The CLAVATA signaling pathway mediating stem cell fate in shoot meristems requires Ca2+ as a secondary cytosolic messenger

CLAVATA1 (CLV1) is a receptor protein expressed in the shoot apical meristem (SAM) that translates perception of a non‐cell‐autonomous CLAVATA3 (CLV3) peptide signal into altered stem cell fate. CLV3 reduces expression of WUSCHEL (WUS) and FANTASTIC FOUR 2 (FAF2) in the SAM. Expression of WUS and FAF2 leads to maintenance of undifferentiated stem cells in the SAM. CLV3 binding to CLV1 inhibits expression of these genes and controls stem cell fate in the SAM through an unidentified signaling pathway. Cytosolic Ca²⁺ elevations, cyclic nucleotide (cGMP)‐activated Ca²⁺ channels, and cGMP have been linked to signaling downstream of receptors similar to CLV1. Hence, we hypothesized that cytosolic Ca²⁺ elevation mediates the CLV3 ligand/CLV1 receptor signaling that controls meristem stem cell fate. CLV3 application to Arabidopsis seedlings results in elevation of cytosolic Ca²⁺ and cGMP. CLV3 control of WUS was prevented in a genotype lacking a functional cGMP‐activated Ca²⁺ channel. In wild‐type plants, CLV3 inhibition of WUS and FAF2 expression was impaired by treatment with either a Ca²⁺ channel blocker or a guanylyl cyclase inhibitor. When CLV3‐dependent repression of WUS is blocked, altered control of stem cell fate leads to an increase in SAM size; we observed a larger SAM size in seedlings treated with the Ca²⁺ channel blocker. These results suggest that the CLV3 ligand/CLV1 receptor system initiates a signaling cascade that elevates cytosolic Ca²⁺, and that this cytosolic secondary messenger is involved in the signal transduction cascade linking CLV3/CLV1 to control of gene expression and stem cell fate in the SAM.     

Dual CLAVATA3 peptides in Arabidopsis shoot stem cell signaling

Plant shoot stem cell pool is constantly maintained by a negative feedback loop through peptide-receptor mediated signaling pathway. CLAVATA3 (CLV3) encode a 96 aminoacid protein which is processed to 12-amino-acid or arabinosylated 13-amino-acid peptides, acting as a ligand signal to regulate stem cell homeostasis in the shoot apical meristem (SAM). Although arabinosylated 13-amino-acid CLV3 peptide (CLV3p) shows more significant binding affinity to its receptors and biological activities in the SAM, the physiological function of two mature forms of CLV3p remained an unresolved puzzle in the past decade due to the technical difficulties of arabinosylation modification in the peptide synthesis. Here, we analyzed the role of two mature CLV3 peptides with newly synthesized arabinosylated peptide. Beside shoot meristem phenotypes, arabinosylated CLV3p showed the conventional trait of CLV2-dependent root growth inhibition. Moreover, both 12-amino-acid and arabinosylated 13-amino-acid CLV3 peptides have analogous activities in shoot stem cell signaling. Notably, we demonstrated that non-arabinosylated 12-amino acid CLV3p can affect shoot stem cell signaling at the physiological level unlike previously suggested (Ohyama et al. 2009; Shinohara and Matsubayashi 2013; Shinohara and Matsubayashi 2015). Therefore, these results support the physiological role of the 12-amino-acid CLV3p in shoot stem cell signaling in the deficient condition of arabinosylated 13-amino-acid CLV3p in Arabidopsis thaliana.     

CLE genes may act in a variety of tissues/cells and involve other signaling cascades in addition to CLV3-WUS-like pathways

CLE, which is the term for the CLV3/ESR-related gene family, is thought to participate in CLAVATA3-WUSCHEL (CLV3-WUS) and CLV3-WUS-like signaling pathways to regulate meristem activity in plant. Although some CLE genes are expressed in meristems, many CLE genes appear to express in a variety of tissues/cells. Here we report that CLE14 and CLE20 express in various specific tissues/cells outside the shoot/root apical meristem (SAM/RAM), including in highly differentiated cells, and at different developmental stages. Overexpressing CLE14 or CLE20 also causes multiple phenotypes, which is consistent with its expression pattern in Arabidopsis. These results suggest that CLE genes may play multiple roles and involve other signaling cascades in addition to the CLV3-WUS and CLV3-WUS-like pathways.Key words: CLE, CLAVATA3-WUSCHEL, cell signaling and development, root apical meristem, arabidopsisIntercellular communication and coordination between adjacent cell populations are critical for cell-fate specification, as well as for meristem organization and maintenance. In the shoot apical meristem (SAM), local signaling, which involves the CLAVATA3-WUSCHEL (CLV3-WUS) negative feedback loop, controls stem cell homeostasis and SAM activity.¹ As well, it has been suggested that a CLV3-WUS-like negative feedback pathway operates to control root apical meristem (RAM) activity. This view is supported by the facts that a WUS-related homeobox gene, WOX5, is expressed in cells of the quiescent center (QC) in the RAM, and that loss-of-function of WOX5 in the QC leads to the differentiation of the adjacent root cap initials (RCI), whereas gain-of-function blocks the differentiation of derivatives of the RCI in the root.² Additional support for the function in the RAM of a CLV3-WUS-like pathway, comes from observations that CLE genes (collectively referred to as the CLV3/ESR-relate gene family) are not only expressed in the RAM,^3,4 but also, that overexpression of some CLE genes triggers premature termination of the RAM.⁵ In this regard it has been recently reported that CLE40, which expresses in the differentiating daughter cells of the distal root stem cells, restricts WOX5 expression and promotes differentiation of stem cells in the RAM.⁶ Taken together these data suggest a CLV3-WUS-like feedback loop acts to negatively regulate RAM activity in plants.Our previous results have shown that CLE14 and CLE20 express in specific cells of roots, and that overexpression of CLE14 or CLE20 in Arabidopsis triggers early termination of the RAM in a CLAVATA1 (CLV1)-independent, but CLAVATA2 (CLV2)-dependent manner.^7,8 We also showed that both CLE14 and CLE20 peptides inhibit, irreversibly, root growth by reducing cell division rates in the RAM.⁷ CLV2 and CRN (a receptor-like protein kinase, also known as SOL2, isolated as a suppressor of root-specific overexpression of CLE19) are required for CLE14 and CLE20 peptide functions in vitro.^9,10 Using computational modeling approaches we further demonstrated that 12-amino-acid CLE14 and CLE20 peptides may function through a potential heterodimer/heterotetramer CLV2-CRN complex.⁷CLV3 expresses exclusively in the stem cells of the SAM, and it has been consistently shown that the CLV3 peptide is required for homeostasis of the stem cells and for the maintenance of the SAM.¹ Although some CLE genes are found to express in meristems, many CLE genes appear to express in an array of tissues and cells, including highly differentiated tissues/cells.^3,4 In this report we show that CLE14 and CLE20 express in specific tissues outside the RAM and SAM of Arabidopsis, including highly differentiated cells, and at different developmental stages. Overexpressing CLE14 or CLE20 also causes multiple phenotypes, which is consistent with its expression pattern in Arabidopsis. These results suggest that CLE genes may play multiple roles in regulating the developmental fate of cells, which includes, but is not limited to, stem cells, and also may be involved in other signaling cascades in addition to the CLV3-WUS pathway.     

Contributions of individual amino acid residues to the endogenous CLV3 function in shoot apical meristem maintenance in Arabidopsis

As a peptide hormone, CLV3 restricts the stem cell number in shoot apical meristem (SAM) by interacting with CLV1/CLV2/CRN/RPK2 receptor complexes. To elucidate how the function of the CLV3 peptide in SAM maintenance is established at the amino acid (AA) level, alanine substitutions were performed by introducing point mutations to individual residues in the peptide-coding region of CLV3 and its flanking sequences. Constructs carrying such substitutions, expressed under the control of CLV3 regulatory elements, were transformed to the clv3-2 null mutant to evaluate their efficiencies in complementing its defects in SAMs in vivo. These studies showed that aspartate-8, histidine-11, glycine-6, proline-4, arginine-1, and proline-9, arranged in an order of importance, were critical, while threonine-2, valine-3, serine-5, and the previously assigned hydroxylation and arabinosylation residue proline-7 were trivial for the endogenous CLV3 function in SAM maintenance. In contrast, substitutions of flanking residues did not impose much damage on CLV3. Complementation of different alanine-substituted constructs was confirmed by measurements of the sizes of SAMs and the WUS expression levels in transgenic plants. These studies established a complete contribution map of individual residues in the peptide-coding region of CLV3 for its function in SAM, which may help to understand peptide hormones in general.     

Dual assay for MCLV3 activity reveals structure-activity relationship of CLE peptides

The dodecapeptide MCLV3 is a functional peptide, derived from the CLV3 precursor protein, which is a candidate ligand of the CLV1/CLV2 receptor complex that restricts the stem cell population in the shoot apical meristem (SAM). MCLV3 can induce shoot and root meristem consumption, the typical phenotype of transgenic plants overexpressing CLV3. We investigated the bioactivities of a series of alanine-substituted MCLV3 and related peptides on the root growth of Arabidopsis. The structure-activity relationship (SAR) of MCLV3 had high similarity with that of tracheary element differentiation inhibitory factor (TDIF). We also evaluated the binding activities of the peptides by a competitive receptor binding assay using tritiated MCLV3 and the membrane fraction of a tobacco BY-2 cell line overexpressing the MCLV3 ectodomain. This dual assay, combining a biological and receptor binding assay for evaluating the activities of MCLV3-related peptides, uncovered the SAR of MCLV3, and indicated that the terminal residues play critical roles in exerting its activity and are important for specific binding to the receptor, CLV1.     

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE‐receptor kinase‐WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant‐parasitic cyst nematodes secrete CLE‐like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR‐LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode‐induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock‐down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants.     

RPK2 is an essential receptor-like kinase that transmits the CLV3 signal in Arabidopsis

The shoot apical meristem (SAM) is the fundamental structure that is located at the growing tip and gives rise to all aerial parts of plant tissues and organs, such as leaves, stems and flowers. In Arabidopsis thaliana, the CLAVATA3 (CLV3) pathway regulates the stem cell pool in the SAM, in which a small peptide ligand derived from CLV3 is perceived by two major receptor complexes, CLV1 and CLV2-CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (SOL2), to restrict WUSCHEL (WUS) expression. In this study, we used the functional, synthetic CLV3 peptide (MCLV3) to isolate CLV3-insensitive mutants and revealed that a receptor-like kinase, RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), also known as TOADSTOOL 2 (TOAD2), is another key regulator of meristem maintenance. Mutations in the RPK2 gene result in stem cell expansion and increased number of floral organs, as seen in the other clv mutants. These phenotypes are additive with both clv1 and clv2 mutations. Moreover, our biochemical analyses using Nicotiana benthamiana revealed that RPK2 forms homo-oligomers but does not associate with CLV1 or CLV2. These genetic and biochemical findings suggest that three major receptor complexes, RPK2 homomers, CLV1 homomers and CLV2-CRN/SOL2 heteromers, are likely to mediate three signalling pathways, mainly in parallel but with potential crosstalk, to regulate the SAM homeostasis.     

Individual amino acid residues in CLV3 peptide contribute to its stability in vitro

CLV3 acts as a peptide ligand to interact with leucine-rich repeat (LRR) receptor kinases in neighboring cells to restrict the size of shoot apical meristems (SAMs) in Arabidopsis. To examine contributions of individual amino acid residues in CLV3 peptide in SAM maintenance, 12 synthetic Ala-substituted CLV3 peptides were applied to clv3-2 seedlings cultured in vitro, and the sizes of SAMs were measured after 9 d. The result showed that Pro-9 and His-11 are the most critical residues, while Val-3 and Ser-5 are the least important ones for CLV3 functions in SAMs in vitro. With MALDI-TOF mass spectrum analyses, we further showed that Ala substitution in His-11 led to a greatly reduced stability of the peptide, leading to a complete degradation of the peptide after cultured with seedlings for only one hour. The substitution of Pro-9 by Ala also led to a complete degradation of the peptides after 2 d incubation. In contrast, Ala substitutions in Val-3 or Ser-5 gave very little changes on peptide stabilities. These results suggested that stabilities of Ala-substituted CLV3 peptides are positively correlated with their activities in SAMs. We thus propose that the stability of CLV3 may partially contribute to its function in SAM maintenance.     

BAM receptors regulate stem cell specification and organ development through complex interactions with CLAVATA signaling

The CLAVATA1 (CLV1) receptor kinase regulates stem cell specification at shoot and flower meristems of Arabidopsis. Most clv1 alleles are dominant negative, and clv1 null alleles are weak in phenotype, suggesting additional receptors functioning in parallel. We have identified two such parallel receptors, BAM1 and BAM2. We show that the weak nature of the phenotype of clv1 null alleles is dependent on BAM activity, with bam clv mutants exhibiting severe defects in stem cell specification. Furthermore, BAM activity in the meristem depends on CLV2, which is required in part for CLV1 function. In addition, clv1 mutants enhance many of the Bam⁻ organ phenotypes, indicating that, contrary to current understanding, CLV1 function is not specific to the meristem. CLV3 encodes a small, secreted peptide that acts as the ligand for CLV1. Mutations in clv3 lead to increased stem cell accumulation. Surprisingly, bam1 and bam2 mutants suppress the phenotype of clv3 mutants. We speculate that in addition to redundant function in the meristem center, BAM1 and BAM2 act to sequester CLV3-like ligands in the meristem flanks.     

FON2 SPARE1 Redundantly Regulates Floral Meristem Maintenance with FLORAL ORGAN NUMBER2 in Rice

CLAVATA signaling restricts stem cell identity in the shoot apical meristem (SAM) in Arabidopsis thaliana. In rice (Oryza sativa), FLORAL ORGAN NUMBER2 (FON2), closely related to CLV3, is involved as a signaling molecule in a similar pathway to negatively regulate stem cell proliferation in the floral meristem (FM). Here we show that the FON2 SPARE1 (FOS1) gene encoding a CLE protein functions along with FON2 in maintenance of the FM. In addition, FOS1 appears to be involved in maintenance of the SAM in the vegetative phase, because constitutive expression of FOS1 caused termination of the vegetative SAM. Genetic analysis revealed that FOS1 does not need FON1, the putative receptor of FON2, for its action, suggesting that FOS1 and FON2 may function in meristem maintenance as signaling molecules in independent pathways. Initially, we identified FOS1 as a suppressor that originates from O. sativa indica and suppresses the fon2 mutation in O. sativa japonica. FOS1 function in japonica appears to be compromised by a functional nucleotide polymorphism (FNP) at the putative processing site of the signal peptide. Sequence comparison of FOS1 in about 150 domesticated rice and wild rice species indicates that this FNP is present only in japonica, suggesting that redundant regulation by FOS1 and FON2 is commonplace in species in the Oryza genus. Distribution of the FNP also suggests that this mutation may have occurred during the divergence of japonica from its wild ancestor. Stem cell maintenance may be regulated by at least three negative pathways in rice, and each pathway may contribute differently to this regulation depending on the type of the meristem. This situation contrasts with that in Arabidopsis, where CLV signaling is the major single pathway in all meristems.     

CLAVATA2 forms a distinct CLE‐binding receptor complex regulating Arabidopsis stem cell specification

CLAVATA1 (CLV1), CLV2, CLV3, CORYNE (CRN), BAM1 and BAM2 are key regulators that function at the shoot apical meristem (SAM) of plants to promote differentiation by limiting the size of the organizing center that maintains stem cell identity in neighboring cells. Previous results have indicated that the extracellular domain of the receptor kinase CLV1 binds to the CLV3‐derived CLE ligand. The biochemical role of the receptor‐like protein CLV2 has remained largely unknown. Although genetic analysis suggested that CLV2, together with the membrane kinase CRN, acts in parallel with CLV1, recent studies using transient expression indicated that CLV2 and CRN from a complex with CLV1. Here, we report detection of distinct CLV2‐CRN heteromultimeric and CLV1‐BAM multimeric complexes in transient expression in tobacco and in Arabidopsis meristems. Weaker interactions between the two complexes were detectable in transient expression. We also find that CLV2 alone generates a membrane‐localized CLE binding activity independent of CLV1. CLV2, CLV1 and the CLV1 homologs BAM1 and BAM2 all bind to the CLV3‐derived CLE peptide with similar kinetics, but BAM receptors show a broader range of interactions with different CLE peptides. Finally, we show that BAM and CLV1 overexpression can compensate for the loss of CLV2 function in vivo. These results suggest two parallel ligand‐binding receptor complexes controlling stem cell specification in Arabidopsis.     

Kip-related protein 3 is required for control of endoreduplication in the shoot apical meristem and leaves of Arabidopsis

The cell cycle plays an important role in the development and adaptation of multicellular organisms; specifically, it allows them to optimally adjust their architecture in response to environmental changes. Kip-related proteins (KRPs) are important negative regulators of cyclin-dependent kinases (CDKs), which positively control the cell cycle during plant development. The Arabidopsis genome possesses seven KRP genes with low sequence similarity and distinct expression patterns; however, why Arabidopsis needs seven KRP genes and how these genes function in cell cycle regulation are unknown. Here, we focused on the characterization of KRP3, which was found to have unique functions in the shoot apical meristem (SAM) and leaves. KRP3 protein was localized to the SAM, including the ground meristem and vascular tissues in the ground part of the SAM and cotyledons. In addition, KRP3 protein was stabilized when treated with MG132, an inhibitor of the 26S proteasome, indicating that the protein may be regulated by 26S proteasome-mediated protein degradation. KRP3-overexpressing (KRP3 OE) transgenic plants showed reduced organ size, serrated leaves, and reduced fertility. Interestingly, the KRP3 OE transgenic plants showed a significant reduction in the size of the SAM with alterations in cell arrangement. In addition, compared to the wild type, the KRP3 OE transgenic plants had a higher DNA ploidy level in the SAM and leaves. Taken together, our data suggest that KRP3 plays important regulatory roles in the cell cycle and endoreduplication in the SAM and leaves.