Two receptor-like protein kinases, MUSTACHES and MUSTACHES-LIKE, regulate lateral root development in Arabidopsis thaliana.

Receptor-like protein kinases (RLKs) play key roles in regulating plant growth, development and stress adaptations. There are at least 610 RLKs (including receptor-like cytoplasmic kinases) in Arabidopsis. The functions of the majority of RLKs have not yet been determined. We previously generated promoter::GUS transgenic plants for all leucine-rich repeat (LRR)-RLKs in Arabidopsis and analyzed their expression patterns during various developmental stages. We found the expression of two LRR-RLKs, MUSTACHES (MUS) and MUSTACHES-LIKE (MUL), are overlapped in lateral root primordia. Independent mutants, mus-3 mul-1 and mus-4 mul-2, show a significantly decreased emerged lateral root phenotype. Our analyses indicate that the defects of the double mutant occur mainly at stage I of lateral root development. Exogenous application of auxin can dramatically enhance the transcription of MUS, which is largely dependent on AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19. MUS and MUL are inactive kinases in vitro but are phosphorylated in planta, possibly by an unknown kinase. The kinase activity of MUS is dispensable for its function in lateral root development. Many cell wall related genes are down regulated in mus-3 mul-1. In conclusion, we identified MUS and MUL, two kinase-inactive RLKs, in controlling the early development of lateral root primordia likely via regulating cell wall synthesis and remodeling.

The Control of Cell Expansion, Cell Division, and Vascular Development by Brassinosteroids: A Historical Perspective.

Steroid hormones are important signaling molecules in plants and animals. The plant steroid hormone brassinosteroids were first isolated and characterized in the 1970s and have been studied since then for their functions in plant growth. Treatment of plants or plant cells with brassinosteroids revealed they play important roles during diverse developmental processes, including control of cell expansion, cell division, and vascular differentiation. Molecular genetic studies, primarily in Arabidopsis thaliana, but increasingly in many other plants, have identified many genes involved in brassinosteroid biosynthesis and responses. Here we review the roles of brassinosteroids in cell expansion, cell division, and vascular differentiation, comparing the early physiological studies with more recent results of the analysis of mutants in brassinosteroid biosynthesis and signaling genes. A few representative examples of other molecular pathways that share developmental roles with brassinosteroids are described, including pathways that share functional overlap or response components with the brassinosteroid pathway. We conclude by briefly discussing the origin and conservation of brassinosteroid signaling.

Scanning for New BRI1 Mutations via TILLING Analysis.

The identification and characterization of a mutational spectrum for a specific protein can help to elucidate its detailed cellular functions. BRASSINOSTEROID INSENSITIVE1 (BRI1), a multidomain transmembrane receptor-like kinase, is a major receptor of brassinosteroids in Arabidopsis (Arabidopsis thaliana). Within the last two decades, over 20 different bri1 mutant alleles have been identified, which helped to determine the significance of each domain within BRI1. To further understand the molecular mechanisms of BRI1, we tried to identify additional alleles via targeted induced local lesions in genomes. Here, we report our identification of 83 new point mutations in BRI1, including nine mutations that exhibit an allelic series of typical bri1 phenotypes, from subtle to severe morphological alterations. We carried out biochemical analyses to investigate possible mechanisms of these mutations in affecting brassinosteroid signaling. A number of interesting mutations have been isolated via this study. For example, bri1-702, the only weak allele identified so far with a mutation in the activation loop, showed reduced autophosphorylation activity. bri1-705, a subtle allele with a mutation in the extracellular portion, disrupts the interaction of BRI1 with its ligand brassinolide and coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1. bri1-706, with a mutation in the extracellular portion, is a subtle defective mutant. Surprisingly, root inhibition analysis indicated that it is largely insensitive to exogenous brassinolide treatment. In this study, we found that bri1-301 possesses kinase activity in vivo, clarifying a previous report arguing that kinase activity may not be necessary for the function of BRI1. These data provide additional insights into our understanding of the early events in the brassinosteroid signaling pathway.

Accelerated rates of protein evolution in barley grain and pistil biased genes might be legacy of domestication.

Traits related to grain and reproductive organs in grass crops have been under continuous directional selection during domestication. Barley is one of the oldest domesticated crops in human history. Thus genes associated with the grain and reproductive organs in barley may show evidence of dramatic evolutionary change. To understand how artificial selection contributes to protein evolution of biased genes in different barley organs, we used Digital Gene Expression analysis of six barley organs (grain, pistil, anther, leaf, stem and root) to identify genes with biased expression in specific organs. Pairwise comparisons of orthologs between barley and Brachypodium distachyon, as well as between highland and lowland barley cultivars mutually indicated that grain and pistil biased genes show relatively higher protein evolutionary rates compared with the median of all orthologs and other organ biased genes. Lineage-specific protein evolutionary rates estimation showed similar patterns with elevated protein evolution in barley grain and pistil biased genes, yet protein sequences generally evolve much faster in the lowland barley cultivar. Further functional annotations revealed that some of these grain and pistil biased genes with rapid protein evolution are related to nutrient biosynthesis and cell cycle/division. Our analyses provide insights into how domestication differentially shaped the evolution of genes specific to different organs of a crop species, and implications for future functional studies of domestication genes.

The receptor-like kinases GSO1 and GSO2 together regulate root growth in Arabidopsis through control of cell division and cell fate specification.

BACKGROUND: The root apical meristem of Arabidopsis is established post-embryonically as the main source of root cells, and its activity is maintained by complex bidirectional signaling between stem cells and mature cells. The receptor-like kinases GASSHO1 (GSO1) and GSO2 have been shown to regulate aerial epidermal function and seedling growth in Arabidopsis. RESULTS: Here we show that gso1; gso2 seedlings also have root growth and patterning defects. Analyses of mutant root morphology indicate abnormal numbers of cells in longitudinal files and radial cell layers, as well as aberrant stem cell division planes. gso1; gso2 double mutants misexpress markers for stem cells and differentiated root cell types. In addition, gso1; gso2 root growth defects, but not marker missexpression or patterning phenotypes, are rescued by growth on media containing metabolizable sugars. CONCLUSIONS: We conclude that GSO1 and GSO2 function together in intercellular signaling to positively regulate cell proliferation, differentiation of root cell types, and stem cell identity. In addition, GSO1 and GSO2 control seedling root growth by modulating sucrose response after germination.

The receptor-like kinases GSO1 and GSO2 together regulate root growth in arabidopsis through control of cell division and cell fate specification.

COVER PHOTOGRAPH: Confocal image of a median optical section through a heart stage Arabidopsis embryo expressing the epidermalmarker pATML1:: HTA6-GFP and counterstained with propidium iodide. From The receptor-like kinases GSO1 and GSO2 together regulate root growth in Arabidopsis through control of cell division and cell fate specification; Racolta et al, Developmental Dynamics 243:257-278.

The tyrosine-sulfated peptide receptors PSKR1 and PSY1R modify the immunity of Arabidopsis to biotrophic and necrotrophic pathogens in an antagonistic manner.

The tyrosine-sulfated peptides PSKα and PSY1 bind to specific leucine-rich repeat surface receptor kinases and control cell proliferation in plants. In a reverse genetic screen, we identified the phytosulfokine (PSK) receptor PSKR1 as an important component of plant defense. Multiple independent loss-of-function mutants in PSKR1 are more resistant to biotrophic bacteria, show enhanced pathogen-associated molecular pattern responses and less lesion formation after infection with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. By contrast, pskr1 mutants are more susceptible to necrotrophic fungal infection with Alternaria brassicicola, show more lesion formation and fungal growth which is not observed on wild-type plants. The antagonistic effect on biotrophic and necrotrophic pathogen resistance is reflected by enhanced salicylate and reduced jasmonate responses in the mutants, suggesting that PSKR1 suppresses salicylate-dependent defense responses. Detailed analysis of single and multiple mutations in the three paralogous genes PSKR1, -2 and PSY1-receptor (PSY1R) determined that PSKR1 and PSY1R, but not PSKR2, have a partially redundant effect on plant immunity. In animals and plants, peptide sulfation is catalyzed by a tyrosylprotein sulfotransferase (TPST). Mutants lacking TPST show increased resistance to bacterial infection and increased susceptibility to fungal infection, mimicking the triple receptor mutant phenotypes. Feeding experiments with PSKα in tpst-1 mutants partially restore the defense-related phenotypes, indicating that perception of the PSKα peptide has a direct effect on plant defense. These results suggest that the PSKR subfamily integrates growth-promoting and defense signals mediated by sulfated peptides and modulates cellular plasticity to allow flexible adjustment to environmental changes.

AtPEPTIDE RECEPTOR2 mediates the AtPEPTIDE1-induced cytosolic Ca(2+) rise, which is required for the suppression of Glutamine Dumper gene expression in Arabidopsis roots.

AtPEPTIDE RECEPTOR2 (AtPEPR2) is a member of leucine-rich repeat receptor-like kinase family and binds to a group of AtPROPEP gene-encoded endogenous peptides, AtPeps. Previously, we found that AtPEPR2 plays a moderate role in the AtPep1-mediated innate immunity responses in Arabidopsis leaf. In this study, we found that AtPEPR2 promoter has strong activity in the vascular tissues of the roots and the atpepr2 mutants showed a moderate but significantly shorter root phenotype. AtPEPR2 partially mediated AtPep1-induced root elongation inhibition. AtPep1-triggered cytosolic Ca(2+) transient rise in roots showed partial dependence on AtPEPR2 and fully on extracellular Ca(2+) ([Ca(2+) ]ext ). Transcriptional profiling analysis found that expression of 75% of AtPep1-modulated genes in roots was fully dependent on AtPEPR2, of which two dramatically induced genes showed partial dependence on the [Ca(2+) ]ext . Arabidopsis genome contains seven Glutamine Dumpers genes (AtGDUs), encoding amino acid exporters. Three of them (AtGDU2, 3, 5) were among the top 10 genes that were downregulated by AtPep1 through AtPEPR2 fully dependent pathway. Treatment with AtPep1 strongly suppressed promoter activity of AtGDU3 in roots, which was relieved by chelating [Ca(2+) ]ext . Arabidopsis overexpressing AtGDU3 showed a shorter root phenotype and decreased sensitivity to the AtPep1-mediated inhibition of root elongation. Taken together, this study demonstrated a significant role of AtPEPR2 in the AtPep1-mediated signaling in the roots.

Synergistic interaction of CLAVATA1, CLAVATA2, and RECEPTOR-LIKE PROTEIN KINASE 2 in cyst nematode parasitism of Arabidopsis.

Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (CLE)-like effector proteins. These proteins act as ligand mimics of plant CLE peptides and are required for successful nematode infection. Previously, we showed that the CLV2/CORYNE (CRN) heterodimer receptor complex is required for nematode CLE signaling. However, there was only a partial reduction in nematode infection when this signaling was disrupted, indicating that there might be additional nematode CLE receptors. In this study, we demonstrate that CLV1 and RECEPTOR-LIKE PROTEIN KINASE 2/TOADSTOOL2 (RPK2), two additional receptors that can transmit the CLV3 signal independent of CLV2/CRN for shoot apical meristem maintenance, also play a role in nematode CLE perception. Localization studies showed that both receptors are expressed in nematode-induced syncytia. Infection assays with clv1 and rpk2 single mutants revealed a decrease in both nematode infection and syncytium size. Significantly, further reduction in nematode infection was observed when rpk2 was combined with clv1 and clv2 mutants. Taken together, our results indicate that parallel signaling pathways involving CLV1, CLV2, and RPK2 are important for nematode parasitism.

PEPR2 is a second receptor for the Pep1 and Pep2 peptides and contributes to defense responses in Arabidopsis.

Pep1 is a 23-amino acid peptide that enhances resistance to a root pathogen, Pythium irregulare. Pep1 and its homologs (Pep2 to Pep7) are endogenous amplifiers of innate immunity of Arabidopsis thaliana that induce the transcription of defense-related genes and bind to PEPR1, a plasma membrane leucine-rich repeat (LRR) receptor kinase. Here, we identify a plasma membrane LRR receptor kinase, designated PEPR2, that has 76% amino acid similarity to PEPR1, and we characterize its role in the perception of Pep peptides and defense responses. Both PEPR1 and PEPR2 were transcriptionally induced by wounding, treatment with methyl jasmonate, Pep peptides, and pathogen-associated molecular patterns. The effects of Pep1 application on defense-related gene induction and enhancement of resistance to Pseudomonas syringae pv tomato DC3000 were partially reduced in single mutants of PEPR1 and PEPR2 and abolished completely in double mutants. Photoaffinity labeling and binding assays using transgenic tobacco (Nicotiana tabacum) cells expressing PEPR1 and PEPR2 clearly demonstrated that PEPR1 is a receptor for Pep1-6 and that PEPR2 is a receptor for Pep1 and Pep2. Our analysis demonstrates differential binding affinities of two receptors with a family of peptide ligands and the corresponding physiological effects of the specific receptor-ligand interactions. Therefore, we demonstrate that, through perception of Peps, PEPR1 and PEPR2 contribute to defense responses in Arabidopsis.

Notes from the underground: receptor-like kinases in Arabidopsis root development.

During plant development, the frequency and context of cell division must be controlled, and cells must differentiate properly to perform their mature functions. In addition, stem cell niches need to be maintained as a reservoir for new cells. All of these processes require intercellular signaling, whether it is a cell relaying its position to other cells, or more mature cells signaling to the stem cell niche to regulate the rate of growth. Receptor-like kinases have emerged as a major component in these diverse roles, especially within the Arabidopsis root. In this review, the functions of receptor-like kinase signaling in regulating Arabidopsis root development will be examined in the areas of root apical meristem maintenance, regulation of epidermal cell fate, lateral root development and vascular differentiation. [Figure: see text] Frans E. Tax (Corresponding author).

RPK1 and TOAD2 are two receptor-like kinases redundantly required for arabidopsis embryonic pattern formation.

Although the basic plant body plan is established during embryogenesis, the molecular basis of embryonic patterning remains to be fully understood. We have identified two receptor-like kinases, RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) and TOADSTOOL2 (TOAD2), required for Arabidopsis embryonic pattern formation. Genetic analysis indicates that RPK1 and TOAD2 have overlapping embryonic functions. The zygotic gene dosage of TOAD2 in an rpk1 background is of critical importance, suggesting that signaling mediated by RPK1 and TOAD2 must be above a threshold level for proper embryo development. The localization of RPK1 and TOAD2 translational fusions to GFP coupled with the analysis of cell-type-specific markers indicate that RPK1 and TOAD2 are redundantly required for both pattern formation along the radial axis and differentiation of the basal pole during early embryogenesis. We propose that RPK1 and TOAD2 receive intercellular signals and mediate intracellular responses that are necessary for embryonic pattern formation.

XYLEM INTERMIXED WITH PHLOEM1, a leucine-rich repeat receptor-like kinase required for stem growth and vascular development in Arabidopsis thaliana.

The regulation of cell specification in plants is particularly important in vascular development. The vascular system is comprised two differentiated tissue types, the xylem and phloem, which form conductive elements for the transport of water, nutrients and signaling molecules. A meristematic layer, the procambium, is located between these two differentiated cell types and divides to initiate vascular growth. We report the identification of a receptor-like kinase (RLK) that is expressed in the vasculature. Histochemical analyses of mutants in this kinase display an aberrant accumulation of highly lignified cells, typical of xylem or fiber cells, within the phloem. In addition, phloem cells are sometimes located adjacent to xylem cells in these mutants. We, therefore, named this RLK XYLEM INTERMIXED WITH PHLOEM 1 (XIP1). Analyses of longitudinal profiles of xip1 mutant stems show malformed cell files, indicating defects in oriented cell divisions or cell morphology. We propose that XIP1 prevents ectopic lignification in phloem cells and is necessary to maintain the organization of cell files or cell morphology in conductive elements.

Two receptor-like kinases required together for the establishment of Arabidopsis cotyledon primordia.

Inter-regional signaling coordinates pattern formation in Arabidopsis thaliana embryos. However, little is known regarding the cells and molecules involved in inter-regional communication. We have characterized two related leucine-rich repeat receptor-like kinases (LRR-RLKs), RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) and TOADSTOOL2 (TOAD2), which are required together for patterning the apical embryonic domain cell types that generate cotyledon primordia. Central domain protoderm patterning defects were always observed subjacent to the defective cotyledon primordia cell types in mutant embryos. In addition, RPK1-GFP and TOAD2-GFP translational fusions were both localized to the central domain protodermal cells when cotyledon primordia were first recognizable. We propose that RPK1 and TOAD2 are primarily required to maintain central domain protoderm cell fate and that the loss of this key embryonic cell type in mutant embryos results in patterning defects in other regions of the embryo including the failure to initiate cotyledon primordia.

SERK Receptor-like Kinases Control Division Patterns of Vascular Precursors and Ground Tissue Stem Cells during Embryo Development in Arabidopsis.

During embryo development, the vascular precursors and ground tissue stem cells divide to renew themselves and produce the vascular tissue, endodermal cells, and cortical cells. However, the molecular mechanisms regulating division of these stem cells have remained largely elusive. In this study, we show that loss of function of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes results in aberrant embryo development. Fewer cortical, endodermal, and vascular cells are generated in the embryos of serk1 serk2 bak1 triple mutants. WUSCHEL-RELATED HOMEOBOX 5 (WOX5) is ectopically expressed in vascular cells of serk1 serk2 bak1 embryos. The first transverse division of vascular precursors in mid-globular embryos and second asymmetric division of ground tissue stem cells in early-heart embryos are abnormally altered to a longitudinal division. The embryo defects can be partially rescued by constitutively activated mitogen-activated protein kinase (MAPK) kinase kinase YODA (YDA) and MAPK kinase MKK5. Taken together, our results reveal that SERK-mediated signals regulate division patterns of vascular precursors and ground tissue stem cells, likely via the YDA-MKK4/5 cascade, during embryo development.

Functional analysis of receptor-like kinases in monocots and dicots.

Receptor-like kinases (RLKs) are signaling proteins that feature an extracellular domain connected via a transmembrane domain to a cytoplasmic kinase. This architecture indicates that RLKs perceive external signals, transducing them into the cell. In plants, RLKs were first implicated in the regulation of development, in pathogen responses, and in recognition events. RLKs comprise a major gene family in plants, with more than 600 encoded in the Arabidopsis genome and more than 1100 found in rice genomes. The greater number of RLKs in rice is mostly attributable to expansions in the clades that are involved in pathogen responses. Recent functional studies in both monocots and dicots continue to identify individual RLKs that have similar developmental and abiotic stress roles. Analysis of closely related RLKs reveals that family members might have overlapping roles but can also possess distinct functions.

A Common Pathway of Root Growth Control and Response to CLE Peptides Through Two Receptor Kinases in Arabidopsis.

Cell-cell communication is essential for plants to integrate developmental programs with external cues that affect their growth. Recent advances in plant signaling have uncovered similar molecular mechanisms in shoot, root, and vascular meristem signaling that involve receptor-like kinases and small, secreted peptides. Here, we report that the receptor-like kinases TOAD2/RPK2 and RPK1 regulate root growth by controlling cell proliferation and affecting meristem size. Two types of developmental alterations were observed upon exogenous CLE peptide application. The first type was detected in all plants treated, and comprise increased proliferative activity of cells in the stem cell niche and a delay of progression in differentiation of daughter cells. The second type was changes specific to the genotypes that are sensitive to CLE-driven root meristem inhibition and include a large decrease in the occurrence of cell divisions in longitudinal files, correlating with shorter meristems and cessation of root growth. The root meristems of toad2/rpk2 mutant plants are insensitive to the inhibitory effect of CLE17 peptide treatment, consistent with TOAD2/RPK2 function as a receptor for CLE peptides. In addition, a strong reduction in the expression of RPK1 protein upon CLE treatment, dependent on TOAD2/RPK2, suggests that these two RLKs mediate CLE signaling in a common pathway to control root growth.

An Allelic Series of bak1 Mutations Differentially Alter bir1 Cell Death, Immune Response, Growth, and Root Development Phenotypes in Arabidopsis thaliana.

Receptor-like kinases (RLKs) mediate cell-signaling pathways in Arabidopsis thaliana, including those controlling growth and development, immune response, and cell death. The RLK coreceptor BRI1-ASSOCIATED KINASE-1 (BAK1) partners with multiple ligand-binding RLKs and contributes to their signaling in diverse pathways. An additional RLK, BAK1-INTERACTING RECEPTOR-1 (BIR1), physically interacts with BAK1, and loss-of-function mutations in BIR1 display constitutive activation of cell death and immune response pathways and dwarfism and a reduction in lateral root number. Here we show that bir1 plants display defects in primary root growth, characterize bir1 lateral root defects, and analyze expression of BIR1 and BAK1 promoters within the root. Using an allelic series of bak1 mutations, we show that loss of BAK1 function in immune response pathways can partially suppress bir1 cell death, immune response, and lateral root phenotypes and that null bak1 alleles enhance bir1 primary root phenotypes. Based on our data, we propose a model in which BIR1 functions to regulate BAK1 participation in multiple pathways.

CLAVATA signaling pathway receptors of Arabidopsis regulate cell proliferation in fruit organ formation as well as in meristems.

The CLAVATA1 (CLV1), CLV2, and CORYNE (CRN) receptors in Arabidopsis thaliana maintain cell proliferation in shoot apical meristems by restricting expression of the transcription factor WUSCHEL (WUS). Previously characterized receptor mutants generate extra fruit and floral organs that are proposed to arise from enlarged floral meristems (FMs). We identified new alleles in clv1, clv2, and crn and found that most mutants produce only extra fruit organs and generate FMs of similar dimensions as wild type. Characterization of gynoecium development in receptor mutants revealed increased cell proliferation and ectopic fruit organ initiation after FM termination. These regions of increased cell division also display expanded expression of the cell proliferation-promoting transcription factor SHOOTMERISTEMLESS (STM), similar to the expansion of WUS expression in the shoot apical meristems of strong clv1 mutants. We also examined genetic interactions between the ERECTA (ER) and BARELY ANY MERISTEM 1 (BAM1) receptor-like kinases and CLV pathway receptors. Our results suggest a model in which CLV1/BAM1 and CLV2/CRN complexes act in separate, parallel pathways in shoot meristems, while the CLV1, CLV2, and CRN receptors function together in a linear pathway during fruit development. These results demonstrate the importance of regulating cell proliferation in plants that undergo organogenesis throughout their life cycle.