Screening And Characterization Of ROP2 Regulators In Arabidopsis Root Hair

Root hairs play important roles in plant water absorption,nutrient uptake and communication with the external environment.It is one of the model systems for exploring the establishment,maintenance and morphogenesis of cell polarity in eukaryotic cells.Therefore,understanding regulation mechanisms of root hair growth has important biological significance.Rho-related GTPases from plants?ROP?are small G proteins unique to plants.ROPs are involved in plant development,biotic and abiotic stress responses,and hormonal signal transduction.It plays a key role in polar growing cells.ROP acts as a key molecular switch during root hair growth.Its"activated form"binds to GTP and its"inactivated form"binds to GDP.ROP-GTP regulates polar growth of root hair through various intracellular activities such as maintaining Ca²⁺gradient,dynamics of cytoskeleton,and ROS production.Early studies have found many regulators of ROPs,such as GEFs,GAPs,GDIs etc.However,their regulatory mechanisms are not fully understood.In this study,we applied reverse-and forward-genetic approaches to discover novel regulators of ROPs during root hair growth.We demonstrated that PLURIPETALA?PLP?and phytochrome B?phyB?mediate the membrane distribution,activity and stability of ROP2 during root hair growth.Results presented here provide a deeper understanding of regulatory mechanisms underlying ROP-mediated root hair growth.The main results and conclusions are as follows:?1?PLP regulates root hair growthPrenylation is an important post-translational lipid modification,in which a C15 of the farnesyl or C20 geranyl geranyl group is linked to a cysteine residue of the C-terminal CAAX motif of a target protein through a thioester bond.Arabidopsis PLP encodes the isoprenyltransferases alpha subunit that catalyzes prenylation.PLP is highly expressed in root hairs during both initation and elongation.Both the length and the density of plp-3 root hairs were significantly reduced compared with those of wild type.Exogenous PLP complemented the defect of plp-3 root hairs.The above results indicated that PLP regulates root hair growth.?2?PLP regulates the plasma membrane?PM?association of ROP2By introducing Pro_E7:GFP-ROP2 into wild-type and plp-3 and by fluorescence imaging,we found that compared with that of wild type,the PM association of ROP2 was significantly reduced.By cell fractionation and western blot analysis,we confirmed the reduced membrane association of ROP in plp-3.These results indicated that PLP regulates membrane association of ROPs in root hairs.?3?ROP signaling is compromised in plp-3 root hairsThe reduced membrane association of ROP2 at the PM suggested weakened ROP signalling.To provide further evidence,we analyzed both the organization of actin MF,the production of ROS and the post-Golgi trafficking in root hairs.To determine actin MF organization,we introduced Pro_35S:ABD2-GFP,expressing an actin MF-specific fluorescent marker,into wild-type and plp-3.Initiating root hairs of plp-3 contained very reduced actin MF meshes at the bulging region.In elongating plp-3 root hairs,longitudinal actin cables penetrated to the apex.However,actin cables are bundled more extensively in plp-3 than in wild-type,possibly a result of reduced actin MF dynamics.We then examined the ROS level in the root hair growth and found that the ROS level was significantly reduced.We introduced a Pro_35S:RFP-RabA4b in wild-type and plp-3 and examined the fluorescence distribution of RFP-RabA4b,and found that RabA4b-positive vesicles dissipated in plp-3both during hair initiation and elongation.To determine endocytosis in plp-3 root hairs,we tested the internalization dynamics of FM4-64.In the plp-3 root hair,the intracellular vesicles labeled with FM4-64 were significantly reduced compared to the wild type.Thus,the loss function of PLP reduces the dynamic organization of actin MF,ROS production and post-Golgi trafficking.?4?PLP regulates the targeting and stability of ROP2 in parallel to GDI1We further analyzed that PLP and GDI1,a ROP regulator,additively mediate ROP2stability and targeting.We generated a double mutant by crossing plp-3 with a GDI1loss-of-function mutant,scn1-1.The introduction of plp-3 significantly suppressed the multiple initiations typical of scn1-1.Interestingly,the distribution and stability of ROP2 in plp-3;scn1-1 reflected a combination of defects observed in both single mutants.The application of the protease inhibitor MG132 substantially restored the protein levels of ROP2in several mutants.The above results indicate that PLP and GDI1 parallel regulate the targeting and stability of ROP2 and suggest that ROP2 is degraded by the 26S proteasome-mediated degradation pathway.?5?PhyB negatively regulates root hair growthIn natural environment,root hairs generally grow in the dark.We found that root hairs became significantly shorter,with reduced density and increased width when simulating natural conditions in which shoot is illuminated and root is dark-grown.Based on this phenomenon,we obtained longer root hair mutants using forward genetics approaches.One of the mutants is defective in PHYB by map-based cloning and was named phyB-11.By analyzing the PHYB promoter-driven GUS transgenic material,we found that PHYB is highly expressed in root hairs.We found that phyB-11 significantly increased root hair length compared to wild type.We quantified the root hair profile and root hair growth speed.Root hairs of phyB-11 are longer than those of wild type due to more rapid elongation.These results suggested that phyB inhibits the tip growth of root hairs.?6?PhyB negatively regulates active ROP2 protein level and membrane localizationROP2 acts as a"molecular switch"in root hair growth while phyB can inhibit the tip growth of root hairs.So we hypothesized that they were genetically related.To do so,we crossed the E7 promoter-driven GFP-ROP2 to phyB-11.We found that the PM localization of ROP2 was enhanced in phyB-11,and the ratio of PM to Cyt increased.The results of biochemical experiments showed that the level of PM-localized ROP2 and active ROP2increased in phyB-11,indicating that phyB negatively regulates the localization and protein activity of ROP2 in the root hair growth.?7?PhyB negatively regulates root hair growth through GEF10 in the cytoplasmThe subcellular localization of PhyB is regulated by light.By analyzing the subcellular localization of phyB,we found that phyB is mainly located in the cytoplasm when root hairs grow in the dark environment no matter whether the shoots are illuminated.This suggests that phyB plays a role in the cytoplasm during root hair growth.We further constructed a phyB mutant,phyB^G767R,which cannot enter into the nuclear form.The phyB^G767R mutant form complemented enhanced root growth phenotype of phyB-11.By Pull down,Y2H and BiFC,we verified the interaction between phyB and GEF10 in the cytoplasm.In addition,the phyB-11;gef10 double mutants exhibits a phenotype equivalent to gef10.The above results indicated that phyB plays a major role in cytoplasm in root hairs when roots grow in the dark,and regulates root hair growth by interacting with GEF10.?8?PhyB and FER competitively combined with GEF10The receptor-like kinase FER regulates ROP signaling by interacting with GEFs.We found that the interaction of FER with GEF10 was significantly reduced when PHYB was expressed.The phyB-11;fer-4 double mutant exhibits a phenotype equivalent to fer-4,indicating that phyB is genetically epistatic to FER.We propose that phyB regulates root hair growth by interfering with the interaction between FER and GEF10.