Studies On The Mechanism Of Arabidopsis GSK3 Family Members AtSK11 And AtSK12 Regulating Root Elongation Under Mild Osmotic Stress

Plant osmotic stress,an important impact factor of plant growth and crop yield,could be aroused by abiotic stresses such as drought,waterlogging,salt damage and low temperature.Most osmotic stresses are harmful to plant growth,but it has been reported that certain degree of drought and PEG mimic mild osmotic stress can facilitate plant root growth.However,the mechanisms of mild osmotic stress promote plant root growth are still unclear.Thus,to study the mild osmotic stress responsive mechanisms can provide an important theoretical reference for germplasm innovation with enhanced stress adaptability,plant water-saving irrigation,and promoting agricultural development.Glycogen synthase kinase 3s(GSK3s)are highly conserved serine/threonine protein kinases which are involved in regulation of many development processes in eukaryotes.In plants,GSK3 s not only are the core component in the Brassinosteroids(BRs)signaling pathway,but also regulate the stress responsive hormone Abscisic acid(ABA)signaling pathway.It is reported that mutating or overexpressing several GSK3 s family members affected the plant osmotic stress response;however,the regulation mechanisms remain elusive.In this study,we focused on the function of GSK3 s in mild osmotic stress response in Arabidopsis and found following results:As root tip is of great importance for water sensing in plant,we first identified the root tip highly expressed Arabidopsis thaliana shaggy-related kinase 11(At SK11),At SK12 and At SK13 by promoter::GUS analysis of GSK3 s members.It was found that compared with Col-0 background wildtype under normal condition,atsk11atsk12 has shorter root length,decreased length of meristematic zone,reduced cell number of meristematic zones,and similar level of mature epidermal cell length,which indicate that At SK11 and At SK12 regulated root growth by promoting cell division in root meristem.In addition,atsk11atsk12 had fewer root hairs than WT,indicating that At SK11 and At SK12 are also involved in the regulation of root hair development.Comparing the root length of Col-0 and At SK11,At SK12,At SK13 related mutant under osmotic condition,it was found that: 1.Compared to the mock condition(-0.25 MPa),the root growth was stimulated under mild osmotic stress(-0.4 MPa)but was repressed under relatively strong osmotic stress(-0.5,-0.6,-0.7 MPa);2.As for the growth stimulation caused by mild osmotic stress(-0.4 MPa),atsk11atsk12 showed more sensitivity than Col-0.Through RNA-seq analysis of Col-0 and atsk11atsk12 under three water potentials(-0.25,-0.4,-0.6 MPa),10 stress responsive genes were identified,8 of them are cell wall related genes including Extensin genes,Proline-rich protein 3(PRP3)and Xyloglucan endotransglucosylase/hydrolase protein gene 14(XTH14).By promoter motif enrichment and transcription factor binding analysis of 10 stress responsive genes in atsk11atsk12,a basic helix-loop-helix(b HLH)family transcription factor LJRHL1-Like 2(LRL2)was identified which might regulate the stress responsive genes.The expression levels of mild stress responsive genes were upregulated in LRL2 mutants and repressed in LRL2 overexpression materials,indicated that LRL2 inhibit the expression of stress responsive genes.In order to study whether GSK3 s function in mild osmotic stress response by regulating LRL2,we first tested the interaction between GSK3 s and LRL2 and found that At SK11,At SK12,and At SK13 can interact with LRL2.In vitro phosphorylation experiments showed that GSK3 s could phosphorylate LRL2.11 potential phosphorylation sites of LRL2 were identified by phosphorylation mass spectrometry.In addition,the Dual-luciferase(Dual-luc)experiments showed that LRL2 could inhibit the promoter activity of the mild stress responsive genes,while At SK11 and At SK12 can inhibit the suppression function of LRL2.Genetic analysis revealed that LRL2 acted downstream of At SK11 and At SK12 in the mild osmotic stress response.In summary,this study found that under normal conditions,At SK11 and At SK12 promote root tip cell division and root hair development;under mild osmotic stress(-0.4 MPa)conditions,the root growth was facilitated and atsk11atsk12 shows more sensitive to mild stress induced root growth;further study suggested At SK11 and At SK12 repress the function of LRL2 to release the transcriptional inhibition of the mild osmotic stress responsive genes and play a negative role in the induction of root growth in response to mild osmotic stress.Our research revealed a new regulation mechanism of mild stress-induced root growth,which could be useful in improving crop adaptation to stress and providing new solutions for crop water-saving irrigation.