Evolutionarily conserved CLE peptide signaling in plant development, symbiosis, and parasitism

Available online at www.sciencedirect.com

ScienceDirect Evolutionarily conserved CLE peptide signaling in plant development, symbiosis, and parasitism Kaori Miyawaki1,3, Ryo Tabata2,4 and Shinichiro Sawa2 Small polypeptides are widely used as signaling molecules in cell-to-cell communication in animals and plants. The CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) gene family is composed of numerous genes that contain conserved CLE domains in various plant species and plant-parasitic nematodes. Here, we review recent progress in our understanding of CLE signaling during stem cell maintenance in Arabidopsis and grasses. We also summarize the roles of CLE signaling in the legumeRhizobium symbiosis and infection by plant-parasitic nematodes. CLE signaling is important for diverse aspects of cell-to-cell signaling and long-distance communication, which are critical for survival, and the basic components of the CLE signaling pathway are evolutionarily conserved in both plants and animals. Addresses 1 Department of Botany and Plant Sciences, Center for Plant Cell Biology (CEPCEB), University of California, Riverside, CA 92521, United States 2 Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan 3 Present address: Shanghai Center for Plant Stress Biology, 3888 Chenhua Road, Shanghai 201602, China. 4 Present address: National Institute for Basic Biology (NIBB), Nishigonaka 38, Myodaiji, Okazaki 444-8585 Aichi, Japan. Corresponding author: Sawa, Shinichiro ([email protected])

Current Opinion in Plant Biology 2013, 16:598–606 This review comes from a themed issue on Cell signalling and gene regulation Edited by Caren Chang and John L Bowman For a complete overview see the Issue and the Editorial Available online 12th September 2013 1369-5266/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.pbi.2013.08.008

Introduction Cell-to-cell communication is critical for various developmental processes in multicellular organisms. Since the characterization of insulin in the early 1920s, peptides have been regarded as an important class of intercellular signaling molecules in animals. More recently, secreted peptides have been identified as critical players in various plant signaling pathways, such as wound responses, pollen incompatibility and stem cell maintenance in shoot and root meristems [1]. CLAVATA3 (CLV3) encodes a 96amino acid protein with an amino-terminal secretion signal and a conserved CLE motif at the carboxyl Current Opinion in Plant Biology 2013, 16:598–606

terminus ([2,3], and Figure 1). Loss-of-function mutations in CLV3 cause enlargement of the shoot apical meristem, indicating that CLV3 negatively regulates stem cell production [2]. Two CLE peptides, CLV3 and tracheary element differentiation inhibitory factor (TDIF), were identified as the first natural CLE peptides [4,5]. The mature forms of CLE proteins are the 12amino acid or 13-amino acid polypeptides, which are derived from the conserved CLE domains [4–6]. For instance, CLV3 is a 13-amino acid arabinosylated glycopeptide. Hydroxyproline (Hyp), which is located at the seventh position in the CLE domain of CLV3, is posttranslationally modified with three residues of L-arabinose ([6], and Figure 1). The Arabidopsis genome contains 32 members of the CLE gene family. Synthetic peptides corresponding to at least 19 of these have been shown to be functional [5,7]. Putative CLE genes have been identified not only in seed plants but also in a lycophyte, a bryophyte, and a green alga [8,9]. Moreover, CLE genes were also found in several plant-parasitic nematodes (reviewed in [10,11]). In this review, we summarize recent studies of CLE signaling with an emphasis on mechanisms underlying stem cell maintenance in Arabidopsis and grasses. We also describe the roles of CLE signaling in rhizobial symbiosis in legumes and the infection of plants by parasitic nematodes. Understanding the similarities and differences in the structural features of CLE peptides and the molecular mechanisms underlying CLE signaling in plants and nematodes will provide insight into the evolution of CLE signaling.

The role of CLE signaling in Arabidopsis stem cell maintenance: CLE-receptor-WOX is a common pathway Stem cells maintain tissue organization through their ability to both self-renew and differentiate into other cells. All aerial parts of plants, including leaves, stems, and flowers, are derived from stem cells in the shoot apical meristem (SAM) or floral meristem. The Arabidopsis SAM is comprised of three domains: the central zone (CZ), the peripheral zone (PZ), and the rib meristem (RM). Pluripotent stem cells, which are located in the apical domain of the CZ, give rise to new stem cells or to the differentiating cells that are displaced into the neighboring PZ. The RM is located beneath the CZ, and a small group of cells in the upper part of the RM is referred to as the organizing center (OC), which provides cues for stem cell specification. The well-characterized CLE gene, CLV3, is www.sciencedirect.com

Evolution of CLE signaling Miyawaki, Tabata and Sawa 599

Figure 1

SP

CLE domain

Variable domain

CLV3

RTVP*SGP*DP*LHHH O OH O

HO O OH

L-arabinose chain

O

HO O OH HO

OH

O

Polyproline OsCLE502 Conserved proline-rich region

HgCLE1

GrCLE4A Current Opinion in Plant Biology

Schematic diagrams of the molecular structures of CLV3, OsCLE502, HgCLE1, and Globodera rostochiensis CLE4A (GrCLE4A). In the schematic model of CLV3, a signal peptide (SP), a variable domain, and the C-terminal CLE domain are depicted as light green, purple, and red rectangles, respectively. The amino acid sequence of mature CLV3 is shown beneath the CLE domain. Asterisks indicate hydroxyproline (Hyp) residues. Hyp at the seventh position in the CLE domain of mature CLV3 is post-translationally modified with linear beta-1,2-linked tri-arabinoside. Gray rectangles indicate polyproline regions in OsCLE502. Turquoise rectangles show the conserved proline-rich region in nematode CLE, HgCLE1 and GrCLE4A.

preferentially expressed in the two outermost layers of the CZ [2]. WUSCHEL (WUS), a homeodomain transcription factor, is expressed in the OC and specifies stem cells [12]. The findings that WUS expression is expanded in clv3 mutants and that overexpression of CLV3 causes downregulation of WUS expression indicate that the CLV3 signal restricts WUS expression in a non-cellautonomous manner [13,14]. Conversely, the WUS protein itself migrates from the OC to the CZ and binds to the promoter region of CLV3 to activate its transcription in a negative feedback loop ([15], and Figure 2). The CLV3 signal is perceived by three types of receptors or receptor complexes, CLAVATA1 (CLV1), the CLAVATA2 (CLV2) and SUPPRESSOR OF LLP1-2 www.sciencedirect.com

(SOL2)/CORYNE (CRN) complex, and RECEPTORLIKE PROTEIN KINASE 2 (RPK2)/TOADSTOOL 2 (TOAD2) (Figure 2 and Table 1). CLV1 is a leucine-rich repeat receptor-like kinase (LRR-RLK) [16]. CLV2 is a LRR-type receptor protein that lacks a kinase domain and acts in concert with SOL2/CRN, which is a membraneassociated pseudokinase [17–19]. RPK2/TOAD2 encodes a LRR-RLK that was identified as a suppressor of CLV3induced SAM termination [20]. Because the phenotypes of the clv1 clv2 rpk2/toad2 triple mutants are additive in severity, the three receptor complexes may function independently. Live imaging and biochemical approaches suggest that CLV1 forms a homodimer, CLV2 interacts with SOL2/CRN to form a heterodimer, and RPK2/ TOAD2 forms a homodimer [20–22]. Furthermore, Current Opinion in Plant Biology 2013, 16:598–606

600 Cell signalling and gene regulation

Figure 2

Arabidopsis CLE

Receptor

Rice FON2

CLV3

CLV2CRN/SOL2

WOX

CLV1

RPK2/ TOAD2

WUS

FON1

?

Stem cell fate

FOS1

FCP1/2

?

?

WOX4

?

?

Stem cell fate

vSAM

Yes

No

Yes

Yes

IM

Yes

No

?

?

FM

Yes

Yes

?

Yes

Current Opinion in Plant Biology

The conserved CLE-receptor-WOX pathways during shoot apical meristem and floral meristem maintenance in Arabidopsis and rice. Whereas a single CLV3 signaling pathway regulates meristem maintenance in Arabidopsis, three CLE signaling pathways regulate meristem maintenance depending on developmental phases in rice. FON2 signaling pathway is active in floral meristems exclusively. FCP1/2 signaling is shown to be active in vegetative SAMs, whereas the function of these peptides remains to be determined in other developmental phases. FOS1-mediated pathway is active in both vegetative SAM and floral meristems, whereas its roles are yet to be understood in inflorescence meristems. FCP1/2 and FOS1 are perceived by unknown receptors that are different from FON1. vSAM, vegetative SAM; IM, inflorescence meristem; and FM, floral meristem. Arrows indicate activation and T-bars represent inhibition.

CLV3 binds to the CLV1 ectodomain [6,23]. CLV3 and CLV2 are also reported to physically interact, but further analyses using an arabinosylated CLV3 peptide remain to be confirmed [24]. Direct binding of CLV3 to RPK2/ TOAD2 remains to be tested. After CLV3 is perceived at the cell surface, the signal is transmitted via a downstream pathway that leads to the repression of WUS transcription (reviewed in [25]). In roots, stem cells are located in the cells surrounding the quiescent center (QC). WUSCHEL-RELATED HOMEOBOX5 (WOX5) is expressed in the QC and maintains the undifferentiated status of the stem cells in a non-cellautonomous fashion [26]. Columella stem cells (CSCs), which are distal to the QC, produce differentiated columella cells. CLE40 transcripts accumulate in the columella cells. CLE40 loss-of-function mutants form an extra layer of CSCs. The WOX5 expression domain is expanded laterally in cle40 mutants and treatment with synthetic CLE40 peptide induces a proximal shift in WOX5 expression, suggesting that CLE40 promotes the differentiation into columella cells by repressing WOX5 [27]. The CLE40 signal is perceived by the membrane-localized receptor-like kinase, ARABIDOPSIS CRINKLY4 (ACR4), and acr4 mutants contain an extra cell layer of CSCs and are insensitive to the treatment with synthetic CLE40 peptide [27]. Recently, Stahl and colleagues Current Opinion in Plant Biology 2013, 16:598–606

reported that CLV1 participates with ACR4 to perceive CLE40. An increase in the number of differentiated columella cells was observed in clv1 mutants compared to the wild type in the presence of synthetic CLE40 peptide, suggesting that CLV1 also functions to buffer the CLE40 signal [27,28]. Plants contain meristematic stem cells not only in roots and shoots, but also in vascular tissues. The vascular meristem (procambium/cambium) is situated between two transporting tissues, the xylem and phloem, and proliferates by periclinal cell division to provide the adjacent differentiated cell layers of the xylem or phloem. TDIF was originally identified in a xylogenic Zinnia cell culture as a factor that suppresses the differentiation of cells into tracheary elements (TEs) [5]. In Arabidopsis, CLE41 and CLE44 encode peptides identical to TDIF. In contrast to other CLE peptides, TDIF did not inhibit SAM or root apical meristem (RAM) development. However, TDIF promotes the accumulation of undifferentiated procambial cells and inhibits their differentiation into xylem. Genetic and biochemical approaches revealed that TDIF RECEPTOR/PHLOEM INTERCALATED WITH XYLEM (TDR/PXY), which is a CLV1-like LRR-RLK, perceives the TDIF signal. Immunolocalization analysis showed that TDIF is localized to phloem and procambial cells, suggesting that www.sciencedirect.com

Evolution of CLE signaling Miyawaki, Tabata and Sawa 601

Table 1 A list of known and putative CLE-receptor-WOX combinations and their roles in various plant development, legume-Rhizobium symbiosis, and plant-parasitic nematode infection. CLE

Receptor

CLV3

CLV1, CLV2-CRN/SOL2, RPK2/TOAD2 ACR4, CLV1 TDR/PXY

CLE40 CLE41 (TDIF)

WOX

Function

Plants/nematodes

References

WUS

SAM maintenance

Arabidopsis thaliana

[2,12,16–20]

Columella stem cell maintenance Vascular stem cell maintenance

Arabidopsis thaliana Arabidopsis thaliana

[26,27,28] [29,30,31,32,33]

Protoxylem vessel formation Floral meristem maintenance Vegetative SAM maintenance

Arabidopsis thaliana Oryza sativa Oryza sativa

[34] [37–39] [40,41]

Oryza sativa indica Zea mays

[42] [43,44]

Lotus japonicus

[46,47,48,49,50]

CLE10 FON2/4 FCP1 FCP2 FOS1 Unknown

CLV2 FON1 Unknown

WOX5 WOX4 WOX14 Unknown Unknown WOX4

Unknown TD1, FEA2

Unknown Unknown

LjCLE-RS1 LjCLE-RS2

HAR1, KLV

Unknown

Meristem maintenance Inflorescence meristem/floral meristem maintenance Autoregulation of nodulation

LjCLV2? SUNN

WOX5

Autoregulation of nodulation

Medicago truncatula

[52,53,55]

GmNARK

Unknown

Autoregulation of nodulation

Glycine max

[51,54]

GmNARK Sym29? Sym28/PtClv2 CLV1, CLV2-CRN/SOL2, RPK2/TOAD2

Unknown WOX5

Autoregulation of nodulation Autoregulation of nodulation

Glycine max Pisum sativum

[51,54] [46,49,55]

Unknown

Infection of soybean cyst nematode Infection of beet cyst nematode

Heterodera glycines

[58,59]

MtCLE12 MtCLE13 GmRIC1 GmRIC2 GmNIC1 Unknown HgCLE HsCLE

TDIF is synthesized in phloem and secreted into procambial cells, where it regulates various aspects of procambium development, such as mitotic activity, the orientation of cell divisions, and the fates of vascular stem cells [29,30]. Analogous to the CLV3-WUS and CLE40WOX5 pathways in SAM and roots, WOX homeodomain transcription factors act downstream of the TDIF-TDR/ PXY pathway. WOX4 is upregulated in response to TDIF application. The WOX4 and TDR promoters are both preferentially active in procambial and cambial cells. Phenotypic analysis of the wox4 mutant showed that WOX4 is required for the proliferation of procambial/ cambial cells, but not for their inhibition of differentiation into xylem in the presence of the TDIF peptide, suggesting that WOX4 functions as a positive regulator exclusively in the procambial/cambial cell proliferation pathway [31,32]. A recent study demonstrated that WOX14 acts redundantly with WOX4 to promote vascular cell division [33]. CLE10 has also been shown to regulate vascular development. The CLE10 peptide inhibited protoxylem vessel formation in Arabidopsis roots by repressing type A-ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR5 and ARR6, which are negative regulators of cytokinin signaling. Mutations in both ARR5 and ARR6 cause defects in protoxylem formation. It has been shown that CLV2 is required for the function of CLE10 [34]. Because cytokinin plays a critical role in the proliferation of vascular stem cells and in the suppression of protoxylem www.sciencedirect.com

Heterodera schachtii

vessel formation, it is likely that CLE10 regulates protoxylem vessel formation by enhancing cytokinin signaling. Thus, CLE-receptor-WOX modules support the stem cell maintenance in all meristems in Arabidopsis (Table 1). It is critical to explore the molecular mechanisms that act downstream of receptors and eventually lead to regulation of WOX genes. Thirty-two Arabidopsis CLE genes have been identified to date, and multiple CLE genes are expressed in various tissues. Mutant analyses of several cle single mutants revealed that CLE genes, except for CLV3 and CLE40, exhibit significant functional redundancy [35]. The Arabidopsis genome encodes 14 functional WOX genes, with each gene exhibiting discrete expression dynamics during plant development [36]. It would be intriguing to explore new CLE-WOX modules, which may be involved in additional developmental processes, other than CLV3-WUS, CLE40-WOX5, and TDIFWOX4/WOX14.

The roles of CLE signaling in meristem maintenance in grasses The CLV/WUS negative feedback loop is important for SAM maintenance in Arabidopsis. Several lines of evidence suggest that CLE signaling is also involved in meristem maintenance in grasses (Figure 2). In Oryza sativa (rice), mutations in FLORAL ORGAN NUMBER 1 (FON1), which encodes a LRR-RLK similar Current Opinion in Plant Biology 2013, 16:598–606

602 Cell signalling and gene regulation

to CLV1, caused enlargement of the floral meristems [37]. FLORAL ORGAN NUMBER 2 (FON2), which is also known as FON4, encodes a CLE peptide that resembles CLV3. FON2 is expressed in the apical domain of the CZ. Floral meristems of fon2 mutants were enlarged, like those of fon1. Because the fon1 fon2 double mutant did not exhibit an additive phenotype and FON1 was found to be required for the function of FON2, FON1 is likely the receptor of FON2 [38,39]. Since fon1 and fon2 exhibit the aberrant phenotypes exclusively during the reproductive phases, alternative pathways are thought to exist for vegetative SAM maintenance. Both the constitutive expression of FON2-LIKE CLE PROTEIN1 (FCP1) and the application of synthetic FCP1 repressed vegetative SAM development independently of FON1 [40]. A recent report revealed that silencing of both FCP1 and FCP2, which is closely related to FCP1, resulted in the expansion of the stem cell region of the vegetative SAM and inhibited of differentiation in the PZ [41]. These data suggest that FCP1/2 negatively regulates stem cell pool size during the vegetative phase. In addition to FON2 and FCP1/2, FON2 SPARE1 (FOS1), which encodes a CLE peptide, was identified as a suppressor of fon2 in indica rice, a subspecies of Oryza sativa. FOS1 has a negative role in both vegetative SAM and floral meristem maintenance [42]. Recently, it was reported that WOX4 acts as a positive regulator of stem cell maintenance in rice [41]. WOX4 expression was negatively regulated by both the constitutive and transient expression of FCP1. RNA silencing of WOX4 reduced the expression of genes specifically expressed in stem cells, such as rice ORYZA SATIVA HOMEOBOX1 (a rice ortholog of SHOOT MERISTEMLESS) and FON2 [41]. Therefore, FCP1/2-WOX4 feedback regulation seems to be a key mechanism in vegetative meristem maintenance. Intriguingly, the expression patterns of FCP1 and WOX4 overlapped in meristems, indicating that WOX4 is not downregulated by FCP1, unlike the repression of WUS by CLV3 in Arabidopsis SAM. Further analysis will reveal the mechanism that regulates WOX4 activity. In maize, mutations in THICK TASSEL DWARF (TD1) and FASCIATED EAR2 (FEA2) predominantly cause a fasciated female inflorescence (ear) and enlarged male inflorescence meristems, which result in an increase in the density of spikelets [43,44]. TD1 encodes a CLV1-like LRR-RLK and FEA2 resembles Arabidopsis CLV2. The td1 fea2 double mutant exhibited an additive phenotype, suggesting that these two putative receptors function in parallel as proposed for their homologs in Arabidopsis [43,44]. Current Opinion in Plant Biology 2013, 16:598–606

The CLE signaling pathway likely represents an evolutionarily conserved mechanism for meristem maintenance in flowering plants. In rice, three redundant pathways have evolved in a unique fashion (Figure 2). However, floral meristems were less affected in rice fon1 and fon2 mutants compared to Arabidopsis clv3 mutants and maize td1 and fea2 mutants, which show severely fasciated meristems [37–39]. The rice genome encodes as many as 47 CLE genes, suggesting that uncharacterized CLE signaling pathways may function redundantly in meristem maintenance. Alternatively, the mechanisms underlying meristem maintenance may differ in rice. Indeed, rice flattened shoot meristem ( f sm) mutants exhibited a decrease in the SAM size, in contrast to the fasciated SAM caused by mutations in FASCIATA1, which is a putative ortholog of FSM in Arabidopsis [45]. Because rice is comprised of many species, it may have evolved different regulatory mechanisms of meristem maintenance during speciation even if the components are conserved among eudicots and other species of monocots. Understanding the mechanisms in other monocots will provide insight into molecular mechanisms of grass meristem maintenance.

The roles of CLE in legume-Rhizobium symbiosis Symbiotic nitrogen fixation is essential for leguminous plants under nitrogen deprivation. Rhizobial invasion results in nodulation, the formation of symbiotic organs, in legume roots. Host legumes have a systemic regulatory system, known as autoregulation of nodulation (AON), to avoid excess nodulation. AON is thought to be a bidirectional system governed by two long-distance signals, a root-derived infection signal and a shoot-derived autoregulation signal. Lotus japonicus HYPERNODULATION ABERRANT ROOT FORMATION 1 (HAR1) and KLAVIER (KLV) encode a CLV1-like LRR-RLK and a RPK2/ TOAD2-like LRR-RLK, respectively [46,47,48]. Both har1 and klv mutants exhibit hypernodulation phenotypes, presumably because of defects in shoot-derived autoregulation. Double mutant and biochemical analyses revealed that HAR1 and KLV interact physically with one another and function in the same pathway [48]. Likewise, mutations in LjCLV2, which encodes a CLV2-like LRR-receptor-like protein, or downregulation of LjCLV2 caused increased nodulation, suggesting that LjCLV2 participates in AON [49]. Two CLE peptides, L. japonicus CLE ROOT SIGNAL1 and 2 (LjCLE-RS1 and LjCLE-RS2, respectively), have been identified as the putative root-derived infection signals. LjCLE-RS1/RS2 expression in roots is induced by inoculation with Rhizobium. LjCLE-RS2 is induced by exogenous treatment with nitrate, which suppresses nodulation. Constitutive expression of LjCLE-RS1/RS2 in roots systemically inhibits the nodulation in a HAR1-KLV-dependent manner [48,50]. These data suggest that LjCLE-RS1/RS2 signals, which are transmitted from roots to shoots, www.sciencedirect.com

Evolution of CLE signaling Miyawaki, Tabata and Sawa 603

systemically repress excess nodulation via perception by the HAR1-KLV receptor complex. Several reports indicate that CLV1-like LRR-RLKs function in a similar manner in other legumes. Glycine max (soybean) NODULE AUTOREGULATION RECEPTOR KINASE (GmNARK), Pisum sativum (pea) SYMBIOSIS29 (Sym29), and Medicago truncatula (barrel medic) SUPER NUMERIC NODULES (SUNN) all participate in shoot-derived inhibitory signaling during AON [46,51,52]. Mutations in PsClv2 (Sym28), which encodes a CLV2-like LRR-receptor-like protein, cause hypernodulation [49]. Recent studies showed that several CLE peptides, that is, Glycine max RHIZOBIAINDUCED CLE 1/2 (GmRIC1 and GmRIC2), Glycine max NITRATE-INDUCED CLE 1 (GmNIC1), and Medicago truncatula CLE12 and CLE13 (MtCLE12 and MtCLE13) are involved in AON in a GmNARK/SUNNdependent manner [53,54]. In barrel medic and pea, WOX5 is expressed during nodule organogenesis [55]. The CLE-receptor-WOX pathway seems to be a conserved mechanism for AON in legumes (Table 1). It remains to be determined whether legume CLE peptides act as a mobile signal for root-to-shoot communication. Interestingly, application of synthetic LjCLE peptides did not suppress nodulation, suggesting that unknown modifications may be required for their function. Arabidopsis CLE1-CLE7 peptides have sequence similarity to legume CLE peptides, which are involved in AON [50]. Whereas overexpression of CLE1-CLE7 genes caused the SAM-consumption phenotypes and longer roots, in planta functions of these genes remain unknown [56]. Further analysis of legume CLE signaling may shed light not only on the molecular mechanism of AON in legumes but also on long-distance communication in other plant species.

The roles of CLE in nematode infection The plant-parasitic nematodes attack the roots of host plants and cause significant crop damage. Nematodes produce various secreted effector proteins, which are stored in the esophageal gland cells and injected into host plant cells during the parasitic cycle. HgCLE was identified as a putative effector gene in a soybean cyst nematode, Heterodera glycines [57]. CLE-like genes have also been cloned from the potato cyst nematode, the beet cyst nematode, and root knot nematodes (reviewed in [11]). Ectopic expression of nematode CLE genes in Arabidopsis, driven by the cauliflower mosaic virus 35S promoter, or the supply of exogenous nematode CLE peptides caused SAM and RAM consumption phenotypes in a CLV2-CRN/SOL2-dependent manner, indicating that nematode CLE peptides are functional in Arabidopsis. Nematode infection assays using clv1, clv2, crn/sol2, and rpk2 mutants showed that either nematode infection or www.sciencedirect.com

syncytium size was reduced in these mutants, suggesting that nematode CLEs can function as ligands that hijack CLE signaling in Arabidopsis to ensure successful infection and syncytium development ([58,59] and Table 1). Immunolocalization studies revealed that HgCLE proteins are delivered to the cytoplasm of syncytial cells after infection, and then translocated to the apoplast, supporting their roles as ligand mimics [60]. Barring the CLE domains, the CLE protein precursors of nematodes have no sequence similarity to those of plants. Additionally, the nematode CLE preproproteins from different nematode species share little sequence similarity ([11], and Figure 1). Thus, nematodes may have acquired CLE genes independently of the plant CLE genes and different nematodes may have evolved their own CLE genes.

CLE structure in the various plants and nematodes Arabidopsis CLE genes share several structural features, including a signal peptide at the N-terminus, a variable domain, and a conserved CLE domain near the C-terminus ([3], and Figure 1). The CLE domain is the most critical for the functional specificity of each CLE peptide [4,5,40]. Single genes encoding multiple CLE domains have been identified in rice, Triticum aestivum (wheat), Medicago truncatula, Selaginella moellendorffii, and nematodes [7,8,11,61]. Indeed, three of the rice CLE genes, OsCLE502, OsCLE504, and OsCLE506, encode CLE precursor proteins with multiple CLE domains separated by polyproline regions between the CLE domains ([7,8] and Figure 1). Since polyproline regions are located directly after the CLE domains, those sequences may be involved in the processing of mature CLE peptides. It is unknown whether the CLE domains of these proteins are processed into active CLE peptides. The presence of multiple CLE domains may have the advantage of facilitating the highly efficient production of CLE peptides. The CLE domain of CLE18 precursor protein is located in the central region of the protein, while a 13-amino acid CLE-like (CLEL) motif exists in the C-terminus [62]. Plants overexpressing full-length CLE18 exhibited the long-root and wavy-root phenotypes [56]. An analysis of the synthetic peptides showed that the CLEL motif of CLE18 precursor protein is responsible for these phenotypes, in contrast to the inhibitory effects of the CLE peptide derived from the CLE domain on root development [5,62]. Further analyses of genes carrying multiple CLE domains may pave the way for deciphering the different signal specificities of CLE and CLEL peptides.

Conclusions CLE signaling has critical roles in both cell-to-cell and long-distance communication during plant development, Rhizobium symbiosis, and nematode infection. Since Current Opinion in Plant Biology 2013, 16:598–606

604 Cell signalling and gene regulation

numerous CLE and LRR-RLK genes exist in plants, ranging from mosses to seed plants, it is probable that the CLE/LRR-RLK module is an evolutionarily conserved component of intracellular and extracellular signaling cascades. Many combinations of CLE peptides and receptors mediate various and precise signals involved in both short-range and long-distance communication, in contrast to other general growth regulators, such as plant hormones that affect cellular features and mitotic activity throughout plant development.

5.

Ito Y, Nakanomyo I, Motose H, Iwamoto K, Sawa S, Dohmae N, Fukuda H: Dodeca-CLE peptides as suppressors of plant stem cell differentiation. Science 2006, 313:842-845.

6.

Ohyama K, Shinohara H, Ogawa-Ohnishi M, Matsubayashi Y: A glycopeptide regulating stem cell fate in Arabidopsis thaliana. Nat Chem Biol 2009, 5:578-580.

7.

Kinoshita A, Nakamura Y, Sasaki E, Kyozuka J, Fukuda H, Sawa S: Gain-of-function phenotypes of chemically synthetic CLAVATA3/ESR-related (CLE) peptides in Arabidopsis thaliana and Oryza sativa. Plant Cell Physiol 2007, 48:1821-1825.

8.

Oelkers K, Goffard N, Weiller G, Gresshoff P, Mathesius U, Frickey T: Bioinformatic analysis of the CLE signaling peptide family. BMC Plant Biol 2008, 8:1.

CLE peptides have diverse roles. Differences in CLE functions could arise due to differences in molecular structure of CLE peptides, substrate specificity and activity of receptors, and spatial expression patterns and environmental responsiveness of CLE genes.

9.

Miwa H, Tamaki T, Fukuda H, Sawa S: Evolution of CLE signaling: origins of the CLV1 and SOL2/CRN receptor diversity. Plant Signal Behav 2009, 4:477-481.

The molecular nature of CLE peptides may vary due to posttranslational modifications, such as cleavage by peptidases and sugar modifications at specific residues. The identification of peptidases or enzymes involved in posttranslational modifications will elucidate the maturation steps of CLE peptides. Substrate specificity can be determined by using a combination of receptors. Live imaging and biochemical approaches will further reveal the nature of functional receptor complexes. Some CLE peptides form gradients and may provide positional cues critical for the growth patterning or responsiveness to environmental conditions, such as nitrate concentration and Rhizobium infection. Understanding the spatial control of CLE gene expression is critical, and thus far WUS is the only transcription factor that has been shown to regulate the promoter activity of CLV3 through direct transcriptional control. Future studies should focus on identifying the factors that regulate the expression patterns of CLE genes.

Acknowledgements We thank Drs. Hiro-Yuki Hirano, G. Venugopala Reddy, and Shingo Nagawa for their valuable comments regarding the manuscript. This work was supported by JSPS Postdoctoral Fellowships for Research Abroad to K.M. from the Japan Society of the Promotion of Science and by KAKENHI (221S0002, 23119517, 23012034, 24114001, 24114009, 24370024, 24657035, and 24658032) to S.S.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Matsubayashi Y, Sakagami Y: Peptide hormones in plants. Annu Rev Plant Biol 2006, 57:649-674.

2.

Fletcher J, Brand U, Running M, Simon R, Meyerowitz E: Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems. Science 1999, 283:1911-1914.

10. Kiyohara S, Sawa S: CLE signaling systems during plant development and nematode infection. Plant Cell Physiol 2012, 53:1989-1999. 11. Mitchum MG, Wang X, Wang J, Davis EL: Role of nematode peptides and other small molecules in plant parasitism. Annu Rev Phytopathol 2012, 50:175-195. 12. Mayer KF, Schoof H, Haecker A, Lenhard M, Jurgens G, Laux T: Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 1998, 95:805-815. 13. Brand U, Fletcher J, Hobe M, Meyerowitz E, Simon R: Dependence of stem cell fate in Arabidopsis on a feedback loop regulated by CLV3 activity. Science 2000, 289:617-619. 14. Schoof H, Lenhard M, Haecker A, Mayer KFX, Ju¨rgens G, Laux T: The stem cell population of Arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 2000, 100:635-644. 15. Yadav R, Perales M, Gruel J, Girke T, Jo¨nsson H, Reddy G:  WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex. Genes Dev 2011, 25:2025-2030. In this work, authors demonstrate translocation of WUS from the OC to the CZ, followed by activation of CLV3 transcription through the direct binding of WUS protein to the promoter region of CLV3. This finding resolves a long-standing question of non-cell-autonomous specification of stem cell fate in SAM. 16. Clark S, Running M, Meyerowitz E: CLAVATA1, a regulator of meristem and flower development in Arabidopsis. Development 1993, 119:397-418. 17. Kayes J, Clark S: CLAVATA2, a regulator of meristem and organ development in Arabidopsis. Development 1998, 125:3843-3851. 18. Mu¨ller R, Bleckmann A, Simon R: The receptor kinase CORYNE of Arabidopsis transmits the stem cell-limiting signal CLAVATA3 independently of CLAVATA1. Plant Cell 2008, 20:934-946. 19. Miwa H, Betsuyaku S, Iwamoto K, Kinoshita A, Fukuda H, Sawa S: The receptor-like kinase SOL2 mediates CLE signaling in Arabidopsis. Plant Cell Physiol 2008, 49:1752-1757. 20. Kinoshita A, Betsuyaku S, Osakabe Y, Mizuno S, Nagawa S, Stahl Y, Simon R, Yamaguchi-Shinozaki K, Fukuda H, Sawa S: RPK2 is an essential receptor-like kinase that transmits the CLV3 signal in Arabidopsis. Development 2010, 137:3911-3920. 21. Bleckmann A, Weidtkamp-Peters S, Seidel C, Simon R: Stem cell signaling in Arabidopsis requires CRN to localize CLV2 to the plasma membrane. Plant Physiol 2010, 152:166-176.

3.

Cock J, McCormick S: A large family of genes that share homology with CLAVATA3. Plant Physiol 2001, 126:939-942.

22. Zhu Y, Wang Y, Li R, Song X, Wang Q, Huang S, Jin J, Liu C, Lin J: Analysis of interactions among the CLAVATA3 receptors reveals a direct interaction between CLAVATA2 and CORYNE in Arabidopsis. Plant J 2010, 61:223-233.

4.

Kondo T, Sawa S, Kinoshita A, Mizuno S, Kakimoto T, Fukuda H, Sakagami Y: A plant peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis. Science 2006, 313:845-848.

23. Ogawa M, Shinohara H, Sakagami Y, Matsubayashi Y: Arabidopsis CLV3 peptide directly binds CLV1 ectodomain. Science 2008, 319:294.

Current Opinion in Plant Biology 2013, 16:598–606

www.sciencedirect.com

Evolution of CLE signaling Miyawaki, Tabata and Sawa 605

24. Guo Y, Han L, Hymes M, Denver R, Clark S: CLAVATA2 forms a distinct CLE-binding receptor complex regulating Arabidopsis stem cell specification. Plant J 2011, 63:889-900. 25. Betsuyaku S, Sawa S, Yamada M: The function of the CLE peptides in plant development and plant–microbe interactions. Arabidopsis Book 2012, 9:e0149. 26. Sarkar A, Luijten M, Miyashima S, Lenhard M, Hashimoto T, Nakajima K, Scheres B, Heidstra R, Laux T: Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature 2007, 446:811-814. 27. Stahl Y, Wink RH, Ingram GC, Simon Rd: A signaling module controlling the stem cell niche in Arabidopsis root meristems. Curr Biol 2009, 19:909-914. 28. Stahl Y, Grabowski S, Bleckmann A, Ku¨hnemuth R, Weidtkamp Peters S, Pinto KG, Kirschner GK, Schmid JB, Wink RH, Hu¨lsewede A et al.: Moderation of Arabidopsis root stemness by CLAVATA1 and ARABIDOPSIS CRINKLY4 receptor kinase complexes. Curr Biol 2013, 23:362-371. Authors report that CLV1, a key receptor kinase in SAM maintenance, also participates in the perception of CLE40 together with ACR4 in the distal root stem cells. Live-imaging analysis revealed that ACR4 and CLV1 form different homomeric and heteromeric complexes depending on subcellular localization. CLV1 also has the function of buffering the excess amount of CLE40 signal for fine-tuned mechanism of root stem cell homeostasis. 29. Hirakawa Y, Shinohara H, Kondo Y, Inoue A, Nakanomyo I, Ogawa M, Sawa S, Ohashi-Ito K, Matsubayashi Y, Fukuda H: Noncell-autonomous control of vascular stem cell fate by a CLE peptide/receptor system. Proc Natl Acad Sci U S A 2008, 105:15208-15213. 30. Fisher K, Turner S: PXY, a receptor-like kinase essential for maintaining polarity during plant vascular-tissue development. Curr Biol 2007, 17:1061-1066. 31. Ji J, Strable J, Shimizu R, Koenig D, Sinha N, Scanlon M: WOX4 promotes procambial development. Plant Physiol 2010, 152:1346-1356. 32. Hirakawa Y, Kondo Y, Fukuda H: TDIF peptide signaling  regulates vascular stem cell proliferation via the WOX4 homeobox gene in Arabidopsis. Plant Cell 2011, 22:2618-2629. This work demonstrates that WOX4 positively regulates the vascular stem cell proliferation in response to phloem-derived TDIF signal. Expression analysis also revealed that WOX4 is expressed in the procambium/ cambium, in which it promotes the cell division, in contrast to noncell-autonomous functions of WUS or WOX5. 33. Etchells J, Provost C, Mishra L, Turner S: WOX4 and WOX14 act  downstream of the PXY receptor kinase to regulate plant vascular proliferation independently of any role in vascular organisation. Development 2013, 140:2224-2234. In this study, authors show that WOX14, together with WOX4, acts redundantly in promoting TDIF-triggered cell division of vascular meristem downstream of TDR/PXY. 34. Kondo Y, Hirakawa Y, Kieber J, Fukuda H: CLE peptides can negatively regulate protoxylem vessel formation via cytokinin signaling. Plant Cell Physiol 2011, 52:37-48. 35. Jun J, Fiume E, Roeder A, Meng L, Sharma V, Osmont K, Baker C, Ha C, Meyerowitz E, Feldman L et al.: Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis. Plant Physiol 2011, 154:1721-1736. 36. Haecker A, Gross-Hardt R, Geiges B, Sarkar A, Breuninger H, Herrmann M, Laux T: Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana. Development 2004, 131:657-668.

39. Chu H, Qian Q, Liang W, Yin C, Tan H, Yao X, Yuan Z, Yang J, Huang H, Luo D et al.: The floral organ number4 gene encoding a putative ortholog of Arabidopsis CLAVATA3 regulates apical meristem size in rice. Plant Physiol 2006, 142:1039-1052. 40. Suzaki T, Yoshida A, Hirano H: Functional diversification of CLAVATA3-related CLE proteins in meristem maintenance in rice. Plant Cell 2008, 20:2049-2058. 41. Ohmori Y, Tanaka W, Kojima M, Sakakibara H, Hirano H:  WUSCHEL-RELATED HOMEOBOX4 is involved in meristem maintenance and is negatively regulated by the CLE gene FCP1 in rice. Plant Cell 2013, 25:229-241. In contrast to several CLE peptides as negative regulators of meristem maintenance, the positive regulators have been unknown in rice. In this work, WOX4 was first identified as a positive regulator for rice meristem maintenance. WOX4 expression was negatively regulated by FCP1, while RNA silencing of WOX4 caused the inhibition of stem cell specification, suggesting that FCP1-WOX4 feedback regulation is critical for meristem maintenance in rice. 42. Suzaki T, Ohneda M, Toriba T, Yoshida A, Hirano H: FON2 SPARE1 redundantly regulates floral meristem maintenance with FLORAL ORGAN NUMBER2 in rice. PLoS Genet 2009, 5:e1000693. 43. Taguchi-Shiobara F, Yuan Z, Hake S, Jackson D: The fasciated ear2 gene encodes a leucine-rich repeat receptor-like protein that regulates shoot meristem proliferation in maize. Genes Dev 2001, 15:2755-2766. 44. Bommert P, Lunde C, Nardmann J, Vollbrecht E, Running M, Jackson D, Hake S, Werr W: thick tassel dwarf1 encodes a putative maize ortholog of the Arabidopsis CLAVATA1 leucine-rich repeat receptor-like kinase. Development 2005, 132:1235-1245. 45. Abe M, Kuroshita H, Umeda M, Itoh J-I, Nagato Y: The rice FLATTENED SHOOT MERISTEM, encoding CAF-1 p150 subunit, is required for meristem maintenance by regulating the cell-cycle period. Dev Biol 2008, 319:384-393. 46. Krusell L, Madsen L, Sato S, Aubert G, Genua A, Szczyglowski K, Duc G, Kaneko T, Tabata S, de Bruijn F et al.: Shoot control of root development and nodulation is mediated by a receptorlike kinase. Nature 2002, 420:422-426. 47. Nishimura R, Hayashi M, Wu G, Kouchi H, Imaizumi-Anraku H, Murakami Y, Kawasaki S, Akao S, Ohmori M, Nagasawa M et al.: HAR1 mediates systemic regulation of symbiotic organ development. Nature 2002, 420:426-429. 48. Miyazawa H, Oka-Kira E, Sato N, Takahashi H, Wu G, Sato S,  Hayashi M, Betsuyaku S, Nakazono M, Tabata S et al.: The receptor-like kinase KLAVIER mediates systemic regulation of nodulation and non-symbiotic shoot development in Lotus japonicus. Development 2010, 137:4317-4325. The klv mutant has been isolated as a hypernodulation mutant. Authors describe that KLV encodes a LRR-RLK, which has highest sequence similarity to RPK2/TOAD2. HAR1 and KLV are required for the function of LjCLE-RS1/2 in AON. Mutant and biochemical analyses revealed HAR1 and KLV function in the same complex of receptors. In addition to hypernodulation phenotypes, klv mutant exhibits the pleiotropic phenotypes including enlarged SAM, reminiscent of clv3 mutants in Arabidopsis. 49. Krusell L, Sato N, Fukuhara I, Koch B, Grossmann C, Okamoto S,  Oka-Kira E, Otsubo Y, Aubert G, Nakagawa T et al.: The Clavata2 genes of pea and Lotus japonicus affect autoregulation of nodulation. Plant J 2011, 65:861-871. In this work, two CLV2-like genes were identified in Lotus japonicus and pea. Mutations in LjCLV2 and PsClv2 resulted in the increased nodulation, suggesting that CLV2 functions as an additional receptor during AON signaling in legume.

37. Suzaki T, Sato M, Ashikari M, Miyoshi M, Nagato Y, Hirano H: The gene FLORAL ORGAN NUMBER1 regulates floral meristem size in rice and encodes a leucine-rich repeat receptor kinase orthologous to Arabidopsis CLAVATA1. Development 2004, 131:5649-5657.

50. Okamoto S, Ohnishi E, Sato S, Takahashi H, Nakazono M, Tabata S, Kawaguchi M: Nod factor/nitrate-induced CLE genes that drive HAR1-mediated systemic regulation of nodulation. Plant Cell Physiol 2009, 50:67-77.

38. Suzaki T, Toriba T, Fujimoto M, Tsutsumi N, Kitano H, Hirano H: Conservation and diversification of meristem maintenance mechanism in Oryza sativa: function of the FLORAL ORGAN NUMBER2 gene. Plant Cell Physiol 2006, 47:1591-1602.

51. Searle IR, Men AE, Laniya TS, Buzas DM, Iturbe-Ormaetxe I, Carroll BJ, Gresshoff PM: Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase. Science 2003, 299:109-112.

www.sciencedirect.com

Current Opinion in Plant Biology 2013, 16:598–606

606 Cell signalling and gene regulation

52. Schnabel E, Journet E-P, Carvalho-Niebel F, Duc G, Frugoli J: The Medicago truncatula SUNN gene encodes a CLV1-like leucine-rich repeat receptor kinase that regulates nodule number and root length. Plant Mol Biol 2005, 58:809-822.

By using synthetic nematode CLE peptides or nematode CLE overexpression lines, combined with nematode infection assays of receptor mutants, authors show that the CLV2/CRN signaling pathway is required for successful nematode infection and syncytium development.

53. Mortier V, Den Herder G, Whitford R, Van de Velde W, Rombauts S,  D’Haeseleer K, Holsters M, Goormachtig S: CLE peptides control Medicago truncatula nodulation locally and systemically. Plant Physiol 2010, 153:222-237. Authors show that two Medicago truncatula CLE genes, MtCLE12 and MtCLE13, which are closely related to LjCLE-RS1/RS2, exhibit the nodulation-related expression pattern. Overexpression of MtCLE12 and MtCLE13 in roots caused a strong suppression of nodulation as well as elongation of petioles systemically. The LRR-RLK, SUNN, is partially required for the function of MtCLE12/13.

59. Replogle A, Wang J, Paolillo V, Smeda J, Kinoshita A, Durbak A,  Tax FE, Wang X, Sawa S, Mitchum MG: Synergistic interaction of CLAVATA1, CLAVATA2, and RECEPTOR-LIKE PROTEIN KINASE 2 in cyst nematode parasitism of Arabidopsis. Mol Plant–Microbe Interact 2013, 26:87-96. Since a partial reduction in nematode numbers and syncytia size in clv2 and crn mutant [58], additional receptors should be required for nematode CLE perception. This study revealed that CLV1 and RPK2/ TOAD2 are important for nematode CLE signaling in parallel with CLV2/CRN.

54. Reid DE, Ferguson BJ, Gresshoff PM: Inoculation- and nitrate induced CLE peptides of soybean control NARK-dependent nodule formation. Mol Plant–Microbe Interact 2011, 24:606-618. This paper describes that overexpression of two rhizobia-induced CLE (GmRIC1 and RIC2) inhibited soybean nodulation in a GmNARK-dependent manner. In contrast, constitutive expression of nitrate-responsive CLE (GmNIC1) resulted in partial reduction of nodulation.

60. Wang J, Lee C, Replogle A, Joshi S, Korkin D, Hussey R, Baum TJ,  Davis EL, Wang X, Mitchum MG: Dual roles for the variable domain in protein trafficking and host-specific recognition of Heterodera glycines CLE effector proteins. New Phytol 2010, 187:1003-1017. In this work, authors describe that the nematode CLE is delivered to the cytoplasm of syncytial cells, but ultimately function in the apoplast. The truncated CLE or domain swapping experiments also revealed the variable domain of HgCLE preprotein is sufficient for trafficking.

55. Osipova M, Mortier V, Demchenko K, Tsyganov V, Tikhonovich I, Lutova L, Dolgikh E, Goormachtig S: Wuschel-related homeobox5 gene expression and interaction of CLE peptides with components of the systemic control add two pieces to the puzzle of autoregulation of nodulation. Plant Physiol 2012, 158:1329-1341. 56. Strabala T, O’donnell P, Smit A, Ampomah-Dwamena C, Martin E, Netzler N, Nieuwenhuizen N, Quinn B, Foote H, Hudson K: Gainof-function phenotypes of many CLAVATA3/ESR genes, including four new family members, correlate with tandem variations in the conserved CLAVATA3/ESR domain. Plant Physiol 2006, 140:1331-1344. 57. Wang X, Allen R, Ding X, Goellner M, Maier T, de Boer JM, Baum TJ, Hussey RS, Davis EL: Signal peptide-selection of cDNA cloned directly from the esophageal gland cells of the soybean cyst nematode Heterodera glycines. Mol Plant– Microbe Interact 2001, 14:536-544. 58. Replogle A, Wang J, Bleckmann A, Hussey R, Baum T, Sawa S,  Davis E, Wang X, Simon R, Mitchum M: Nematode CLE signaling in Arabidopsis requires CLAVATA2 and CORYNE. Plant J 2012, 65:430-440.

Current Opinion in Plant Biology 2013, 16:598–606

61. Lu S-W, Chen S, Wang J, Yu H, Chronis D, Mitchum MG, Wang X: Structural and functional diversity of CLAVATA3/ESR (CLE)like genes from the potato cyst nematode Globodera rostochiensis. Mol Plant–Microbe Interact 2009, 22:1128-1142. 62. Meng L, Buchanan B, Feldman L, Luan S: CLE-like (CLEL)  peptides control the pattern of root growth and lateral root development in Arabidopsis. Proc Natl Acad Sci U S A 2012, 109:1760-1765. Authors describe that overexpression of the full-length CLE18 genes resulted in long-root phenotype in Arabidopsis, which is the opposite phenotype caused by application of the synthetic CLE18 peptide derived from the CLE domain. In CLE18 precursor protein, the CLE domain is located in the central region whereas the CLEL domain exists in the C terminus region. Exogenous application of the synthetic peptide derived from the CLEL domain induced the long roots, suggesting that CLEL domain is responsible for the phenotype that is caused by overexpression of CLE18. These data indicate that only a single motif is functional even if the precursor protein contains the multiple CLE or CLEL motifs.

www.sciencedirect.com