Target Of Rapamycin Regulates Root Hair Growth In Arabidopsis

Root hair plays an important role in planting soil,adapting to the environment and absorbing nutrients.It is important to study plant root hair development to regulate plant growth and development.Target of rapamycin(TOR)is a highly conserved serine/threonine protein kinase in eukaryotes.TOR can regulate cell autophagy,metabolism and protein synthesis by phosphorylation and dephosphorylation of downstream substrates in response to external nutrition,energy and amino acid signals.At present,it has been found that the development of plant root hair is regulated by a complex molecular network.Several key transcription factors play an important role in cell fate determination,root hair development,root hair growth and maturation,however,further research is needed on how upstream energy nutrition signals regulate root hair growth and development.The mechanism by which TOR as a plant energy nutrition stress signal sensor regulates root hair development remains unclear.Studying the molecular mechanism of TOR regulation of root hair development is of great significance for improving the molecular regulation network of plant root hair.In this paper,phenotypic analysis,transcriptome sequencing analysis,genetics and molecular biology were used to study the important functions of TOR signaling pathway in Arabidopsis root hair development,and the function of TOR-TAP46-RHD6 signaling pathway in Arabidopsis root hair development regulation was preliminarily elucidated.The results of this study are as follows:(1)In order to investigate the effect of TOR on root hair development,TOR inhibitor RAP was used to treat WT and BP12-2 plants.The plant itself's FKBP12 is not sensitive to RAP,and BP12-2 is a transgenic line that is very sensitive to RAP obtained by transforming yeast FKBP12 into Arabidopsis.It was found that the root hair length of BP12-2 was significantly inhibited compared with control WT,indicating that TOR activity was inhibited by inhibiting root hair development.The expression level of root hair development related genes was detected after RAP treatment,and the expression level of transcription factor RSL4 was down-regulated.The transcriptome data of rhd6rsl1 VS WT and BP12-2 in DMSO vs RAP were analyzed.It was found that RHD6,an upstream regulator of RSL4,may play an important role in TOR regulation of Arabidopsis root hair development.At the same time,qPCR results showed that RHD6 transcription levels were not affected by TOR inhibitors.This suggests that RHD6 may be regulated by protein levels.(2)By cloning the Arabidopsis RHD6 and RSL4 genes,the overexpression vector was constructed and the target gene was transformed into Arabidopsis thaliana BP12-2 to obtain Arabidopsis thaliana transgenic plants.Transgenic plants were treated with RAP.After culture,overexpression of RHD6 and RSL4 could partially restore the phenotype of BP12-2 inhibition of root hair growth during TOR inhibitor treatment,suggesting that TOR may regulate root hair development through RHD6 and RSL4.(3)Based on the above results,the TOR and its substrate were studied by yeast two-hybrid assay with RHD6.The results showed that there may be interaction between TOR's direct substrate TAP46 and RHD6.Further use of bimolecular fluorescence complementation experiments(BiFC)and co-immunoprecipitation experiments(Co-IP)demonstrated that TAP46 does interact with RHD6.This indicates that TOR may regulate RHD6 through its substrate TAP46 to regulate root hair development.At the same time,bioinformatics analysis found that the six genes of the RHD6 family have a high degree of homology in evolution.Further,it was found by yeast two-hybrid that multiple members of the RHD6 subfamily may interact with TAP46.(4)In addition,TAP46 overexpression vector was also constructed in this study,which was transformed into Arabidopsis wild type and successfully obtained transgenic plants.Arabidopsis thaliana transgenic plants were treated with AZD,and the root hair development of the transgenic plants was found to be significantly better than that of the wild type control,indicating that TAP46 has a promoting effect on Arabidopsis root hair development.The results of this study showed that root hair development was also inhibited after TOR of Arabidopsis thaliana was inhibited.Quantitative analysis and transcriptome analysis revealed that RHD6 may be a key gene for TOR regulation of root hair development,while RSL4 is a direct target of RHD6,transcription of RHD6 is not regulated by TOR,and transcriptional level of RSL4 is affected by TOR;phenotypic experiments found that overexpression of TAP46,RHD6 and RSL4 could restore root hair growth restriction after TOR inhibition;further protein interaction experiments demonstrated between TAP46 and RHD6 there are protein interactions;this study demonstrates that TOR regulates Arabidopsis root hair development via RHD6 based on the above results.