE domain at the C-terminal region and a hydrophobic signal peptide at the N-terminal region. Most CLE genes are transcribed in multiple tissues during development (Sharma et al. 2003; Strabala et al. 2006), suggesting that these proteins may function as signaling molecules in various aspects of morphogenesis. However, no loss-offunction cle mutant other than clv3 has been reported to date. Tracheary element (TE) differentiation inhibitory factor (TDIF) has been isolated as an inhibitor of TE differentiation in a Zinnia elegans L. xylogenic culture system (Fukuda 1997) and is known to function as a dodecapeptide with two hydroxyproline residues (HEVPhSGPhNPISN) (Ito et al. 2006). Interestingly, the active form of TDIF corresponds to a functional CLV3 peptide; both are dodecapeptides derived from the CLE domain, and the fourth and seventh prolines are hydroxylated. This suggests that a typical length and amino acid modification are conserved in some CLE proteins. So far, TDIF and CLV3 are the only CLE proteins that show a specific function. Although the function of CLE proteins is mostly unclear, gain-of-function analyses help us to speculate on their function. Strabala et al. (2006) examined overexpression phenotypes of CLV3 and 17 CLE genes and identified ten CLE genes that arrest SAM growth and seven CLE genes that inhibit root growth. Additionally, synthetic peptide treatment assays using conserved 12amino acid sequences of 26 CLE peptides, corresponding to putative mature forms of the 31 Arabidopsis CLE gene products, revealed that ten CLE peptides, including CLV3, have a strong effect on the SAM, and 19 CLE peptides, including CLV3, regulate the RAM identity (Kinoshita et al. 2007; Fig. 1b, c). These data suggest that CLE proteins are, in part, redundant in the SAM and RAM. Because the clv3 mutant exhibits no phenotype in the RAM (Clark et al. 1995), it is possible that other CLE genes are expressed in the RAM and redundantly regulate its stem cell identity. Post-translational regulation of CLE peptide is suggested to be important for CLE function. For example, overexpression of the CLE1 gene induces short root phenotypes (Strabala et al. 2006), whereas application of a synthetic CLE1 peptide does not induce any visible phenotypes (Kinoshita et al. 2007), suggesting that posttranslational modification of the CLE protein may be essential for CLE1 function in plants.

Receptor kinases that act in the SAM and RAM Besides CLV1 and CLV2, the receptor-like kinases that function in SAM maintenance in Arabidopsis are CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (SOL2), BARELY

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ANY MERISTEM 1 (BAM1), BAM2, BAM3, and ERECTA (Table 1). The sol2 mutant was isolated as a suppressor of the CLE19 overexpression phenotype in RAM development (Casamitjana-Martı´nez et al. 2003), and crn was identified as a suppressor of the CLV3 overexpression phenotype in SAM development (Mu¨ller et al. 2008). Molecular genetic studies on SOL2 and CRN showed that both SOL2 and CRN encode the same receptor-like kinase with a short extracellular domain (Miwa et al. 2008; Mu¨ller et al. 2008). In shoot development, the crn/sol2 mutant shows an enlarged SAM and is defective in floral organ development. This phenotype is similar to that of clv mutants, and, therefore, the action of CRN/SOL2 is implicated in the same signaling pathway of CLV–WUS. Genetic studies have revealed that wus is epistatic with crn/sol2, suggesting that CRN/SOL2 acts upstream of WUS (Mu¨ller et al. 2008). Spatial expression analysis by mRNA in situ hybridization revealed that the CLV3 and WUS expression domains were expanded in the crn/sol2 mutant. The expression of CLV3 and WUS also expanded in the clv mutants, in comparison with that in the wild-type. Therefore, it is postulated that CLV and CRN/SOL2 act closely to repress the WUS expression in the SAM. When carpel number is used as an indicator of the CLV signaling activity, the clv1 and crn mutations have an additive effect on carpel number, but the clv2 crn double mutant has carpel number similar to each single mutant. This suggests that CRN and CLV2 act together but independently of CLV1. Taken together, these results suggest that CRN/SOL2 mediates CLV signaling, together with CLV2, and balances cell proliferation and differentiation in the SAM in parallel with CLV1 (Mu¨ller et al. 2008). The function of CRN/SOL2 and CLV2 in RAM maintenance has been implicated by the application of synthetic 12-amino acid peptides on seedlings. In a root growth inhibition assay with the 26 CLE peptides, sol2 showed different levels of resistance to the various peptides, and the spectrum of peptide resistance was quite similar to that of clv2 (Miwa et al. 2008). RAM consumption caused by various CLE peptides depends mostly on CLV2 and CRN/SOL2, indicating that these two receptor proteins work together for the same CLE signaling pathway in the RAM. The effect of the CLE peptides on root growth is not affected by the clv1 mutations. This suggests that CLV1 is not involved in the CLE signaling pathway in roots or gene(s) with functions redundant to CLV1 operating in the roots (Miwa et al. 2008). Structurally, the CRN/SOL2 protein has a cytoplasmic kinase domain and a short extracellular domain, whereas CLV2 has an extracellular LRR domain and a short intracellular domain. One explanation is that CRN/ SOL2 and CLV2 act cooperatively in the same receptor

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complex, and that the kinase domain of CRN/SOL2 complements the lack of an intracellular domain in CLV2. Phylogenetic analysis identified BAM1, BAM2, and BAM3 receptor kinases, which are classified into the same monophyletic group with CLV1 (DeYoung et al. 2006; Table 1). Although none of these single mutants shows a phenotype, the bam1 bam2 double mutants and the bam1 bam2 bam3 triple mutants show reductions in shoot meristem size. This phenotype is opposite to that of the clv1 mutant, suggesting the opposite role of BAM proteins in stem cell regulation (DeYoung et al. 2006). On the other hand, weak phenotypes of clv1 null alleles were enhanced by the bam mutations, resulting in enlarged SAM formation (DeYoung and Clark 2008). Taking these findings together, the authors suggested that the BAM receptors function to sequester unknown CLE peptide ligands produced in the PZ and that the effects of BAM1 and BAM2 on stem cell homeostasis are integral to the CLV signaling pathway and do not represent a separate pathway regulating meristem development (DeYoung and Clark 2008). ERECTA encodes an LRR-RLK, which functions in SAM development (Torii et al. 1996). The er mutants are defective in shoot organ development, showing a phenotype of short and thick inflorescence stem and increased numbers of flower buds, and blunt, short, and wider siliques. When clv1 alleles are crossed to other Arabidopsis accessions, enhancement of the clv1 phenotype is observed in the progeny crossed by Ler accession. This suggests functional overlap between CLV1 and ER in SAM homeostasis (Die´vart et al. 2003). In the SAM, some RLKs have been shown to function differently to regulate meristem maintenance, but the molecular mechanism for how each receptor-like kinase integrates the CLE signals remains to be solved. Although no obvious root phenotypes have been identified in these receptor-like kinase mutants, this does not rule out a potential role for these genes in regulating root meristem function. Root phenotype might be too subtle to be detected, or other LRR-RLK proteins might function in the RAM redundantly. A genome-wide collection of receptorlike protein gene transferred DNA (T-DNA) insertion mutants has been reported (Wang et al. 2008), and this collection would be helpful for further analyses to determine the roles of receptor genes in root development.

WUS and WOX5 as master regulators in the SAM and RAM WUS is a member of the WUSCHEL-related homeobox (WOX) family homeodomain transcription factor and acts as a master regulator to specify stem cell identity (Fig. 1a, b; Laux et al. 1996; Mayer et al. 1998). One important role

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of WUS in the regulation of stem cells is to activate CLV3 in a non-cell-autonomous manner through unknown intercellular signaling factors (Lenhard and Laux 2003). CLV3 is secreted from the L1 and L2 layers to suppress WUS expression non-cell-autonomously (Fig. 1a, b), giving rise to a negative feedback loop. This feedback regulation in SAM maintenance allows the SAM to balance stem cell division in the central zone and cell differentiation in the peripheral zone (Schoof et al. 2000). When WUS is expressed ectopically in roots, leaves are developed in the RAM region, suggesting that WUS is sufficient to redirect root cells to other developmental pathways such as leaf cells (Gallois et al. 2004). The regulation system for stem cell maintenance seems partly conserved in shoot and roots. The WUS-expressing organization center cells in the SAM correspond to the quiescent center (QC) cells in roots. The QC cells, surrounded by stem cells, express the WUS homolog, WOX5 (Fig. 1c). A loss-of-function mutation in the WOX5 gene causes terminal differentiation, with enlarged cells at the QC and in columella stem cells. Conversely, WOX5 overexpression causes repression of differentiation in the columella cells and overproduces the columella initial cells. When WOX5 is expressed under the WUS promoter in the wus mutant, phenotypes in an indeterminate inflorescence meristem are restored. In contrast, WUS expression under the WOX5 promoter restores the QC and columella abnormalities in the wox5 mutant. These results suggest that WOX5 and WUS are interchangeable in stem cell control, and WUS and WOX5 function in stem cell maintenance in shoots or roots respectively (Sarkar et al. 2007). Recent studies have identified several signaling components involved in the WUS signaling pathway. BRCA1associated RING domain 1 (BARD1) encodes a protein containing two tandem BRCA1 C-terminal (BRCT) domains, which function in phosphorylation-dependent protein–protein interactions, and a RING domain, thought to be involved in DNA repair. Severe SAM defects in the bard1 mutant have been observed, and the bard1 wus double mutant shows the wus mutant phenotype. Direct interaction between BARD proteins and the WUS promoter region has been shown by chip analysis. Together with molecular genetic analyses that showed that BARD gene overexpression induced the wus mutant phenotypes, BARD1 is suggested to be responsible for the regulation of WUS gene expression in the CLV signaling pathway (Han et al. 2008). OBERON1 (OBE1) and OBE2 encode homeodomain finger proteins. CLV3, WUS, and WOX5 gene expressions are dramatically reduced in the obe1 obe2 double mutant. The obe1 obe2 wus triple mutant phenotype is similar to that of the obe1 obe2 double mutant. Thus, OBE1 and

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OBE2 are suggested to be responsible for plant cells reaching an appropriate state for the establishment and maintenance of the meristem by the action of meristem genes, rather than by the specification of the apical meristems directly (Saiga et al. 2008). HANABA TARANU (HAN) encodes a GATA-3-like transcription factor and functions in meristem formation in a non-cell-autonomous manner (Zhao et al. 2004). HAN expression in vascular tissues and cells separating the meristem from organ primordial cells controls the number and correct positioning of WUS-expressing cells (Tucker and Laux 2007). ULTRAPETALA1 (ULT1) encodes a SAND-domain transcription factor that restricts SAM activity by negatively regulating WUS (Carles et al. 2004, 2005). ULT1 and a close homolog, ULT2, are expressed in embryonic shoot apical meristems and in developing stamens, carpels and ovules. The ult1 wus double mutant shows additive phenotypes, and ULT1 is suggested to have WUS-independent functions in maintaining SAM activity, converging with the CLV pathway primarily at the point of limiting the lateral expansion of the WUS-expressing cell population (Carles et al. 2005). Mutations in the homeobox gene, STIP/WOX9 reduce CLV3 and WUS expression in the SAM, resulting in the wus mutant-like phenotypes. Genetic analysis has revealed that the loss of STIP function completely suppresses the clv3 phenotype. On other hand, overexpression of STIP enhances clv3 mutant phenotypes, implying that STIP is a positive regulator that functions in the WUS pathway (Wu et al. 2005).

Conservation of CLV signaling pathway in various plants Recent bioinformatics analysis has identified many CLE genes from various plant species in addition to Arabidopsis. Database searches revealed 15 CLE genes from Glycine max, 16 from Medicago truncatula, 26 from Populus trichocarpa, and 47 from Oryza sativa (Kinoshita et al. 2007; Oelkers et al. 2008; Sawa et al. 2008). CLE genes have also been found in Chlamydomonas reinhardtii and Physcomitrella patens, suggesting that the CLE peptide signaling pathway is conserved in plants (Oelkers et al. 2008). Besides plants, the cyst nematode Heterodera glycines has been shown to contain one CLE gene that may function in the process of infecting host plants (Wang et al. 2001). Genetic studies of rice and maize have shown that the CLV signaling pathway is conserved in monocotyledons. In rice, mutation in either FLORAL ORGAN NUMBER 1 (FON1) or FON2 causes enlarged floral meristems, leading

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to an increase in the number of floral organs (Nagasawa et al. 1996; Suzaki et al. 2004, 2006; Table 1). FON1 encodes an LRR receptor kinase, which is the rice counterpart of CLV1. FON2 and FON2-LIKE CLE PROTEIN1 (FCP1)/OsCLE402 encode small secretary proteins containing a CLE domain, closely related to CLV3 (Chu et al. 2006; Suzaki et al. 2006, 2008). Unlike FON1 and FON2, which regulate the maintenance of the flower and inflorescence meristem, FCP1 appears to regulate the maintenance of the vegetative SAM and RAM (Suzaki et al. 2008). These two CLE proteins may have diversified functions to regulate the different types of meristems in rice. In maize, the thick tassel dwarf 1 (td1) mutant and the fasciated ear 2 (fea2) mutant show massive over-proliferation of female inflorescence meristems, resulting in the fasciated ear. These mutants exhibit the abnormality in floral meristems and an increased number of floral organs (Taguchi-Shiobara et al. 2001; Bommert et al. 2005). Whereas the clv1 mutant shows defects in all aerial meristems, td1 and fea2 do not show any defects in meristem development in the vegetative phase. Genetic studies show that TD1 and FEA2 encode orthologous proteins of CLV1 and CLV2, respectively (Taguchi-Shiobara et al. 2001; Bommert et al. 2005; Table 1). TD1 expression is observed in leaf primordia and in leaves, but it is absent in vegetative meristems, and its expression pattern is different from CLV1 expression in Arabidopsis. In the reproductive stage, TD1 gene expression is observed in the outer layers of the inflorescence meristem, whereas, in Arabidopsis, CLV1 gene expression is observed in the inner layers. These results indicate that the CLV signaling pathways in meristem maintenance systems are relatively similar but that there are some diversified molecular mechanisms between eudicotyledonous and monocotyledonous plants. In legumes CLV1 orthologs have been identified, such as HYPERNODULATION ABERRANT ROOT 1 (HAR1) in Lotus japonicus, NODULE AUTOREGULATION RECEPTOR KINASE (NARK) in soybean, SUPERNUMERIC NODULES (SUNN) in Medicago truncatula, and SYM29 in pea (Krusell et al. 2002; Nishimura et al. 2002; Searle et al. 2003; Schnabel et al. 2005; Table 1). Mutations in these genes induce increased numbers of nodules of root endosymbiosis with soil bacteria, but these mutants do not show apparent abnormalities in their shoot meristems. This suggests that, even within eudicotyledons, there is a divergence of CLV1 function in meristem maintenance and regulation of nodule formation. The downstream target of the CLV signaling pathway is the WUS transcription factor in Arabidopsis. WUS gene expression is negatively regulated by CLV signaling and promotes stem cell proliferation in Arabidopsis. So far, WUS orthologs have been identified in rice (OsWUS), maize (ZmWUS1 and ZmWUS2), Antirrhinum majus

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(ROSRATA), Petunia hybrida (TERMINATOR), citrus (CsWUS), and in Brachypodium distachyon (BdWUS) (Stuurman et al. 2002; Kieffer et al. 2006; Nardmann and Werr 2006; Nardmann et al. 2007; Tan and Swain 2007). The function of WUS in maintaining the SAM appears to be conserved, at least in eudicotyledons, because both the Petunia ter mutant and the Antirrhinum roa mutant show shoot meristem termination, which is similar to that of the Arabidopsis wus mutant. In contrast, the spatial expression pattern and ectopic expression of OsWUS suggest that this gene is not involved in the promotion of stem cell proliferation (Nardmann and Werr 2006; Hirano, personal communication). In the root, the WUS-type homeobox gene QHB is highly expressed in the center of the cells in the RAM in rice (Kamiya et al. 2003). Ectopic expression of QHB leads to the development of multiple shoots from ectopic SAMs with malformed leaves, suggesting that the WUS-type homeobox gene is involved in the specification and maintenance of stem cells in the RAM, by a mechanism similar to that for WUS in the SAM. In conclusion, the regulatory mechanisms mediated by CLE peptides and LRR-RLKs seem conserved in the SAM and RAM in various eudicotyledonous and monocotyledonous plants. It has been indicated that the transcriptional regulation of meristem homeostasis by the WOX gene family is basically conserved in both shoot and root apical meristems in various vascular plants. However, recent genetic analyses of rice mutants has also indicated that there may be species-specific signaling pathways that control meristem maintenance independently of the CLV/ FON pathway. Further studies by genetic and biochemical analyses will provide new opportunities to broaden our understanding of the signaling components involved in plant meristem maintenance. Acknowledgment We would like to thank Shigeyuki Betsuyaku and Yuki Kobayashi for helping us to prepare the figure. We appreciate Hiroyuki Hirano’s critical reading of this manuscript. This work was supported by the Sumitomo Foundation; the Fuji Foundation; a grant-in aid for Creative Scientific Research; a grant-in-aid for Young Scientists (19677001) from the Japan Society for the Promotion of Science; a grant-in-aid for Scientific Research for Priority Areas from the Ministry of Education, Culture, Sports, Science (19060009 to H.F., 20061004, and 19060016) and Technology, and a Program of Basic Research Activities for Innovative Biosciences from the Biooriented Technology Research Advancement Institution.

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