Receptor-like Kinase SRH1 Function In The Regulation Of Root Hair Growth In Arabidopsis Thaliana

Root hair is a tubular structure formed by the outward bulge of root epidermal cell,which connects the root system with the soil and plays an important role in nutrient uptake,interaction with microorganisms,and helping plant anchoring.It is also an excellent model system for studying cell fate determination and polar growth,so the study of regulation mechanism of root hair growth is of great biological significance.Receptor-like kinases(RLKs)are localized on cell membrane to sense and transmit signals.Receptor-like cytoplasmic kinases(RLCKs)is a special RLK without an extracellular ligand binding domain,which can receive signals from the external environment earlier.It transmits signals by interacting with RLKs and phosphorylating downstream target proteins to regulate plant growth,development,response to stress and other life processes.The growth and development of root hair are affected by many factors,and the strict and complex gene regulatory network is the key for plants to respond flexibly to external environmental signals.We identified and analyzed the expression and subcellular localization of SRH1,which encodes a member of RLCKs family,and found that the root hairs of its mutants became shorter.The interaction proteins of SRH1 were screened by the split-ubiquitin yeast two-hybrid membrane system,and the expression patterns and functions of PIP3 A and HRL were preliminarily explored.The specific results and conclusions are as follows:(1)SRH1 regulates the growth of root hairsBy analyzing the T-DNA insertion mutant of SRH1,it was found that srh1 has a shorter root hair than the wild type,and SRH1 can complement the short phenotype of srh1 root hairs.In order to futher support the function of SRH1 during root hair growth,we observed the expression pattern of SRH1 by GUS staining and found that SRH1 was constitutively expressed and highly expressed in root hairs.The results of subcellular localization showed that SRH1 was localized to the plasma membrane and partly in the cytoplasm.The above results imply that SRH1 regulates the growth of root hair.(2)Screening and verificationof SRH1 interacting proteinsTo further reveal the molecular mechanism of SRH1 regulating root hair growth,we screen the interacting proteins of SRH1 by split-ubiquitin membrane system.We screened a total of 27 proteins that may interact with SRH1,and we selected 5 candidate genes that were highly or specifically expressed in root hairs for further study.The results of yeast two-hybrid and Split-LUC experiments showed that SRH1 interacted with PIP3 A and HRL,but not with MRH1,MRH5 and PAE7.(3)Phenotypic analysis of root hair of PIP3 mutantPIPs encode plasma membrane intrinsic protein and play an important role in regulating the transport of water and molecules.PIP3 A belongs to the PIP family.We performed phylogenetic tree analysis and homologous sequence alignment of PIPs in Arabidopsis,PIP3 A was found to have a highly homologous protein PIP3 B.The bioinformatics results showed that PIP3 A and PIP3 B were expressed in various tissues,and the expression were higher in roots.We created transgenic plants of PIP3 A and PIP3 B fused with a green fluorescent protein,and found that PIP3 A and PIP3 B were localized on the plasma membrane and also in the cytoplasm.Using CRISPR/Cas9 technology,we obtained PIP3 A and PIP3B’s double mutant pip3a;pip3b,and performed a phenotypic analysis of the mutants.The root hairs of pip3a;pip3b were shorter than those of wild type.(4)Phenotypic analysis of root hair of HRL mutantHRL is an unidentified protein that may be involved in hypersensitivity reactions and induce programmed cell death.We created GUS report transgenic plants driven by HRL self-promoter and analyzed by GUS staining.It was found that HRL was expressed in all tissues and was highly expressed in all stages of root hair growth.We created transgenic plants containing HRL fused with a green fluorescent protein.Using confocal microscopy,we found that HRL was located in the cytoplasm.We ordered two T-DNA insertion mutants of HRL.Through flanking sequence alignment,we found that both T-DNA were inserted in the 5’-UTR region of HRL.qRT-PCR detection results showed that the expression of HRL in these two mutants was significantly down-regulated.Compared with the wild type,the root hairs of the two mutants had no significant change.In summary,SRH1 is a regulator of root hair growth in Arabidopsis.We screened the interacting proteins of SRH1 by the yeast two-hybrid membrane system,and verified the interaction between SRH1 and candidate proteins by a variety of methods.The preliminary functional study of the interacting proteins PIP3 A and HRL showed that the loss of PIP3 function affected the growth of root hair,while the function of HRL remains to be futher studied.These results are of reference value for us to analyze the root hair growth regulatory network and deeply understand the role of receptor-like kinase family in cell polar growth and other life activities.