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.     

Heterotrimeric G proteins control stem cell proliferation through CLAVATA signaling in Arabidopsis

Cell-to-cell communication is a fundamental mechanism for coordinating developmental and physiological events in multicellular organisms. Heterotrimeric G proteins are key molecules that transmit extracellular signals; similarly, CLAVATA signaling is a crucial regulator in plant development. Here, we show that Arabidopsis thaliana Gβ mutants exhibit an enlarged stem cell region, which is similar to that of clavata mutants. Our genetic and cell biological analyses suggest that the G protein beta-subunit1 AGB1 and RPK2, one of the major CLV3 peptide hormone receptors, work synergistically in stem cell homeostasis through their physical interactions. We propose that AGB1 and RPK2 compose a signaling module to facilitate meristem development.     

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.     

CLV3 is localized to the extracellular space,where it activates the Arabidopsis CLAVATA stem cell signaling pathway

Plant growth and development depends on the activity of a continuously replenished pool of stem cells within the shoot apical meristem to supply cells for organogenesis. In Arabidopsis, the stem cell-specific protein CLAVATA3 (CLV3) acts cell nonautonomously to restrict the size of the stem cell population, but the hypothesis that CLV3 acts as an extracellular signaling molecule has not been tested. We used genetic and immunological assays to show that CLV3 localizes to the apoplast and that export to the extracellular space is required for its function in activating the CLV1/CLV2 receptor complex. Apoplastic localization allows CLV3 to signal from the stem cell population to the organizing center in the underlying cells.     

Nematode CLE signaling in Arabidopsis requires CLAVATA2 and CORYNE

Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ESR (CLE)-like effector proteins. These proteins have been shown to act as ligand mimics of plant CLE peptides and are required for successful nematode infection; however, the receptors for nematode CLE-like peptides have not been identified. Here we demonstrate that CLV2 and CORYNE (CRN), members of the receptor kinase family, are required for nematode CLE signaling. Exogenous peptide assays and overexpression of nematode CLEs in Arabidopsis demonstrated that CLV2 and CRN are required for perception of nematode CLEs. In addition, promoter-reporter assays showed that both receptors are expressed in nematode-induced syncytia. Lastly, infection assays with receptor mutants revealed a decrease in both nematode infection and syncytium size. Taken together, our results indicate that perception of nematode CLEs by CLV2 and CRN is not only required for successful nematode infection but is also involved in the formation and/or maintenance of nematode-induced syncytia.     

Signals derived from YABBY gene activities in organ primordia regulate growth and partitioning of Arabidopsis shoot apical meristems

Shoot apical meristems (SAMs) are self-sustaining groups of cells responsible for the ordered initiation of all aerial plant tissues, including stems and lateral organs. The precise coordination of these processes argues for crosstalk between the different SAM domains. The products of YABBY (YAB) genes are limited to the organ primordium domains, which are situated at the periphery of all SAMs and which are separated by a margin of three to seven cells from the central meristem zone marked by WUSCHEL and CLAVATA3 expression. Mutations in the two related YAB1 genes, FILAMENTOUS FLOWER and YABBY3 (YAB3), cause an array of defects, including aberrant phyllotaxis. We show that peripheral YAB1 activity nonautonomously and sequentially affects the phyllotaxis and growth of subsequent primordia and coordinates the expression of SAM central zone markers. These effects support a role for YAB1 genes in short-range signaling. However, no evidence was found that YAB1 gene products are themselves mobile. A screen for suppression of a floral YAB1 overexpression phenotype revealed that the YAB1-born signals are mediated in part by the activity of LATERAL SUPPRESSOR. This GRAS protein is expressed at the boundary of organ primordia and the SAM central zone, distinct from the YAB1 expression domain. Together, these results suggest that YAB1 activity stimulates signals from the organs to the meristem via a secondary message or signal cascade, a process essential for organized growth of the SAM.     

Salt modulates gravity signaling pathway to regulate growth direction of primary roots in Arabidopsis

Plant root architecture is highly plastic during development and can adapt to many environmental stresses. The proper distribution of roots within the soil under various conditions such as salinity, water deficit, and nutrient deficiency greatly affects plant survival. Salinity profoundly affects the root system architecture of Arabidopsis (Arabidopsis thaliana). However, despite the inhibitory effects of salinity on root length and the number of roots, very little is known concerning influence of salinity on root growth direction and the underlying mechanisms. Here we show that salt modulates root growth direction by reducing the gravity response. Exposure to salt stress causes rapid degradation of amyloplasts in root columella cells of Arabidopsis. The altered root growth direction in response to salt was found to be correlated with PIN-FORMED2 (PIN2) messenger RNA abundance and expression and localization of the protein. Furthermore, responsiveness to gravity of salt overly sensitive (sos) mutants is substantially reduced, indicating that salt-induced altered gravitropism of root growth is mediated by ion disequilibrium. Mutation of SOS genes also leads to reduced amyloplast degradation in root tip columella cells and the defects in PIN2 gene expression in response to salt stress. These results indicate that the SOS pathway may mediate the decrease of PIN2 messenger RNA in salinity-induced modification of gravitropic response in Arabidopsis roots. Our findings provide new insights into the development of a root system necessary for plant adaptation to high salinity and implicate an important role of the SOS signaling pathway in this process.     

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.     

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.     

Functional identification of AtSKIP as a regulator of the cell cycle signaling pathway in Arabidopsis thaliana

Light is a key environmental cue controlling plant development, which involves meristemic activation by cell proliferation and differentiation. Here, we identify one gene, AtSKIP, associated with cell cycle-regulated root and leaf growth processes in Arabidopsis. The spatial pattern of β-glucuronidase (GUS) activity indicated that AtSKIP is expressed in the leaf primodia, root meristem region and root vascular system, and can be activated by light. Ectopic expression of AtSKIP resulted in enhanced leaf development but suppressed root elongation in Arabidopsis, whereas AtSKIP^DD seedlings displayed retarded leaf growth and normal root growth. Moreover, AtSKIP cells displayed enhanced sensitivity to a cytokinin in a callus induction assay, further demonstrated that AtSKIP expression altered endogenous cell cycle-regulated signaling in plants. Together, these data indicate that AtSKIP participates in cell cycle-mediated growth of leaf and root.     

Beta 3-adrenergic receptors regulate retinal endothelial cell migration and proliferation

Sympathetic nerves may play a role in vascular disorders of the eye. In the present study, we hypothesized that activation of beta3-adrenergic receptors on retinal endothelial cells would promote migration and proliferation of these cells, two markers of an angiogenic phenotype. We show, for the first time, expression of beta3-adrenergic receptors on cultured retinal endothelial cells. Activation of these receptors with BRL37344, a specific beta3-adrenergic receptor agonist, promoted migration that was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K), the mitogen activated protein kinase component MEK, and matrix metalloproteinases (MMPs) 2 and 9. BRL37344 stimulated proliferation, which could be blocked by inhibitors of Src, PI3K, and MEK. These cells also express the beta1-adrenergic receptor with no beta2-adrenergic receptor expression observed. Stimulation of the beta1-adrenergic receptor with xamoterol, a specific partial agonist, did not promote proliferation or migration. These results support the hypothesis that beta3-adrenergic receptors play a role in proliferation and migration of cultured human retinal endothelial cells.     

Autonomy of cell proliferation and developmental programs during Arabidopsis aboveground organ morphogenesis

Elaboration of size and shape in multicellular organisms involves coordinated cell division and cell growth. In higher plants, continuity of cell layer structures exists from the shoot apical meristem (SAM), where organ primordia arise, to mature aboveground organs. To unravel the extent of inter-cell layer coordination during SAM and aboveground organ development, cell division in the epidermis was selectively restricted by expressing two cyclin-dependent kinase inhibitor genes, KRP1/ICK1 and KRP4, driven by the L1 layer-specific AtML1 promoter. The transgenes conferred reduced plant size with striking, distorted lateral organ shape. While epidermal cell division was severely inhibited with compensatory cell size enlargement, the underlying mesophyll/cortex layer kept normal cell numbers and resulted in small, packed cells with disrupted cell files. Our results demonstrate the autonomy of cell number checkpoint in the underlying tissues when epidermal cell division is restricted. Finally, the L1 layer-specific expression of both KRP1/ICK1 and KRP4 showed no effects on the structure and function of the SAM, suggesting that the effects of these cyclin-dependent kinase inhibitors are context dependent.