Rho Of Plants Research Articles

Plant Rho GTPase ROP6 Is Essential for Manganese Homeostasis in Arabidopsis.

Manganese (Mn) is an indispensable mineral for plant growth and development. However, plants cultivated in acidic and poorly drained soils are vulnerable to Mn2+ toxicity due to its heightened increased bioavailability. Despite the crucial roles of the Rho of plant (ROP) GTPases in various cellular processes, their precise function in regulating Mn homeostasis remains elusive. In this study, we unveil a novel ROP6 GTPase signalling pathway that profoundly influences Mn phytotoxicity tolerance in Arabidopsis. Remarkably, the rop6 and dominant-negative ROP6 (rop6DN) mutant plants displayed a dramatically sensitive phenotype to Mn toxicity, whereas ROP6-overexpression and constitutively activated ROP6 (rop6CA) lines exhibited enhanced Mn stress tolerance. Immunoblot analysis corroborated that the ROP6 protein, especially the active form of ROP6, increased in abundance in the presence of high Mn levels. Further, we identified that ROP6 physically interacted and colocalized with Metal Tolerance Protein 8 (MTP8) in vivo. Mn transport complementation assays in yeast, combined with biochemical analyses, emphasized the essentiality of ROP6 for MTP8's transport activity. In addition, genetic analyses indicated that ROP6 acted upstream of MTP8 in the regulatory cascade. Collectively, our findings elucidate that ROP6 GTPase signalling positively modulates and enhances Mn stress tolerance in plants.

RHO OF PLANTS signalling and the activating ROP GUANINE NUCLEOTIDE EXCHANGE FACTORS: specificity in cellular signal transduction in plants.

Every cell constantly receives signals from its neighbours or the environment. In plants, most signals are perceived by RECEPTOR-LIKE KINASEs (RLKs) and then transmitted into the cell. The molecular switches RHO OF PLANTS (ROP) are critical proteins for polar signal transduction and regulate multiple cell polarity processes downstream of RLKs. Many ROP-regulating proteins and scaffold proteins of the ROP complex are known. However, the spatiotemporal ROP signalling complex composition is not yet understood. Moreover, how specificity is achieved in different ROP signalling pathways within one cell still needs to be determined. This review gives an overview of recent advances in ROP signalling and how specificity by downstream scaffold proteins can be achieved. The composition of the ROP signalling complexes is discussed, focusing on the possibility of the simultaneous presence of ROP activators and inactivators within the same complex to balance ROP activity. Furthermore, this review highlights the function of plant-specific ROP GUANINE NUCLEOTIDE EXCHANGE FACTORS polarizing ROP signalling and defining the specificity of the initiated ROP signalling pathway.

Crosstalk between Rho of Plants GTPase signalling and plant hormones.

Rho of Plants (ROPs) constitute a plant-specific subset of small guanine nucleotide-binding proteins within the Cdc42/Rho/Rac family. These versatile proteins regulate diverse cellular processes, including cell growth, cell division, cell morphogenesis, organ development, and stress responses. In recent years, the dynamic cellular and subcellular behaviours orchestrated by ROPs have unveiled a notable connection to hormone-mediated organ development and physiological responses, thereby expanding our knowledge of the functions and regulatory mechanisms of this signalling pathway. This review delineates advancements in understanding the interplay between plant hormones and the ROP signalling cascade, focusing primarily on the connections with auxin and abscisic acid pathways, alongside preliminary discoveries in cytokinin, brassinosteroid, and salicylic acid responses. It endeavours to shed light on the intricate, coordinated mechanisms bridging cell- and tissue-level signals that underlie plant cell behaviour, organ development, and physiological processes, and highlights future research prospects and challenges in this rapidly developing field.

ROP GTPases with a geranylgeranylation motif modulate alkaloid biosynthesis in Catharanthus roseus.

Rho of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpenoid indole alkaloids (MIA) in Madagascar periwinkle (Catharanthus roseus), indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular, and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C-terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, their transcripts showed higher expression in most parts than other CrROPs. Protein-protein interaction studies revealed that CrROP3 and CrROP5, but not ΔCrROP3, ΔCrROP5, and CrROP2 lacking the CSIL motif, interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to the plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of ΔCrROP3 and ΔCrROP5 mutants devoid of sequence coding for the CSIL motif failed to enhance MIA biosynthesis. These results implicate that CrROP3 and CrROP5 have a positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus.

RHO of plant signaling was established early in streptophyte evolution

The algal ancestors of land plants underwent a transition from a unicellular to a multicellular body plan.1 This transition likely took place early in streptophyte evolution, sometime after the divergence of the Chlorokybophyceae/Mesostigmatophyceae lineage, but before the divergence of the Klebsormidiophyceae lineage.2 How this transition was brought about is unknown; however, it was likely facilitated by the evolution of novel mechanisms to spatially regulate morphogenesis. In land plants, RHO of plant (ROP) signaling plays a conserved role in regulating polarized cell growth and cell division orientation to orchestrate morphogenesis.3,4,5,6,7,8 ROP constitutes a plant-specific subfamily of the RHO GTPases, which are more widely conserved throughout eukaryotes.9,10 Although the RHO family originated in early eukaryotes,11,12 how and when the ROP subfamily originated had remained elusive. Here, we demonstrate that ROP signaling was established early in the streptophyte lineage, sometime after the divergence of the Chlorokybophyceae/Mesostigmatophyceae lineage, but before the divergence of the Klebsormidiophyceae lineage. This period corresponds to when the unicellular-to-multicellular transition likely took place in the streptophytes. In addition to being critical for the complex morphogenesis of extant land plants, we speculate that ROP signaling contributed to morphological evolution in early streptophytes.

Two subtypes of GTPase-activating proteins coordinate tip growth and cell size regulation in Physcomitrium patens

The establishment of cell polarity is a prerequisite for many developmental processes. However, how it is achieved during tip growth in plants remains elusive. Here, we show that the RHO OF PLANTs (ROPs), ROP GUANINE NUCLEOTIDE EXCHANGE FACTORs (RopGEFs), and ROP GTPASE-ACTIVATING PROTEINs (RopGAPs) assemble into membrane domains in tip-growing cells of the moss Physcomitrium patens. The confinement of membrane domains requires redundant global inactivation of ROPs by PpRopGAPs and the PLECKSTRIN HOMOLOGY (PH) domain-containing RenGAP PpREN. Unexpectedly, PpRopGAPs and PpREN exert opposing effects on domain size and cell width upon overexpression. Biochemical and functional analyses indicate that PpRopGAPs are recruited to the membrane by active ROPs to restrict domain size through clustering, whereas PpREN rapidly inactivates ROPs and inhibits PpRopGAP-induced clustering. We propose that the activity- and clustering-based domain organization by RopGAPs and RenGAPs is a general mechanism for coordinating polarized cell growth and cell size regulation in plants.

RHO GTPase of plants regulates polarized cell growth and cell division orientation during morphogenesis.

Cell polarity-broadly defined as the asymmetric distribution of cellular activities and subcellular components within a cell-determines the geometry of cell growth and division during development. RHO GTPase proteins regulate the establishment of cell polarity and are conserved among eukaryotes. RHO of plant (ROP) proteins are a subgroup of RHO GTPases that are required for cellular morphogenesis in plants. However, how ROP proteins modulate the geometry of cell growth and division during the morphogenesis of plant tissues and organs is not well understood. To investigate how ROP proteins function during tissue development and organogenesis, we characterized the function of the single-copy ROP gene of the liverwort Marchantia polymorpha (MpROP). M.polymorpha develops morphologically complex three-dimensional tissues and organs exemplified by air chambers and gemmae, respectively. Mprop loss-of-function mutants form defective air chambers and gemmae, indicating ROP function is required for tissue development and organogenesis. During air chamber and gemma development in wild type, the MpROP protein is enriched to sites of polarized growth at the cell surface and accumulates at the expanding cell plate of dividing cells. Consistent with these observations, polarized cell growth is lost and cell divisions are misoriented in Mprop mutants. We propose that ROP regulates both polarized cell growth and cell division orientation in a coordinated manner to orchestrate tissue development and organogenesis in land plants.

RHO OF PLANT proteins are essential for pollen germination in Arabidopsis.

Pollen germination is a process of polarity establishment, through which a single and unique growth axis is established. Although most of the intracellular activities associated with pollen germination are controlled by RHO OF PLANTs (ROPs) and increased ROP activation accompanies pollen germination, a critical role of ROPs in this process has not yet been demonstrated. Here, by genomic editing of all four Arabidopsis (Arabidopsis thaliana) ROPs that are preferentially expressed in pollen, we showed that ROPs are essential for polarity establishment during pollen germination. We further identified and characterized two ROP effectors in pollen germination (REGs) through genome-wide interactor screening, Boundary of ROP domain (BDR) members BDR8 and BDR9, whose functional loss also resulted in no pollen germination. BDR8 and BDR9 were distributed in the cytosol and the vegetative nucleus of mature pollen grains but re-distributed to the plasma membrane (PM) of the germination site and to the apical PM of growing pollen tubes. We demonstrated that the PM re-distribution of BDR8 and BDR9 during pollen germination relies on ROPs but not vice versa. Furthermore, enhanced expression of BDR8 partially restored germination of rop1 pollen but had no effects on that of the quadruple rop pollen, supporting their genetic epistasis. Results presented here demonstrate an ROP signaling route essential for pollen germination, which supports evolutionarily conserved roles of Rho GTPases in polarity establishment.

Physcomitrium patens PpRIC, an ancestral CRIB-domain ROP effector, inhibits auxin-induced differentiation of apical initial cells.

RHO guanosine triphosphatases are important eukaryotic regulators of cell differentiation and behavior. Plant ROP (RHO of plant) family members activate specific, incompletely characterized downstream signaling. The structurally simple land plant Physcomitrium patens is missing homologs of key animal and flowering plant RHO effectors but contains a single CRIB (CDC42/RAC interactive binding)-domain-containing RIC (ROP-interacting CRIB-containing) protein (PpRIC). Protonemal P. patens filaments elongate based on regular division and PpROP-dependent tip growth of apical initial cells, which upon stimulation by the hormone auxin differentiate caulonemal characteristics. PpRIC interacts with active PpROP1, co-localizes with this protein at the plasma membrane at the tip of apical initial cells, and accumulates in the nucleus. Remarkably, PpRIC is not required for tip growth but is targeted to the nucleus to block caulonema differentiation downstream of auxin-controlled gene expression. These observations establish functions of PpRIC in mediating crosstalk between ROP and auxin signaling, which contributes to the maintenance of apical initial cell identity.

ROP Interactive Partners are Involved in the Control of Cell Division Patterns in Arabidopsis Leaves.

Animal Rho GTP-binding proteins and their plant counterparts, Rho of plants (ROPs), regulate cell polarity, but they do so through different effector proteins. A class of ROP effectors, interactor of constitutive active ROPs (ICRs)/ROP interactive partners (RIPs), has been implicated in diverse biological processes; however, there are limited analyses of RIP loss-of-function mutants. Here, we report an analysis of the functions of the Arabidopsis thaliana RIPs in the leaf epidermis. Green Fluorescent Protein (GFP) fusion proteins of all the RIPs colocalized to cortical microtubules. RIP1, RIP3 and RIP4, but not RIP2 and RIP5, colocalized with the preprophase band (PPB), spindles and phragmoplasts. RIP2 and RIP5 did not colocalize with the PPB, spindles or phragmoplasts even when they were expressed under a promoter active in proliferative cells, indicating that there are differences among RIP protein properties. The overexpression of RIP1 or RIP4 resulted in the fragmentation of cortical microtubules, and the rip1 2 3 4 5 quintuple mutant showed increased growth rate of microtubules at their plus ends compared with the wild type. The rip1 2 3 4 5 mutant leaves and petals were narrow, which was explained by the decreased cell number along the transverse axis compared with that of the wild type. The rip1 2 3 4 5 mutant leaf epidermis possessed fewer PPBs oriented close to the long axis of the leaf compared with wild type, indicating the involvement of RIPs in cell division plane regulation and leaf shape determination.

Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination.

Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.

MxRop1-MxrbohD1 interaction mediates ROS signaling in response to iron deficiency in the woody plant Malus xiaojinensis

Iron (Fe) deficiency affects crop production and quality. Rho of plants (ROPs) involves in multiple physiological processes in plants. While it has not been well characterized under Fe deficiency, especially in perennial woody plants. In our study, we cloned ROP homologous gene MxRop1 from Malus xiaojinenesis, then overexpressed it in Arabidopsis, showing enhanced plant tolerance to Fe deficiency, which demonstrated its gene function during this stress. Overexpression of MxRop1 also increased reactive oxygen species (ROS) levels. Moreover, active state of MxRop1 (CA-MxRop1) interacted with N-terminal region of MxrbohD1, one ROS synthesis gene. When MxrbohD1 was overexpressed in apple calli, it showed significantly increased H2O2 content, fresh weight and FCR activity, while ROS inhibitor application dramatically inhibited FCR activity, demonstrating ROS produced by MxrbohD1 regulated Fe deficiency responses. Furthermore, using Agrobacterium rhizogenes transformation, MxrbohD1 was overexpressed in apple roots, with increased expression of Fe deficiency-induced genes and increased root FCR activity. Under Fe deficiency, it exhibited slight leaf yellowing phenotype. Co-expression of CA-MxRop1 and MxrbohD1 significantly induced ROS generation. Finally, we proposed that MxRop1 interacted with MxrbohD1 to modulate ROS mediated Fe deficiency adaptive responses in Malus xiaojinensis, which will provide a guidance of cultivation of Fe-deficiency tolerant apple plant.

Formation of self-organizing functionally distinct Rho of plants domains involves a reduced mobile population.

Rho family proteins are central to the regulation of cell polarity in eukaryotes. Rho of Plants-Guanyl nucleotide Exchange Factor (ROPGEF) can form self-organizing polar domains following co-expression with an Rho of Plants (ROP) and an ROP GTPase-Activating Protein (ROPGAP). Localization of ROPs in these domains has not been demonstrated, and the mechanisms underlying domain formation and function are not well understood. Here we show that six different ROPs form self-organizing domains when co-expressed with ROPGEF3 and GAP1 in Nicotiana benthamiana or Arabidopsis (Arabidopsis thaliana). Domain formation was associated with ROP-ROPGEF3 association, reduced ROP mobility, as revealed by time-lapse imaging and Fluorescence Recovery After Photobleaching beam size analysis, and was independent of Rho GTP Dissociation Inhibitor mediated recycling. The domain formation depended on the ROPs' activation/inactivation cycles and interaction with anionic lipids via a C-terminal polybasic domain. Coexpression with the microtubule-associated protein ROP effector INTERACTOR OF CONSTITUTIVELY ACTIVE ROP 1 (ICR1) revealed differential function of the ROP domains in the ability to recruit ICR1. Taken together, the results reveal mechanisms underlying self-organizing ROP domain formation and function.

GTPase ROP6 negatively modulates phosphate deficiency through inhibition of PHT1;1 and PHT1;4 in Arabidopsis thaliana.

Phosphorus, an essential macroelement for plant growth and development, is a major limiting factor for sustainable crop yield. The Rho of plant (ROP) GTPase is involved in regulating multiple signal transduction processes in plants, but potentially including the phosphate deficiency signaling pathway remains unknown. Here, we identified that the rop6 mutant exhibited a dramatic tolerant phenotype under Pi-deficient conditions, with higher phosphate accumulation and lower anthocyanin content. In contrast, the rop6 mutant was more sensitive to arsenate (As(V)) toxicity, the analog of Pi. Immunoblot analysis displayed that the ROP6 protein was rapidly degraded through ubiquitin/26S proteasome pathway under Pi-deficient conditions. In addition, pull-down assay using GST-RIC1 demonstrated that the ROP6 activity was decreased obviously under Pi-deficient conditions. Strikingly, protein-protein interaction and two-voltage clamping assays demonstrated that ROP6 physically interacted with and inhibited the key phosphate uptake transporters PHT1;1 and PHT1;4 in vitro and in vivo. Moreover, genetic analysis showed that ROP6 functioned upstream of PHT1;1 and PHT1;4. Thus, we conclude that GTPase ROP6 modulates the uptake of phosphate by inhibiting the activities of PHT1;1 and PHT1;4 in Arabidopsis.

Competition between anthocyanin and kaempferol glycosides biosynthesis affects pollen tube growth and seed set of Malus

Flavonoids play important roles in regulating plant growth and development. In this study, three kaempferol 3-O-glycosides were identified and mainly accumulated in flowers but not in leaves or fruits of Malus. In Malus, flower petal color is normally white, but some genotypes have red flowers containing anthocyanin. Anthocyanin biosynthesis appears to be in competition with kaempferol 3-O-glycosides production and controlled by the biosynthetic genes. The white flower Malus genotypes had better-developed seeds than the red flower genotypes. In flowers, the overexpression of MYB10 in Malus domestica enhanced the accumulation of anthocyanin, but decreased that of kaempferol 3-O-glycosides. After pollination the transgenic plants showed slower pollen tube growth and fewer developed seeds. Exogenous application of different flavonoid compounds suggested that kaempferol 3-O-glycosides, especially kaempferol 3-O-rhamnoside, regulated pollen tube growth and seed set rather than cyanidin or quercetin 3-O-glycosides. It was found that kaempferol 3-O-rhamnoside might regulate pollen tube growth through effects on auxin, the Rho of plants (ROP) GTPases, calcium and the phosphoinositides signaling pathway. With the inhibition of auxin transport, the transcription levels of Heat Shock Proteins (HSPs) and ROP GTPases were downregulated while the levels were not changed or even enhanced when blocking calcium signaling, suggesting that HSPs and ROP GTPases were downstream of auxin signaling, but upstream of calcium signaling. In summary, kaempferol glycoside concentrations in pistils correlated with auxin transport, the transcription of HSPs and ROP GTPases, and calcium signaling in pollen tubes, culminating in changes to pollen tube growth and seed set.

Interaction of OsRopGEF3 Protein With OsRac3 to Regulate Root Hair Elongation and Reactive Oxygen Species Formation in Rice (Oryza sativa).

Root hairs are tip-growing cells that emerge from the root epidermis and play a role in water and nutrient uptake. One of the key signaling steps for polar cell elongation is the formation of Rho-GTP by accelerating the intrinsic exchange activity of the Rho-of-plant (ROP) or the Rac GTPase protein; this step is activated through the interaction with the plant Rho guanine nucleotide exchange factor (RopGEFs). The molecular players involved in root hair growth in rice are largely unknown. Here, we performed the functional analysis of OsRopGEF3, which is highly expressed in the root hair tissues among the OsRopGEF family genes in rice. To reveal the role of OsRopGEF3, we analyzed the phenotype of loss-of-function mutants of OsRopGEF3, which were generated using the CRISPR-Cas9 system. The mutants had reduced root hair length and increased root hair width. In addition, we confirmed that reactive oxygen species (ROS) were highly reduced in the root hairs of the osropgef3 mutant. The pairwise yeast two-hybrid experiments between OsRopGEF3 and OsROP/Rac proteins in rice revealed that the OsRopGEF3 protein interacts with OsRac3. This interaction and colocalization at the same subcellular organelles were again verified in tobacco leaf cells and rice root protoplasts via bimolecular functional complementation (BiFC) assay. Furthermore, among the three respiratory burst oxidase homolog (OsRBOH) genes that are highly expressed in rice root hair cells, we found that OsRBOH5 can interact with OsRac3. Our results demonstrate an interaction network model wherein OsRopGEF3 converts the GDP of OsRac3 into GTP, and OsRac3-GTP then interacts with the N-terminal of OsRBOH5 to produce ROS, thereby suggesting OsRopGEF3 as a key regulating factor in rice root hair growth.

The small GTP-binding protein TaRop10 interacts with TaTrxh9 and functions as a negative regulator of wheat resistance against the stripe rust

Small GTP-binding proteins, also known as ROPs (Rho of Plants), are a subfamily of the Ras superfamily of signaling G-proteins and are required for numerous signaling processes, ranging from growth and development to biotic and abiotic signaling. In this study, we cloned and characterized wheat TaRop10, a homolog of Arabidopsis ROP10 and member of the class II ROP, and uncovered a role for TaRop10 in wheat response to Puccinia striiformis f. sp. tritici (Pst). TaRop10 was downregulated by actin depolymerization and was observed to be differentially induced by abiotic stress and the perception of plant hormones. A combination of yeast two-hybrid and bimolecular fluorescence complementation assays revealed that TaRop10 interacted with a h-type thioredoxin (TaTrxh9). Knocking-down of TaRop10 and TaTrxh9 was performed using the BSMV-VIGS (barley stripe mosaic virus-based virus-induced gene silencing) technique and revealed that TaRop10 and TaTrxh9 play a role in the negative regulation of defense signaling in response to Pst infection. In total, the data presented herein further illuminate our understanding of how intact plant cells accommodate fungal infection structures, and furthermore, support the function of TaRop10 and TaTrxh9 in negative modulation of defense signaling in response to stripe rust infection.

No Time for Transcription-Rapid Auxin Responses in Plants.

Auxin regulates the transcription of auxin-responsive genes by the TIR1/AFBs-Aux/IAA-ARF signaling pathway, and in this way facilitates plant growth and development. However, rapid, nontranscriptional responses to auxin that cannot be explained by this pathway have been reported. In this review, we focus on several examples of rapid auxin responses: (1) the triggering of changes in plasma membrane potential in various plant species and tissues, (2) inhibition of root growth, which also correlates with membrane potential changes, cytosolic Ca2+ spikes, and a rise of apoplastic pH, (3) the influence on endomembrane trafficking of PIN proteins and other membrane cargoes, and (4) activation of ROPs (Rho of plants) and their downstream effectors such as the cytoskeleton or vesicle trafficking. In most cases, the signaling pathway triggering the response is poorly understood. A role for the TIR1/AFBs in rapid root growth regulation is emerging, as well as the involvement of transmembrane kinases (TMKs) in the activation of ROPs. We discuss similarities and differences among these rapid responses and focus on their physiological significance, which remains an enigma in most cases.

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.

A positive feedback circuit for ROP-mediated polar growth

Tip growth is a special type of polarized growth in which a single and unique polarization site is established and maintained. Rho of Plants (ROP) proteins, which represent the only class of Rho GTPases in plants, regulate tip growth. The dynamic and asymmetric distribution of ROPs is critical for the establishment and maintenance of tip growth, and requires at least one positive feedback loop, which is still elusive. Here, we report a positive feedback circuit essential for tip growth of root hairs, in which ROPs, ROP activators and effectors, and AGC1.5 subfamily kinases are interconnected by sequential oligomerization and phosphorylation. AGC1.5 subfamily kinases interact with and phosphorylate two guanine nucleotide exchange factors (GEFs) of ROPs, RopGEF4 and RopGEF10. They also interact with two ROP effectors, ICR2/RIP3 and MIDD1/RIP4, which bridge active ROPs with AGC1.5. Functional loss of the AGC1.5 subfamily kinases or ICR2 and MIDD1 compromised root hair growth due to reduced ROP signaling. We found that asymmetric targeting of RopGEF4 and RopGEF10 is controlled by AGC1.5-dependent phosphorylation. Interestingly, we discovered that the ROP effectors recruit AGC1.5 to active ROP domains at the plasma membrane during root hair growth and are critical for AGC1.5-dependent phosphorylation of RopGEFs. Given the large number of AGC kinases in plants, this positive feedback circuit may be a universal theme for plant cell polar growth.