The CLE33 peptide represses phloem differentiation via autocrine and paracrine signaling in Arabidopsis.

Plant meristems require a constant supply of photoassimilates and hormones to the dividing meristematic cells. In the growing root, such supply is delivered by protophloem sieve elements. Due to its preeminent function for the root apical meristem, protophloem is the first tissue to differentiate. This process is regulated by a genetic circuit involving in one side the positive regulators DOF transcription factors, OCTOPUS (OPS) and BREVIX RADIX (BRX), and in the other side the negative regulators CLAVATA3/EMBRYO SURROUNDING REGION RELATED (CLE) peptides and their cognate receptors BARELY ANY MERISTEM (BAM) receptor-like kinases. brx and ops mutants harbor a discontinuous protophloem that can be fully rescued by mutation in BAM3, but is only partially rescued when all three known phloem-specific CLE genes, CLE25/26/45 are simultaneously mutated. Here we identify a CLE gene closely related to CLE45, named CLE33. We show that double mutant cle33cle45 fully suppresses brx and ops protophloem phenotype. CLE33 orthologs are found in basal angiosperms, monocots, and eudicots, and the gene duplication which gave rise to CLE45 in Arabidopsis and other Brassicaceae appears to be a recent event. We thus discovered previously unidentified Arabidopsis CLE gene that is an essential player in protophloem formation.

Deeper genomic insights into tomato CLE genes repertoire identify new active peptides.

BACKGROUND: In eukaryotes, cell-to-cell communication relies on the activity of small signaling peptides. In plant genomes, many hundreds of genes encode for such short peptide signals. However, only few of them are functionally characterized and due to the small gene size and high sequence variability, the comprehensive identification of such peptide-encoded genes is challenging. The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-RELATED (CLE) gene family encodes for short peptides that have a role in plant meristem maintenance, vascular patterning and responses to environment. The full repertoire of CLE genes and the role of CLE signaling in tomato (Solanum lycopersicum)- one of the most important crop plants- has not yet been fully studied. RESULTS: By using a combined approach, we performed a genome-wide identification of CLE genes using the current tomato genome version SL 4.0. We identified 52 SlCLE genes, including 37 new non annotated before. By analyzing publicly available RNAseq datasets we could confirm the expression of 28 new SlCLE genes. We found that SlCLEs are often expressed in a tissue-, organ- or condition-specific manner. Our analysis shows an interesting gene diversification within the SlCLE family that seems to be a result of gene duplication events. Finally, we could show a biological activity of selected SlCLE peptides in the root growth arrest that was SlCLV2-dependent. CONCLUSIONS: Our improved combined approach revealed 37 new SlCLE genes. These findings are crucial for better understanding of the CLE signaling in tomato. Our phylogenetic analysis pinpoints the closest homologs of Arabidopsis CLE genes in tomato genome and can give a hint about the function of newly identified SlCLEs. The strategy described here can be used to identify more precisely additional short genes in plant genomes. Finally, our work suggests that the mechanism of root-active CLE peptide perception is conserved between Arabidopsis and tomato. In conclusion, our work paves the way to further research on the CLE-dependent circuits modulating tomato development and physiological responses.

Understanding the root xylem plasticity for designing resilient crops.

Xylem is the main route for transporting water, minerals and a myriad of signalling molecules within the plant. With its onset during early embryogenesis, the development of the xylem relies on hormone gradients, the activity of unique transcription factors, the distribution of mobile microRNAs, and receptor-ligand pathways. These regulatory mechanisms are often interconnected and together contribute to the plasticity of this water-conducting tissue. Environmental stresses, such as drought and salinity, have a great impact on xylem patterning. A better understanding of how the structural properties of the xylem are regulated in normal and stress conditions will be instrumental in developing crops of the future. In addition, vascular wilt pathogens that attack the xylem are becoming increasingly problematic. Further knowledge of xylem development in response to these pathogens will bring new solutions against these diseases. In this review, we summarize recent findings on the molecular mechanisms of xylem formation that largely come from Arabidopsis research with additional insights from tomato and monocot species. We emphasize the impact of abiotic factors and pathogens on xylem plasticity and the urgent need to uncover the underlying mechanisms. Finally, we discuss the multidisciplinary approach to model xylem capacities in crops.

BAM1/2 receptor kinase signaling drives CLE peptide-mediated formative cell divisions in Arabidopsis roots.

Cell division is often regulated by extracellular signaling networks to ensure correct patterning during development. In Arabidopsis, the SHORT-ROOT (SHR)/SCARECROW (SCR) transcription factor dimer activates CYCLIND6;1 (CYCD6;1) to drive formative divisions during root ground tissue development. Here, we show plasma-membrane-localized BARELY ANY MERISTEM1/2 (BAM1/2) family receptor kinases are required for SHR-dependent formative divisions and CYCD6;1 expression, but not SHR-dependent ground tissue specification. Root-enriched CLE ligands bind the BAM1 extracellular domain and are necessary and sufficient to activate SHR-mediated divisions and CYCD6;1 expression. Correspondingly, BAM-CLE signaling contributes to the restriction of formative divisions to the distal root region. Additionally, genetic analysis reveals that BAM-CLE and SHR converge to regulate additional cell divisions outside of the ground tissues. Our work identifies an extracellular signaling pathway regulating formative root divisions and provides a framework to explore this pathway in patterning and evolution.

A Cellular Insulator against CLE45 Peptide Signaling.

Plants continuously elaborate their bodies through post-embryonic, reiterative organ formation by apical meristems [1]. Meristems harbor stem cells, which produce daughter cells that divide repeatedly before they differentiate. How transitions between stemness, proliferation, and differentiation are precisely coordinated is not well understood, but it is known that phytohormones as well as peptide signals play important roles [2-7]. For example, in Arabidopsis thaliana root meristems, developing protophloem sieve elements (PPSEs) express the secreted CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 45 (CLE45) peptide and its cognate receptor, the leucine-rich repeat receptor kinase (LRR-RK) BARELY ANY MERISTEM 3 (BAM3). Exogenous CLE45 application or transgenically increased CLE45 dosage impairs protophloem formation, suggesting autocrine inhibition of PPSE differentiation by CLE45 signaling. Since CLE45 and BAM3 are expressed throughout PPSE development, it remains unclear how this inhibition is eventually overcome. The OCTOPUS (OPS) gene is required for proper PPSE differentiation and therefore the formation of continuous protophloem strands. OPS dosage increase can mend the phenotype of other mutants that display protophloem development defects in association with CLE45-BAM3 hyperactivity [8, 9]. Here, we provide evidence that OPS protein promotes differentiation of developing PPSEs by dampening CLE45 perception. This markedly quantitative antagonism is likely mediated through direct physical interference of OPS with CLE45 signaling component interactions. Moreover, hyperactive OPS confers resistance to other CLE peptides, and ectopic OPS overexpression triggers premature differentiation throughout the root. Our results thus reveal a novel mechanism in PPSE transition toward differentiation, wherein OPS acts as an "insulator" to antagonize CLE45 signaling.

A novel Ca2+-binding protein that can rapidly transduce auxin responses during root growth.

Signaling cross talks between auxin, a regulator of plant development, and Ca2+, a universal second messenger, have been proposed to modulate developmental plasticity in plants. However, the underlying molecular mechanisms are largely unknown. Here, we report that in Arabidopsis roots, auxin elicits specific Ca2+ signaling patterns that spatially coincide with the expression pattern of auxin-regulated genes. We have identified the single EF-hand Ca2+-binding protein Ca2+-dependent modulator of ICR1 (CMI1) as an interactor of the Rho of plants (ROP) effector interactor of constitutively active ROP (ICR1). CMI1 expression is directly up-regulated by auxin, whereas the loss of function of CMI1 associates with the repression of auxin-induced Ca2+ increases in the lateral root cap and vasculature, indicating that CMI1 represses early auxin responses. In agreement, cmi1 mutants display an increased auxin response including shorter primary roots, longer root hairs, longer hypocotyls, and altered lateral root formation. Binding to ICR1 affects subcellular localization of CMI1 and its function. The interaction between CMI1 and ICR1 is Ca2+-dependent and involves a conserved hydrophobic pocket in CMI1 and calmodulin binding-like domain in ICR1. Remarkably, CMI1 is monomeric in solution and in vitro changes its secondary structure at cellular resting Ca2+ concentrations ranging between 10-9 and 10-8 M. Hence, CMI1 is a Ca2+-dependent transducer of auxin-regulated gene expression, which can function in a cell-specific fashion at steady-state as well as at elevated cellular Ca2+ levels to regulate auxin responses.

Perception of root-active CLE peptides requires CORYNE function in the phloem vasculature.

Arabidopsis root development is orchestrated by signaling pathways that consist of different CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide ligands and their cognate CLAVATA (CLV) and BARELY ANY MERISTEM (BAM) receptors. How and where different CLE peptides trigger specific morphological or physiological changes in the root is poorly understood. Here, we report that the receptor-like protein CLAVATA 2 (CLV2) and the pseudokinase CORYNE (CRN) are necessary to fully sense root-active CLE peptides. We uncover BAM3 as the CLE45 receptor in the root and biochemically map its peptide binding surface. In contrast to other plant peptide receptors, we found no evidence that SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) proteins act as co-receptor kinases in CLE45 perception. CRN stabilizes BAM3 expression and thus is required for BAM3-mediated CLE45 signaling. Moreover, protophloem-specific CRN expression complements resistance of the crn mutant to root-active CLE peptides, suggesting that protophloem is their principal site of action. Our work defines a genetic framework for dissecting CLE peptide signaling and CLV/BAM receptor activation in the root.

Phosphosite charge rather than shootward localization determines OCTOPUS activity in root protophloem.

Polar cellular localization of proteins is often associated with their function and activity. In plants, relatively few polar-localized factors have been described. Among them, the plasma membrane-associated Arabidopsis proteins OCTOPUS (OPS) and BREVIS RADIX (BRX) display shootward and rootward polar localization, respectively, in developing root protophloem cells. Both ops and brx null mutants exhibit defects in protophloem differentiation. Here we show that OPS and BRX act genetically in parallel in this process, although OPS dosage increase mends defects caused by brx loss-of-function. OPS protein function is ancient and conserved in the most basal angiosperms; however, many highly conserved structural OPS features are not strictly required for OPS function. They include a BRASSINOSTEROID INSENSITIVE 2 (BIN2) interaction domain, which supposedly mediates gain-of-function effects obtained through ectopic OPS overexpression. However, engineering an increasingly positive charge in a critical phosphorylation site, S318, progressively amplifies OPS activity. Such hyperactive OPS versions can even complement the severe phenotype of brx ops double mutants, and the most active variants eventually trigger gain-of-function phenotypes. Finally, BRX-OPS as well as OPS-BRX fusion proteins localize to the rootward end of developing protophloem cells, but complement ops mutants as efficiently as shootward localized OPS. Thus, our results suggest that S318 phosphorylation status, rather than a predominantly shootward polar localization, is a primary determinant of OPS activity.

CLAVATA 1-type receptors in plant development.

A fundamental aspect of plant development is the coordination of growth through endogenous signals and its integration with environmental inputs. Similar to animals, plants frequently use cell surface-localized receptors to monitor such stimuli, for instance through plasma membrane-integral receptor-like kinases (RLKs). Compared to other organisms, plants possess a large number of RLKs (more than 600 in Arabidopsis thaliana), which implies that ligand-receptor-mediated molecular mechanisms regulate a wide range of processes during plant development. Here, we focus on A. thaliana RLKs of the CLAVATA 1 (CLV1) type, which orchestrate key steps during plant development, including the regulation of meristem maintenance, anther development, vascular tissue formation, and root system architecture. These receptors are regulated by small signalling peptides that belong to the family of CLE (CLV3 / EMBRYO SURROUNDING REGION) ligands. We discuss different aspects of plant development that are regulated by these receptors in light of their molecular mechanism of action. As so often, the intensive research on this group of plant RLKs has raised many intriguing questions, which remain to be answered.

Bimodal regulation of ICR1 levels generates self-organizing auxin distribution.

Auxin polar transport, local maxima, and gradients have become an important model system for studying self-organization. Auxin distribution is regulated by auxin-dependent positive feedback loops that are not well-understood at the molecular level. Previously, we showed the involvement of the RHO of Plants (ROP) effector INTERACTOR of CONSTITUTIVELY active ROP 1 (ICR1) in regulation of auxin transport and that ICR1 levels are posttranscriptionally repressed at the site of maximum auxin accumulation at the root tip. Here, we show that bimodal regulation of ICR1 levels by auxin is essential for regulating formation of auxin local maxima and gradients. ICR1 levels increase concomitant with increase in auxin response in lateral root primordia, cotyledon tips, and provascular tissues. However, in the embryo hypophysis and root meristem, when auxin exceeds critical levels, ICR1 is rapidly destabilized by an SCF(TIR1/AFB) [SKP, Cullin, F-box (transport inhibitor response 1/auxin signaling F-box protein)]-dependent auxin signaling mechanism. Furthermore, ectopic expression of ICR1 in the embryo hypophysis resulted in reduction of auxin accumulation and concomitant root growth arrest. ICR1 disappeared during root regeneration and lateral root initiation concomitantly with the formation of a local auxin maximum in response to external auxin treatments and transiently after gravitropic stimulation. Destabilization of ICR1 was impaired after inhibition of auxin transport and signaling, proteasome function, and protein synthesis. A mathematical model based on these findings shows that an in vivo-like auxin distribution, rootward auxin flux, and shootward reflux can be simulated without assuming preexisting tissue polarity. Our experimental results and mathematical modeling indicate that regulation of auxin distribution is tightly associated with auxin-dependent ICR1 levels.

The Arabidopsis Rho of plants GTPase AtROP6 functions in developmental and pathogen response pathways.

How plants coordinate developmental processes and environmental stress responses is a pressing question. Here, we show that Arabidopsis (Arabidopsis thaliana) Rho of Plants6 (AtROP6) integrates developmental and pathogen response signaling. AtROP6 expression is induced by auxin and detected in the root meristem, lateral root initials, and leaf hydathodes. Plants expressing a dominant negative AtROP6 (rop6(DN)) under the regulation of its endogenous promoter are small and have multiple inflorescence stems, twisted leaves, deformed leaf epidermis pavement cells, and differentially organized cytoskeleton. Microarray analyses of rop6(DN) plants revealed that major changes in gene expression are associated with constitutive salicylic acid (SA)-mediated defense responses. In agreement, their free and total SA levels resembled those of wild-type plants inoculated with a virulent powdery mildew pathogen. The constitutive SA-associated response in rop6(DN) was suppressed in mutant backgrounds defective in SA signaling (nonexpresser of PR genes1 [npr1]) or biosynthesis (salicylic acid induction deficient2 [sid2]). However, the rop6(DN) npr1 and rop6(DN) sid2 double mutants retained the aberrant developmental phenotypes, indicating that the constitutive SA response can be uncoupled from ROP function(s) in development. rop6(DN) plants exhibited enhanced preinvasive defense responses to a host-adapted virulent powdery mildew fungus but were impaired in preinvasive defenses upon inoculation with a nonadapted powdery mildew. The host-adapted powdery mildew had a reduced reproductive fitness on rop6(DN) plants, which was retained in mutant backgrounds defective in SA biosynthesis or signaling. Our findings indicate that both the morphological aberrations and altered sensitivity to powdery mildews of rop6(DN) plants result from perturbations that are independent from the SA-associated response. These perturbations uncouple SA-dependent defense signaling from disease resistance execution.

RAC/ROP GTPases and auxin signaling.

Auxin functions as a key morphogen in regulating plant growth and development. Studies on auxin-regulated gene expression and on the mechanism of polar auxin transport and its asymmetric distribution within tissues have provided the basis for realizing the molecular mechanisms underlying auxin function. In eukaryotes, members of the Ras and Rho subfamilies of the Ras superfamily of small GTPases function as molecular switches in many signaling cascades that regulate growth and development. Plants do not have Ras proteins, but they contain Rho-like small G proteins called RACs or ROPs that, like fungal and metazoan Rhos, are regulators of cell polarity and may also undertake some Ras functions. Here, we discuss the advances made over the last decade that implicate RAC/ROPs as mediators for auxin-regulated gene expression, rapid cell surface-located auxin signaling, and directional auxin transport. We also describe experimental data indicating that auxin-RAC/ROP crosstalk may form regulatory feedback loops and theoretical modeling that attempts to connect local auxin gradients with RAC/ROP regulation of cell polarity. We hope that by discussing these experimental and modeling studies, this perspective will stimulate efforts to further refine our understanding of auxin signaling via the RAC/ROP molecular switch.

An auxin regulated positive feedback loop integrates Rho modulated cell polarity with pattern formation.

In plants, polar transport of the hormone auxin between cells connects cell polarity and pattern formation and is thus required for plant development. The direction of auxin transport is determined by polar localization of PIN auxin efflux transporters. The dynamic polar localization of PIN proteins depends on constitutive endocytic recycling to and from the plasma membrane. However, it was not well understood how PIN polarization is connected to regulators of cell polarity. In a paper that was published in the January issue of PLoS Biology, 2010 we described the involvement of the ROP GTPase associated scaffold Interactor of Constitutive active ROP 1 (ICR1) in recruitment of PIN proteins to polar domains in the plasma membrane. icr1 mutant plants and embryos have severe developmental aberrations that are caused by abnormal auxin distribution. ICR1 functions at or close to the plasma membrane where it is required for exocytosis. ICR1 transcription is quickly induced by auxin but is post-transcriptionally repressed at the site of stable auxin maximum in embryos and roots. Thus, ICR1 integrates auxin-regulated transcription with ROP modulated cell polarity.

A rho scaffold integrates the secretory system with feedback mechanisms in regulation of auxin distribution.

Development in multicellular organisms depends on the ability of individual cells to coordinate their behavior by means of small signaling molecules to form correctly patterned tissues. In plants, a unique mechanism of directional transport of the signaling molecule auxin between cells connects cell polarity and tissue patterning and thus is required for many aspects of plant development. Direction of auxin flow is determined by polar subcellular localization of PIN auxin efflux transporters. Dynamic PIN polar localization results from the constitutive endocytic cycling to and from the plasma membrane, but it is not well understood how this mechanism connects to regulators of cell polarity. The Rho family small GTPases ROPs/RACs are master regulators of cell polarity, however their role in regulating polar protein trafficking and polar auxin transport has not been established. Here, by analysis of mutants and transgenic plants, we show that the ROP interactor and polarity regulator scaffold protein ICR1 is required for recruitment of PIN proteins to the polar domains at the plasma membrane. icr1 mutant embryos and plants display an a array of severe developmental aberrations that are caused by compromised differential auxin distribution. ICR1 functions at the plasma membrane where it is required for exocytosis but does not recycle together with PINs. ICR1 expression is quickly induced by auxin but is suppressed at the positions of stable auxin maxima in the hypophysis and later in the embryonic and mature root meristems. Our results imply that ICR1 is part of an auxin regulated positive feedback loop realized by a unique integration of auxin-dependent transcriptional regulation into ROP-mediated modulation of cell polarity. Thus, ICR1 forms an auxin-modulated link between cell polarity, exocytosis, and auxin transport-dependent tissue patterning.

A novel ROP/RAC GTPase effector integrates plant cell form and pattern formation.

ROPs/RACs are the only known signaling Ras superfamily small GTPases in plants. As such they have been suggested to function as central regulators of diverse signaling cascades. The ROP/RAC signaling networks are largely unknown, however, because only few of their effector proteins have been identified. In a paper that was published in the June 5, 2007 issue of Current Biology we described the identification of a novel ROP/RAC effector designated ICR1 (Interactor of Constitutive active ROPs 1). We demonstrated that ICR1 functions as a scaffold that interacts with diverse but specific group of proteins including SEC3 subunit of the exocyst vesicle tethering complex. ICR1-SEC3 complexes can interact with ROPs in vivo and are thereby recruited to the plasma membrane. ICR1 knockdown or silencing leads to cell deformation and loss of the root stem cells population, and ectopic expression of ICR1 phenocopies activated ROPs/RACs. ICR1 presents a new paradigm in ROP/RAC signaling and integrates mechanisms regulating cell form and pattern formation at the whole plant level.

A Novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking.

ROP/RAC GTPases are master regulators of cell polarity in plants, implicated in the regulation of diverse signaling cascades including cytoskeleton organization, vesicle trafficking, and Ca(2+) gradients [1-8]. The involvement of ROPs in differentiation processes is yet unknown. Here we show the identification of a novel ROP/RAC effector, designated interactor of constitutive active ROPs 1 (ICR1), that interacts with GTP-bound ROPs. ICR1 knockdown or silencing leads to cell deformation and loss of root stem-cell population. Ectopic expression of ICR1 phenocopies activated ROPs, inducing cell deformation of leaf-epidermis-pavement and root-hair cells [3, 5, 6, 9]. ICR1 is comprised of coiled-coil domains and forms complexes with itself and the exocyst vesicle-tethering complex subunit SEC3 [10-13]. The ICR1-SEC3 complexes can interact with ROPs in vivo. Plants overexpressing a ROP- and SEC3-noninteracting ICR1 mutant have a wild-type phenotype. Taken together, our results show that ICR1 is a scaffold-mediating formation of protein complexes that are required for cell polarity, linking ROP/RAC GTPases with vesicle trafficking and differentiation.