Functions And Molecular Mechanisms Of SAUR15 In Regulating Development Of Plant Lateral And Adventitious Roots

Zygophyllum xanthoxylum,widely distributing throughout the desert region of northwestern China,has remarkable vitality to thrive under adverse drought condition.Forming well-developed root system dominated by lateral and adventitious roots to absorb water and nutrients from soil is one of the important strategies for Z.xanthoxylum to cope with drought stress.However,the regulation mechanisms of Z.xanthoxylum lateral and adventitious root development has not been revealed.Root development is accurately regulated by auxin and brassinosteroids(BRs).In previous study,a large number of Small Auxin-Up RNAs(SAURs)were screened via analyzing transcriptome datasets of-0.5 MPa-treated Z.xanthoxylum roots,which is a downstream response gene family of auxin and BR signaling pathway and play important roles in cell expansion-induced growth and development.However,their functions and mechanisms in lateral and adventitious root development driven by cell expansion remains unclear.Therefore,to figure out functions of these genes in lateral and adventitious root development,we chose Unigene40989_All(ZxSA UR15),a member of the ZxSAUR family induced by osmotic stress significantly,as the candidate gene,and characterized function of ZxSAUR15 in root development.In addition,molecular mechanisms of AtSAUR15,the homolog of ZxSAUR15 in Arabidopsis thaliana,in regulating auxin-and BR-mediated root development was also explored.The main results are as follows:1.ZxSAUR15 played positive roles in lateral and adventitious root developmentThe full-length coding sequence of ZxSAUR15 was cloned based on the sequence of Unigene40989_All.The length of ZxSAUR15 coding sequence is 306 bp,encoding 101 amino acids.Bioinformation analysis showed that ZxSAUR15 was the homologous gene of SAUR15 from other plants and contained 13 predicted phosphorylation sites,3 O-N-acetylglucosamine glycosylation sites and 1 ubiquitination site,which indicated the activity,localization and stability of ZxSAUR15 maybe influenced by phosphorylation,glycosylation and ubiquitination.Subcellular localization analysis showed that ZxSAUR15 was localized on the plasma membrane.The promoter sequence of ZxSAUR15 was amplified by chromosome walking method and the length of the sequence was 1564 bp.The results of cis-element analysis showed that the ZxSAUR15 promoter contained elements related to plant hormones such as auxin and environmental factors such as drought.The plant expression vector pBIB-BASTA-pZxSA UR15::GUS was transformed into Z.xanthoxylum and Arabidopsis seperately.GUS staining analysis showed that the promoter of ZxSAUR15 possessed transcription activity in lateral roots of Z.xanthoxylum as well as lateral and adventitious roots of Arabidopsis.ZxSAUR15 plant expression vector was constructed and the homozygous ZxSA UR15-overexpression Arabidopsis lines were obtained.ZxSAUR15 overexpression promoted root development of transgenic seedlings.Compared to wildtype Col-0,the primary root length,number of lateral root and lateral root primordia,number of adventitious root and adventitious root primordia of transgenic seedlings were increased for about 33%and 23%,66%and 48%,89%and 49%,59%and 31%,95%and 49%,seperately,indicating that ZxSAUR15 plays positive roles in root development.2.In auxin signaling pathway,AtSAUR15 promoted lateral and adventitious root development via regulating plasma membrane H+-ATPase activity and auxin biosynthesisAuxin sensitivity assay suggested that the exogenous auxin treatment significantly induced the expression of AtSAUR15 and rescued lateral and adventitious root defects of saur15-1.RT-qPCR showed that the response of AtSAUR15 to auxin treatment was altered in loss-of-function mutants and overexpression lines of AUXIN RESPONSE FACTOR6(ARF6)and ARF7.Besides,ChIP-qPCR and luciferase activity assay indicated that ARF6 and ARF7 bind to the auxin response elements in AtSAUR15 promoter directly,which can be enhanced by auxin.Meanwhile,overexpression of AtSAUR15 can restore root phenotype of ARF6-and ARF7-related mutants.All these data indicated that,in auxin signaling pathway,AtSAUR15 regulated lateral and adventitious root development as the direct target gene of ARF6 and ARF7.Protein interaction analysis and phosphatase activity assay suggested that AtSAUR15 interacted and inhibited D-clade type 2C protein phosphatases(PP2C-Ds)on plasma membrane.The genetic assays indicated that PP2C-D2 and PP2C-D5 played negative roles in AtSAUR15-regulated root development.Pysiological and biochemical assays showed that the activity of plasma membrane H+-ATPase decreased in saur15-1 but increased in AtSAUR15 overexpression seedlings.Besides,genetic experiments suggested that plasma membrane H+-ATPase played positive roles in AtSAUR15related root development.Considering that PP2C-Ds interact with plasma membrane H+-ATPase directly and inhibits its activity,all these results demonstrated that AtSAUR15 activated plasma membrane H+-ATPases via inhibiting PP2C-Ds to facilitate lateral and adventitious root development.AtSAUR15 overexpression seedlings exhibited auxin overproduce phenotype.Additionally,comparing to Col-0,DR5 promoter activity and free IAA concentration were increased in AtSAUR15 overexpression seedlings but decreased in saur15-1.Besides,comparing to Col-0,expression of genes encoding the key auxin biosynthesis enzymes in AtSAUR15 overexpression seedlings were also upregulated,which were downregulated in saur15-1.These data demonstrated that AtSAUR15 positively regulated auxin synthesis via inducing auxin biosynthesis genes,which may activate auxin signaling and promote lateral and adventitious root formation in a positive feedback loop.3.In BR signaling pathway,AtSAUR15 promoted plasma membrane H+ATPase via activating BRI1 to regulated lateral and adventitious root developmentComparing to Col-0,the BR sensitivity of saur15-1 was decreased but increased in AtSAUR15 overexpression lines.On the contrary,the BRZ inhibition on saur15-1 was increased but decreased on AtSAUR15 overexpression lines.Genetic experiments showed that AtSAUR15 overexpression rescued rosette,hypocotyl and root defects of cpd91,a weak impaired allele of BR biosynthesis enzyme CONSTITUTIVE PHOTOMORPHOGENIC DWARF(CPD),and bril-301,a weak impaired allele of BR receptor BRASSINOSTEROID-INSENSITIVE1(BRI1),but had no effect on null alleles,cpd and bril-701.These results indicated that AtSAUR15 played positive roles in BR signaling pathway which was dependent on BR-activated BRI1.According to yeast two hybrid assay and bimolecular fluorescence complementation analysis,AtSAUR15 bond to BRI1 on plasma membrane.However,co-immunoprecipitation assays found little or no interaction between AtSAUR15 and BRI1 in the absence of BL,and BL treatment promoted the interaction between them.In addition,biochemical expreiments showed that AtSAUR15 elevated the phosphorylation status of BRI1 and promoted the interaction between BRI1 and its co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1(BAK1).These results demonstrated that AtSAUR15 interacted and activated BRI1.Comparing to Col-0,changes in CPD、DWARF4(DWF4)expression level as well as BRASSINAZOLE-RESISTANT1(BZR1)phosphorylation status in response to exogenous BL treatment was not altered in AtSAUR15-related seedlings.Besides,AtSAUR15 overexpression also compensated phenotype defects of bin2-1,a gain-offunction mutant of BRASSINOSTEROID INSENSITIVE 2(BIN2),the negative regulator in BR signaling pathway.These results indicated that AtSAUR15-BRI1 and BRI1-BIN2 had separated functions in the BR signaling pathway.Genetic and biochemical experiments further suggested that AtSAUR15-activated BRI1 directly stimulated plasma membrane H+-ATPase via phosphorylation,which induced cell expansion and consequently promoted various plant organs growth and development including lateral and adventitious root development.In conclusion,in this study,functions and molecular mechanisms of SAUR15 in auxin-and BR-mediated lateral and adventitious root development are elucidated,which provides a theoretical basis for clarifying the regulation mechanisms of the developed Z.xanthoxylum root system formation.