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.Key words: Rho, auxin, root development, vesicle trafficking, RAC, ROP, polarity, Arabidopsis, exocyst     

RAC/ROP GTPases: 'hubs' for signal integration and diversification in plants

RAC/ROP GTPases are a family of plant-specific signaling molecules solely representing the Ras and Rho family of Ras-related G proteins in plants. RAC/ROPs potentially interact with cell surface-associated signal perception apparatus for a broad range of extracellular stimuli, including hormones, pathogen elicitors and abiotic stress, and mediate diverse cellular pathways in response to these signals. They are also known to interact with multiple effectors, affecting cellular and biochemical systems that regulate actin dynamics, reactive oxygen species production, proteolysis, and gene expression. RAC/ROPs are, thus, ideally suited as integrators for multiple signals and as coordinators of diverse cellular pathways to control growth, differentiation, development and defense responses. Recent findings that suggest how RAC/ROP signaling activity is regulated and how functional specificity can be achieved are discussed here.     

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.     

ROP3 GTPase Contributes to Polar Auxin Transport and Auxin Responses and Is Important for Embryogenesis and Seedling Growth in Arabidopsis

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.     

ROPs:植物细胞内多种信号通路的分子开关

植物RHO相关蛋白GTPases(RHO-related GTPases of plants, ROPs)是广泛存在于植物中的一类信号转导G蛋白(又称GTP结合蛋白),其通过结合GDP或GTP在非活性和活性状态间进行切换,进而在细胞极性控制、形态发育、激素水平调控、逆境反应等诸多植物生命活动的信号转导过程中扮演重要的分子开关角色。本文对ROP蛋白的结构域及基于蛋白质结构分类进行了介绍,并对拟南芥、玉米、水稻和大麦中的ROP家族蛋白质进行了系统进化分析。分析结果表明,这些植物中的ROP蛋白根据蛋白质结构域组成可分为Ⅰ类(typeⅠ)和Ⅱ类(typeⅡ)两种类型,而根据蛋白质序列的保守性可将其在植物中的ROP蛋白划分为4个进化枝。本综述不但对ROP蛋白作为分子开关在细胞内调控各种信号通路的机制进行了叙述,还对ROP在花粉管、根毛及植物表皮铺盖细胞极性发育,以及其他抗逆反应中的具体作用和机制及研究进展进行了阐述。本文还对ROP蛋白在ABA、IAA、BR等植物激素信号传导过程中的调控作用及研究进展进行了阐述。本文对植物ROP蛋白研究过程中尚未解决的问题,例如不同的ROP蛋白在同一个信号通路中的作用为何如此不同,以及ROP是如何协调不同的信号通路以共同调控一个植物发育或者生理过程等问题进行了总结,并在此基础上对未来的研究方向进行了展望。     

Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus

Small monomeric G-proteins of the plant ras (rat sarcome oncogene product) related C3 botulinum toxin substrate (RAC)/Rho of plants (ROP) family are molecular switches in signal transduction of many cellular processes. RAC/ROPs regulate hormone effects, subcellular gradients of Ca2+, the organisation of the actin cytoskeleton and the production of reactive oxygen intermediates. Therefore, we followed a genetic bottom-up strategy to study the role of these proteins during the interaction of barley (Hordeum vulgare L.) with the fungal biotrophic pathogen Blumeria graminis f.sp. hordei (Bgh). We identified six barley RAC/ROP proteins and studied their gene expression. Five out of six Rac/Rop genes were expressed constitutively in the leaf epidermis, which is the site of interaction with Bgh. None of the genes showed enhancement of mRNA abundance after inoculation with Bgh. After microprojectile mediated transformation of single barley epidermal cells with constitutively activated mutant RAC/ROP proteins, we found an RAC/ROP-specific enhancement of pathogen accessibility, tagging HvRACB, HvRAC3 and HvROP6 as host proteins potentially involved in the establishment of susceptibility to Bgh. Confocal laser scanning microscopy (CLSM) of green fluorescent protein (GFP):HvRAC/ROP-transformed cells revealed varying strengths of plasma membrane association of barley RAC/ROPs. The C-terminal CAAX motif for presumable prenylation or the C-terminal hypervariable region (HVR), respectively, were required for membrane association of the RAC/ROPs. Proper intracellular localisation was essential for HvRACB and HvRAC3 function. Together, our data support the view that different paths of host signal transduction via RAC/ROP G-proteins are involved in processes supporting parasitic entry into epidermal host cells.     

A Theoretical Model for ROP Localisation by Auxin in Arabidopsis Root Hair Cells

Background

Local activation of Rho GTPases is important for many functions including cell polarity, morphology, movement, and growth. Although a number of molecules affecting Rho-of-Plants small GTPase (ROP) signalling are known, it remains unclear how ROP activity becomes spatially organised. Arabidopsis root hair cells produce patches of ROP at consistent and predictable subcellular locations, where root hair growth subsequently occurs.

Methodology/Principal Findings

We present a mathematical model to show how interaction of the plant hormone auxin with ROPs could spontaneously lead to localised patches of active ROP via a Turing or Turing-like mechanism. Our results suggest that correct positioning of the ROP patch depends on the cell length, low diffusion of active ROP, a gradient in auxin concentration, and ROP levels. Our theory provides a unique explanation linking the molecular biology to the root hair phenotypes of multiple mutants and transgenic lines, including OX-ROP, CA-rop, aux1, axr3, tip1, eto1, etr1, and the triple mutant aux1 ein2 gnom ^eb.

Conclusions/Significance

We show how interactions between Rho GTPases (in this case ROPs) and regulatory molecules (in this case auxin) could produce characteristic subcellular patterning that subsequently affects cell shape. This has important implications for research on the morphogenesis of plants and other eukaryotes. Our results also illustrate how gradient-regulated Turing systems provide a particularly robust and flexible mechanism for pattern formation.     

A RHOse by any other name： a comparative analysis of animal and plant Rho GTPases

Rho GTPases are molecular switches that act as key regulators of a many cellular processes,including cell movement,morphogenesis,host defense,cell division and gene expression.Rho GTPases are found in all eukaryotic kingdoms.Plantslack clear homologs to conventional Rho GTPases found in yeast and animals;instead,they have over time developeda unique subfamily,ROPs,also known as RAC.The origin of ROP-like proteins appears to precede the appearance ofland plants.This review aims to discuss the evolution of ROP/RAC and to compare plant ROP and animal Rho GTPases,focusing on similarities and differences in regulation of the GTPases and their downstream effectors.     

Molecular basis for the substrate specificity of plant guanine nucleotide exchange factors for ROP

Plant G proteins of the ROP/RAC family regulate cellular processes including cytoskeletal rearrangement in polar growth. Activation of the ROP molecular switch is triggered by guanine nucleotide exchange factors. Plant-specific RopGEFs are exclusively active on ROPs despite their high homology to animal Rho proteins. Based on a sequence comparison of ROPs vs. animal Rho proteins together with structural data on distinct ROPs, we identified unique substrate determinants of RopGEF specificity by mutational analysis: asparagine 68 next to switch II, arginine 76 of a putative phosphorylation motif and the Rho insert are essential for substrate recognition by RopGEFs. These data also provide first evidence for a function of the Rho insert in interactions with GEFs.     

Cell polarity: ROPing the ends together

Cell polarity plays an important role in plant development, but the mechanisms that first establish polarity cues remain obscure. By contrast, a flurry of information has recently emerged on the elaboration of cell shape from such unknown initial cell-polarity cues. Recent studies suggest that Rho-related GTPases in plants (ROPs), and their effector targets among the ROP-interactive CRIB motif-containing proteins (RICs), mediate two antagonistic pathways that have opposing action on cell polarization. ROP proteins appear to interact directly with upstream regulators of the ARP2/3 complex, which are conserved modulators of the actin cytoskeleton. ROP function is dependent on the class 1 ADP-ribosylation factors (ARFs), which are core components of the vesicle transport machinery that are also involved in the polar localization of PIN-FORMED (PIN) family auxin efflux facilitators.     

ROP GTPases act with the receptor-like protein PAN1 to polarize asymmetric cell division in maize

Plant Rho family GTPases (ROPs) have been investigated primarily for their functions in polarized cell growth. We previously showed that the maize (Zea mays) Leu-rich repeat receptor-like protein PANGLOSS1 (PAN1) promotes the polarization of asymmetric subsidiary mother cell (SMC) divisions during stomatal development. Here, we show that maize Type I ROPs 2 and 9 function together with PAN1 in this process. Partial loss of ROP2/9 function causes a weak SMC division polarity phenotype and strongly enhances this phenotype in pan1 mutants. Like PAN1, ROPs accumulate in an asymmetric manner in SMCs. Overexpression of yellow fluorescent protein-ROP2 is associated with its delocalization in SMCs and with aberrantly oriented SMC divisions. Polarized localization of ROPs depends on PAN1, but PAN1 localization is insensitive to depletion and depolarization of ROP. Membrane-associated Type I ROPs display increased nonionic detergent solubility in pan1 mutants, suggesting a role for PAN1 in membrane partitioning of ROPs. Finally, endogenous PAN1 and ROP proteins are physically associated with each other in maize tissue extracts, as demonstrated by reciprocal coimmunoprecipitation experiments. This study demonstrates that ROPs play a key role in polarization of plant cell division and cell growth and reveals a role for a receptor-like protein in spatial localization of ROPs.     

ROP GTPase-dependent actin microfilaments promote PIN1 polarization by localized inhibition of clathrin-dependent endocytosis

Cell polarization via asymmetrical distribution of structures or molecules is essential for diverse cellular functions and development of organisms, but how polarity is developmentally controlled has been poorly understood. In plants, the asymmetrical distribution of the PIN-FORMED (PIN) proteins involved in the cellular efflux of the quintessential phytohormone auxin plays a central role in developmental patterning, morphogenesis, and differential growth. Recently we showed that auxin promotes cell interdigitation by activating the Rho family ROP GTPases in leaf epidermal pavement cells. Here we found that auxin activation of the ROP2 signaling pathway regulates the asymmetric distribution of PIN1 by inhibiting its endocytosis. ROP2 inhibits PIN1 endocytosis via the accumulation of cortical actin microfilaments induced by the ROP2 effector protein RIC4. Our findings suggest a link between the developmental auxin signal and polar PIN1 distribution via Rho-dependent cytoskeletal reorganization and reveal the conservation of a design principle for cell polarization that is based on Rho GTPase-mediated inhibition of endocytosis.     

Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis

Auxin is a multifunctional hormone essential for plant development and pattern formation. A nuclear auxin-signaling system controlling auxin-induced gene expression is well established, but cytoplasmic auxin signaling, as in its coordination of cell polarization, is unexplored. We found a cytoplasmic auxin-signaling mechanism that modulates the interdigitated growth of Arabidopsis leaf epidermal pavement cells (PCs), which develop interdigitated lobes and indentations to form a puzzle-piece shape in a two-dimensional plane. PC interdigitation is compromised in leaves deficient in either auxin biosynthesis or its export mediated by PINFORMED 1 localized at the lobe tip. Auxin coordinately activates two Rho GTPases, ROP2 and ROP6, which promote the formation of complementary lobes and indentations, respectively. Activation of these ROPs by auxin occurs within 30 s and depends on AUXIN-BINDING PROTEIN 1. These findings reveal Rho GTPase-based auxin-signaling mechanisms, which modulate the spatial coordination of cell expansion across a field of cells.     

Auxin dynamics: the dazzling complexity of a small molecule’s message

The phytohormone auxin is a potent regulator of plant development. Since its discovery in the beginning of the twentieth century many aspects of auxin biology have been extensively studied, ranging from biosynthesis and metabolism to the elucidation of molecular components of downstream signaling. With the identification of the F-box protein TIR1 as an auxin receptor a major breakthrough in understanding auxin signaling has been achieved and recent modeling approaches have shed light on the putative mechanisms underlying the establishment of auxin gradients and maxima essential for many auxin-regulated processes. Here, we review these and other recent advances in unraveling the entanglement of biosynthesis, polar transport and cellular signaling events that allow small auxinic molecules to facilitate their complex regulatory action.     

Function of membrane domains in rho-of-plant signaling

In a crowded environment, establishing interactions between different molecular partners can take a long time. Biological membranes have solved this issue, as they simultaneously are fluid and possess compartmentalized domains. This nanoscale organization of the membrane is often based on weak, local, and multivalent interactions between lipids and proteins. However, from local interactions at the nanoscale, different functional properties emerge at the higher scale, and these are critical to regulate and integrate cellular signaling. Rho of Plant (ROP) proteins are small guanosine triphosphate hydrolase enzymes (GTPases) involved in hormonal, biotic, and abiotic signaling, as well as fundamental cell biological properties such as polarity, vesicular trafficking, and cytoskeleton dynamics. Association with the membrane is essential for ROP function, as well as their precise targeting within micrometer-sized polar domains (i.e. microdomains) and nanometer-sized clusters (i.e. nanodomains). Here, we review our current knowledge about the formation and the maintenance of the ROP domains in membranes. Furthermore, we propose a model for ROP membrane targeting and discuss how the nanoscale organization of ROPs in membranes could determine signaling parameters like signal specificity, amplification, and integration.

The nanoscale organization of Rho of Plant proteins creates emergent properties that determine cellular signaling.     

The small GTPase AtRAC2/ROP7 is specifically expressed during late stages of xylem differentiation in Arabidopsis

The RAC/ROP family of small GTPases are central regulators of important cellular processes in plants. AtRAC2/ROP7 is an ancient member of the RAC/ROP gene family in Arabidopsis thaliana whose functions are generally unknown. In order to study the spatial expression pattern of the AtRAC2/ROP7 gene, transgenic plants expressing GUS or GFP under the control of the AtRAC2/ROP7 promoter were analysed. Functional analysis of AtRAC2/ROP7 was done using transgenic plants overexpressing wild-type and constitutively activated AtRAC2/ROP7 (Val15Gly), and an AtRAC2/ROP7T-DNA insertion mutant. The AtRAC2/ROP7 promoter directs a highly specific xylem-specific expression in the root, hypocotyl, stem, and leaves. The expression is developmentally limited to the late stages of xylem differentiation, and coincides with the formation of secondary cell walls. Leaf epidermal cells of transgenic plants overexpressing constitutively active AtRAC2/ROP7 exhibited highly impaired lobe formation, suggesting that AtRAC2/ROP7 is able to regulate polar cell expansion. Finally, GFP-AtRAC2/ROP7 fusion proteins were localized to the plasma membrane. The results indicate a role for AtRAC2/ROP7 in the development of secondary cell walls of xylem vessels.     

ROPGEF1 and ROPGEF4 are functional regulators of ROP11 GTPase in ABA-mediated stomatal closure in Arabidopsis

ROPs constitute a family of plant-specific, RHO-like small GTPases that serve as molecular switches in a wide range of signaling pathways. The activities of ROPs are regulated by guanine nucleotide exchange factors (GEFs). ROP11, a member of the ROP GTPase family in Arabidopsis, is a negative regulator of multiple ABA responses. In this study, we show that ROPGEF1 and ROPGEF4 interact with ROP11 on plasma membranes in guard cells. Furthermore, our analyses of ROPGEF1/4 knockout mutants and overexpressing lines suggested that ROPGEF1 and ROPGEF4 are specific regulators of ROP11 function in ABA-mediated stomatal closure.