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.     

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.     

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.     

The CLAVATA1-related BAM1, BAM2 and BAM3 receptor kinase-like proteins are required for meristem function in Arabidopsis

Organ formation at shoot and flower meristems in plants requires the maintenance of a population of centrally located stem cells and the differentiation of peripherally located daughter cells. The CLAVATA (CLV) gene products in Arabidopsis, including the CLV1 receptor-kinase, regulate this process by promoting the differentiation of stem cells on the meristem flanks. Here, we have analyzed the developmental roles of the CLV1-related BAM1 (derived from barely any meristem 1), BAM2 and BAM3 receptor-like kinases. Loss-of-function alleles of these receptors lead to phenotypes consistent with the loss of stem cells at the shoot and flower meristem, suggesting that their developmental role is opposite to that of CLV1. These closely related receptors are further distinguished from CLV1, whose expression and function is highly specific, by having broad expression patterns and multiple developmental roles. These include a requirement for BAM1, BAM2 and BAM3 in the development of high-ordered vascular strands within the leaf and a correlated control of leaf shape, size and symmetry. In addition, BAM1, BAM2 and BAM3 are required for male gametophyte development, as well as ovule specification and function. Significantly, the differing roles of CLV1 and BAM receptors in meristem and organ development are largely driven by differences in expression patterns.     

POLTERGEIST encodes a protein phosphatase 2C that regulates CLAVATA pathways controlling stem cell identity at Arabidopsis shoot and flower meristems

BACKGROUND: Receptor kinases are a large gene family in plants and have more than 600 members in Arabidopsis. Receptor kinases in plants regulate a broad range of developmental processes, including steroid hormone perception, organ elongation, self-incompatibility, and abscission. Intracellular signaling components for receptor kinases in plants are largely unknown. The CLAVATA 1 (CLV1) receptor kinase in Arabidopsis regulates stem cell identity and differentiation through its repression of WUSCHEL (WUS) expression. Mutations at the POLTERGEIST (POL) gene were previously described as phenotypic suppressors of mutations within the CLV1 gene. Genetic evidence placed POL as a downstream regulator of CLAVATA1 signaling.RESULTS: We provide evidence that POL functions in both the CLV1-WUS pathway and a novel WUS-independent CLV1 pathway regulating stem cell identity. We demonstrate that POL encodes a protein phosphatase 2C (PP2C) with a predicted nuclear localization sequence, indicating that it has a role in signal transduction downstream of the CLV1 receptor. The N terminus of POL has a possible regulatory function, and the C terminus has PP2C-like phosphatase catalytic activity. Although the POL catalytic domain is conserved in other PP2Cs, the POL protein represents a unique subclass of plant PP2Cs. POL is broadly expressed throughout the plant.CONCLUSIONS: POL represents a novel component of the CLV1 receptor kinase signaling pathway. The ubiquitous expression of POL and pol phenotypes outside the meristem suggest that POL may be a common regulator of many signaling pathways.     

CLAVATA3-like genes are differentially expressed in grape vine (Vitis vinifera) tissues

The CLAVATA3 (CLV3)/endosperm surrounding region [(ESR) CLE] peptides function as intercellular signaling molecules that regulate various physiological and developmental processes in diverse plant species. We identified five CLV3-like genes from grape vine (Vitis vinifera var. Pinot Noir): VvCLE 6, VvCLE 25-1, VvCLE 25-2, VvCLE 43 and VvCLE TDIF. These CLV3-like genes encode short proteins containing 43–128 amino acids. Except VvCLE TDIF, grape vine CLV3-like proteins possess a consensus amino acid sequence known as the CLE domain. Phylogenic analysis suggests that the VvCLE 6, VvCLE25-1, VvCLE25-2 and VvCLE43 genes have evolved from a single common ancestor to the Arabidopsis CLV3 gene. Expression analyses showed that the five grape CLV3-like genes are expressed in leaves, stems, roots and axillary buds with significant differences in their levels of expression. For example, while all of them were strongly expressed in axillary buds, VvCLE6 and VvCLE43 expression prevailed in roots, and VvCLE25-1, VvCLE25-2 and VvCLE TDIF expression in stems. The differential expression of the five grape CLV3-like peptides suggests that they play different roles in different organs and developmental stages.     

A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro

Plant miRNAs, the critical regulator of gene expression, involve many development processes in vivo. However, the roles of miRNAs in plant cell proliferation and redifferntiation in vitro remain unknown. To determine better the molecular mechanism of these processes, we have recently reported that a set of miRNAs with different expression patterns between cells of totipotent and non-totipotent Arabidopsis calli. Some of these were specifically up- or downregulated during callus formation or shoot regeneration, and other development. Among them, miR160, and one of its target genes, ARF10, regulated Arabidopsis in vitro shoot regeneration via WUS, CLV3 and CUC1/2. The miR160-overexpressing, 35S transgenic lines, exhibited reduced shoot regeneration efficiency. The mARF10, a miR160-resistant form of ARF10, showed a high level of shoot regeneration ability. In the transgenic, expression of the above shoot meristem-specific genes was elevated, which is consistent with the improved shoot regeneration. In contrast, the ARF10 deficient knockout mutant produced fewer regenerated shoot. However, overexpressors of ARF10 were only marginally more efficient than the wild type with the respect to shoot regeneration. Our observation strongly supports that proper shoot regeneration from in vitro cultured cells requires the miR160-directed negative influence of ARF10. The enhanced expression of ARF10 is likely to have contributed to the improved regeneration ability.     

Cytokinin and auxin regulates WUS induction and inflorescence regeneration in vitro in Arabidopsis

Inflorescence regeneration in vitro provides a simplified approach for the study of inflorescence development. In this study, high frequency of regenerated inflorescences was established using Arabidopsis stage-10 pistil as the explants on the inducing medium containing the 2 mg/L zeatin and 0.01 mg/L indole-3-acetic acid. TERMINAL FLOWER 1 (TFL1) expression was detected in callus at 6 days after transferred to inducing medium, and LEAFY (LFY) expression was detectable subsequently, suggesting that both genes play important roles as they function on inflorescence development in the plant. To investigate the formation of the stem cell organizing center, we examined the WUSCHEL (WUS) and CLAVATA3 (CLV3) expression within callus during inflorescence regeneration. WUS signals start to accumulate on callus at 4 days after induction, and then, the CLV3 signals are induced on callus at 5 days on the inflorescence-inducing medium. The expression domain of WUS is below that of CLV3, indicating that the patterns of the organizing center and stem cell formation are similar to that in zygotic and somatic embryogenesis. However, more cells of the organizing center were observed within callus than pro-embryo, suggesting that inflorescence differentiation requires more cells of the organizing center. Furthermore, it was found that the WUS expression is controlled by the ratio of cytokinin with auxin. The results suggest that other factors besides WUS and CLV3 are required for inflorescence regeneration.     

Centering the Organizing Center in the Arabidopsis thaliana Shoot Apical Meristem by a Combination of Cytokinin Signaling and Self-Organization

Plants have the ability to continously generate new organs by maintaining populations of stem cells throught their lives. The shoot apical meristem (SAM) provides a stable environment for the maintenance of stem cells. All cells inside the SAM divide, yet boundaries and patterns are maintained. Experimental evidence indicates that patterning is independent of cell lineage, thus a dynamic self-regulatory mechanism is required. A pivotal role in the organization of the SAM is played by the WUSCHEL gene (WUS). An important question in this regard is that how WUS expression is positioned in the SAM via a cell-lineage independent signaling mechanism. In this study we demonstrate via mathematical modeling that a combination of an inhibitor of the Cytokinin (CK) receptor, Arabidopsis histidine kinase 4 (AHK4) and two morphogens originating from the top cell layer, can plausibly account for the cell lineage-independent centering of WUS expression within SAM. Furthermore, our laser ablation and microsurgical experiments support the hypothesis that patterning in SAM occurs at the level of CK reception and signaling. The model suggests that the interplay between CK signaling, WUS/CLV feedback loop and boundary signals can account for positioning of the WUS expression, and provides directions for further experimental investigation.     

Analysis of interactions among the CLAVATA3 receptors reveals a direct interaction between CLAVATA2 and CORYNE in Arabidopsis

In Arabidopsis, CORYNE (CRN), a new member of the receptor kinase family, was recently isolated as a key player involved in the CLAVATA3 (CLV3) signaling pathway, thereby playing an important role in regulating the development of shoot and root apical meristems. However, the precise relationships among CLAVATA1 (CLV1), CLAVATA2 (CLV2), and CRN receptors remain unclear. Here, we demonstrate the subcellular localization of CRN and analyze the interactions among CLV1, CLV2, and CRN using firefly luciferase complementation imaging (LCI) assays in both Arabidopsis mesophyll protoplasts and Nicotiana benthamiana leaves. Fluorescence targeting showed that CRN was localized to the plasma membrane. The LCI assays coupled with co‐immunoprecipitation assays demonstrated that CLV2 can directly interact with CRN in the absence of CLV3. Additional LCI assays showed that CLV1 did not interact with CLV2, but can interact weakly with CRN. We also found that CLV1 can interact with CLV2–CRN heterodimers, implying that these three proteins may form a complex. Moreover, CRN, rather than CLV1 and CLV2, was able to form homodimers without CLV3 stimulation. Taken together, our results add direct evidence to the newly proposed two‐parallel receptor pathways model and therefore provide new insights into the CLV3 signaling pathway.     

PHOSPHATIDYLSERINE SYNTHASE1 is Required for Inflorescence Meristem and Organ Development in Arabidopsis

<正>Phosphatidylserine(PS),a quantitatively minor membrane phospholipid,is involved in many biological processes besides its role in membrane structure.One PS synthesis gene,PHOSPHATIDYLSERINE SYNTHASE1(PSS1),has been discovered to be required for microspore development in Arabidopsis thaliana L.but how PSS1 affects postembryonic development is still largely unknown.Here,we show that PSS1 is also required for inflorescence meristem and organ development in Arabidopsis.Disruption of PSS1 causes severe dwarfism,smaller lateral organs and reduced size of inflorescence meristem. Morphological and molecular studies suggest that both cell division and cell elongation are affected in the pss1-1 mutant.RNA in situ hybridization and promoter GUS analysis show that expression of both WUSCHEL(WUS) and CLAVATA3(CLV3) depend on PSS1.Moreover,the defect in meristem maintenance is recovered and the expression of WUS and CLV3 are restored in the pss1-1 clv1-1 double mutant. Both SHOOTSTEMLESS(STM) and BREVIPEDICELLUS(BP) are upregulated,and auxin distribution is disrupted in rosette leaves of pss1-1.However,expression of BP,which is also a regulator of internode development,is lost in the pss1-1 inflorescence stem.Our data suggest that PSS1 plays essential roles in inflorescence meristem maintenance through the WUS-CLV pathway,and in leaf and internode development by differentially regulating the class I KNOX genes.