Expression And Functional Analysis Of MicroRNA319 In Tomato Response To Temperature Stress

Tomato(Solarium lycopersicum L.),an herbaceous plant,belongs to the Solanum of Solanaceae.As one of the most important worldwide vegetables,tomato is widely cultivated and consumed all over the world.Tomato is warm-season vegetable and sensitive to both low and high temperature.Tomato is often subjected to the chilling and heat stress during plant growth.MicroRNAs(miRNAs),a kind of important gene expression regulator,play crucial roles not only in plant growth and development,but also in stress responses.Some miRNAs have been identified from tomato for their responses to chilling or heat stress.However,the mechanisms of miRNA in regulating tomato chilling and heat stress tolerance are largely unknown.S.habrochaites is one kind of wild tomato with higher chilling-tolerance ability than other genotypes.In our previous work,chilling-responsive miRNA library was produced from 'LA1777'(S.habrochaites L.)seedlings.In this study,sha-miR319d was selected from chilling-responsive miRNA library,for it was strongly induced by chilling stress.Take chilling-responsive sha-miR319d as the entry point,the responses mode of miR319 in S.lycopersicum and S.habrochaites to chilling and heat stress were explored.Sha-miR319d was overexpressed(35S::sha-miR319d)in 'Micro-Tom'(S.lycopersicum L.)to further investigate its role in regulating chilling stress tolerance,heat stress tolerance,and growth and development of tomato.The major contents and results are as follows:1.Cloning of miR319 family members in tomato.The homologous miRNAs of sha-miR319d in S.lycopersicum('LA2683','Pole red Siberian','LA3475','Stupice' and'Micro-Tom')and in S.pimpiellifolium('LA2093')were identified using Stem-loop RT-PCR.This result confirmed that sha-miR319d was a new member of miR319 family in tomato plants.Base on the known miR319 family members in tomato and our chilling-responsive miRNA library of 'LA1777',4 precursors of sha-miR319 were cloned from 'LA 1777' and we named them sha-MIR319a,sha-MIR319b,sha-MIR319c and sha-MIR319d,respectively.Quantitative RT-PCR(qPCR)analysis results showed that sha-miR319a,b,c,and d were expressed in various organs,especially in stems.Stress-,hormone signals-,cell precess-and light-related cis-elements found in the promoter regions of the miR319 family members indicated a potential role of miR3 19 in regulation of stress tolerance and development processes in tomato plants.2.The expression of miR319 family members in tomato responses to chilling and heat stress.The putative target genes Teosinte branched/cycloidea/PCF transcription factors(TCPs)coding genes TCP3 and TCP29 of miR319 in 'LA1777' were validated using 5'-RLM-RACE.Five tomato genotypes('LA1777','LA2683','Pole red Siberian','LA3475',and 'Stupice')that exhibit different degrees of tolerance to chilling and heat stress were selected to characterize expression patterns of miR319 and its target genes under temperature stress conditions.We found that the involvement of miR319 in the chilling tolerance of tomato might be mediated by the repression of TCP3 and TCP29 expression.Initial stages(1 h)of heat stress up-regulated miR319a,b,and d and led to a decrease in the expression of TCP3,TCP29,and TCP2,whereas miR319c,which was down-regulated in the later stages of heat stress,may regulate TCP3,TCP29,and TCP2 expression.3.Overexpression of sha-miR319d confers chilling and heat stress tolerance in S.lycopersicum.Under chilling or heat stress,compared with the wild type(WT),35S::sha-miR319d transgenic plants had less membrane damage,higher amountamounts of chlorophyll and values maximum quantum efficiency of photosystem ? photochemistry(Fv/Fm)values,lower O2-generation and H2O2 concentration,as well as improved the activities of superoxide dismutase(SOD)and catalase(CAT).Moreover,overexpression of sha-miR319d increased expression levels of C-repeat binding factors(CBFs)coding gene CBF1,heat shock transcription factors(Hsf)coding genes HsfA1a and HsfA1b,antioxidant enzyme coding genes CuZnSOD,FeSOD,CAT,APX1,APX2,C2H2-type zinc finger transcription factors coding genes ZAT12 and ZAT10,and decreased expression levels of MYB83 and heat shock proteins(Hsp)coding gene Hsp90.Silencing of GAMYB-likel and Kelch-type3 genes(putative targets of sha-miR319d)by virus-induced gene silencing(VIGS)were performed to uncover the effects of GAMYB-like1-and Kelch-type3-silencing on tomato under chilling stress conditions.The result showed that silencing of GAMYB-likel and Kelch-type3 increased tomato chilling tolerance but decreased heat tolerance.The relative expression levels of CBF1,CuZnSOD,CAT,APX1,APX2,ZAT12 and ZAT10 were increased in VGAMYB-likel and VKelch-type3 plants.These results indicated that sha-miR319d regulates tomato chilling tolerance,possibly by inhibiting expression of GAMYB-like1 and Kelch-type3.4.Overexpression of sha-miR319d affects tomato growth,leaf and fruit development.Overexpression of sha-miR319d resulted in reduced plant height,increased dry and fresh weight,leaf crinkling and reduced both horizontal and vertical diameters of fruit(shape index was not changed),reduced weight of per fruit and fewer seeds in each fruit.Silencing of TCP3 and TCP29 using VIGS resulted in leaf crinkling like 35S::sha-miR319d transgenic lines.The flower morphology was also changed in TCP3-and TCP29-silencing plants.The interaction between TCP3 and TCP29 were indicated by Bimolecular fluorescence complementation(BiFC),indicating that TCP3 and TCP29 may have co-operation in some biological processes.The expression levels of No apical meristem 2(NAM2),NAM3,and NAM,ATAF1/2,CUC2(NAC1)were higher in 35S::sha-miR319d,VTCP3,and VTCP29 plants than those in WT.Moreover,miR164 was lower in 35S::sha-miR319d,VTCP3,and VTCP29 plants than that in WT.In conclusion,overexpression of sha-miR319d had effects on tomato growth.The expression of TCP3 and TCP29 regulated by sha-miR319d further mediated the expression of downstream miR164 and its target genes,participating in the regulation of leaf morphology.