Molecular Mechanism Of Wheat TIFY Transcription Factor TaTIFY10b In Regulating Grain Development

Wheat(Triticum aestivum L.)is one of the largest food crops in the world,and its planting area,total grain yield and the proportion in the international trade all rank first.Wheat production is facing a series of challenges in China because of increasing population,reduced arable land and increased production costs in the scenario of climate change.It is the most effective way to improve grain yield and ensure food security by exploration of the genetic characteristics of wheat germplasms and elite gene resources.TIFY transcription factor is an important component of JA signaling pathway,involved in regulating plant growth and development and response to various stress.In this study,we characterized the functions of TaTIFY10 b through gene expression analysis,DNA sequence polymorphism assays,functional marker development,association analysis and protein interaction experiments.Our results indicated that TaTIFY10 b was implicated in regulation of grain weight,and the Hap-2A-1was an elite haplotype for high thousand kernel weight(TKW).Furthermore,we preliminarily discovered the signaling pathway of TaSnRK2.4 controlling grain size through phosphorylation of TaTIFY10 b.The main findings are as follows:(1)The genomic sequences of wheat TaTIFY10b-2A,-2B and-2D were cloned,which contains a conserved TIFY domain and a CCT-2 domain.Secondary structure prediction showed TaTIFY10 b had four α helices and two β sheets.(2)Multiple cis-acting elements were identified in the promoter region of TaTIFY10 bs,including root specific expression elements as-1,ABA response elements ABRE,low temperature stress response elements LTR and methyl-jasmonate response elements CGTCA/TGACG motif,etc.However,the number and distributions of cis-acting elements varied significantly in different promoters of TaTIFY10 bs.(3)Gene expression analysis showed that TaTIFY10 b was expressed in root,stem,leaf and ear of wheat at jointing,heading and flowering stages,and the expression level was relatively higher in spikes compare to other tissues.TaTIFY10b-2A was induced by high salt,drought,exogenous ABA,low temperature and Me JA,but its expression patterns varied greatly under different abiotic stress conditions.(4)Sequence polymorphism assays showed that there were three SNPs in the non-coding region of TaTIF10b-2A,one SNP in the non-coding region of TaTIF10b-2B,and no variation in TaTIF10b-2D.Based on the variations,three molecular markers were developed accordingly,i.e.SNP-A-1,SNP-A-3and SNP-B-1.(5)Association analysis indicated that both SNP-A-1 and SNP-A-3 of TaTIFY10b-2A were significantly associated with TKW under multiple environments.There are three main haplotypes of TaTIFY10b-2A in natural population P1,among which Hap-2A-1 is an elite haplotype for higher TKW,and has been positively selected in wheat breeding in China.(6)Two candidate interacting proteins of TaTIFY10 b were obtained by screening the wheat c DNA library in yeast cells.Further yeast two hybridization and LCI(Luciferase complementation imaging)experiments proved that TaTIFY3 was an interactor of TaTIFY10 b.Subcellular localization results showed that both TaTIFY10 b and TaTIFY3 were localized in the nucleus,and the Bi FC experiments revealed that their interaction occurred in the nucleus.(7)Yeast two hybridization and LCI experiments revealed that TaSnRK2.4,TaTIFY10 b and TaTIFY3 could interact in pairs.Tissue specific expression showed that their transcriptional levels were relative high in wheat spikes,suggesting that they might form complex and be implicated in controlling the growth and development of wheat grains.(8)Protein phosphorylation results showed that TaSnRK2.4 could phosphorylate TaTIFY10 b.Given both of them are involved in regulating grain size,it was speculated that TaSnRK2.4 might control grain size by regulating the biological activity of TaTIFY10 bs in wheat.