Mechanism Of MiR164 Targeting SlNAM3 To Regulate Ethylene-Mediated Cold Tolerance In Tomato

Cold stress,which seriously affects the yield and quality of thermophilic crops such as tomato(Solanum lycopersicum L.),has become one of the important limiting factors for year-round production of facility vegetables.Therefore,analyzing the cold response mechanism of vegetable crops and using environmental regulation,gene editing,plant growth regulators and other means to improve the cold resistance of vegetable crops have become significant for ensuring the yield and quality of vegetable crops.In this study,tomato,as the main vegetable crop cultivated in facilities,was taken as the research object.Using plant physiology,biochemistry,molecular biology,genetics and other methods,we explored the mechanism of miR164/Sl NAM3 module and phytohormone ethylene in regulating cold tolerance in tomato.The main findings are as follows:1.Key genes of Sl NAC transcription factors in cold response were identified,the roles of Sl-miR164a/b-5p and Sl NAM3 in improving cold tolerance in tomato was explored.By RT-q PCR and bioinformatics analysis,Sl NAM3,which targeted by miRNA and induced by cold,was screened.Further,multiple methods such as 5’ RLMRACE and dual luciferase assays confirmed that Sl-miR164a/b-5p cleaved transcript and repressed the accummulation of Sl NAM3 transcript.By constructing Sl-miR164a/b-5p gene-silenced plants(STTM164a/b-5p),Sl NAM3-overexpression transgenic lines(OE-NAM3#6)and nam3 mutants(nam3#2,4),it was cleared that Sl-miR164a/b-5p and Sl NAM3 negatively and positively regulated the cold resistance in tomato respectively.At the same time,in both wild-type plants(WT)and STTM164a/b-5p plants,silencing of Sl NAM3 resulted in increased sensitivity to cold stress,and there was no significant difference in cold resistance when silencing of Sl NAM3 in these two genotypes.Above all,it was indicated that Sl-miR164a/b-5p was dependent on Sl NAM3 to jointly regulate cold resistance in tomato.2.The mechanism of the miR164/Sl NAM3 module in improving cold tolerance in tomato was deeply explored,the role of the phytohormone ethylene in cold tolerance induced by the miR164/Sl NAM3 module was analyzed.The positive regulation of ethylene in cold tolerance in tomato was confirmed by using nor mutant and WT plants with exogenous ACC treatment.By determinating ethylene synthesis in STTM164a/b-5p,OE-NAM3#6 and nam3 mutants,it was confirmed that Sl-miR164a/b-5p and Sl NAM3 negatively and positively regulated ethylene synthesis,respectively.Further,it was observed that cold tolerance was recovered in nam3 mutants by exogenous ACC treatment.The above results showed that ethylene regulated the cold resistance induced by the miR164/Sl NAM3 module.3.The regulatory mechanism of miR164/Sl NAM3 module on ethylene biosynthesis was explored.Through detecting of RT-q PCR and cold tolerance of related gene-silenced plants,the ethylene synthesis genes Sl ACO1,Sl ACO4,Sl ACS1 A and Sl ACS1 B were screened,which positively regulated cold resistance in tomato.By measuring the expression of the above gene in STTM164a/b-5p,OE-NAM3#6 and nam3 mutants,it was confirmed that the miR164/Sl NAM3 module activated the gene expression of Sl ACO1,Sl ACO4,Sl ACS1 A and Sl ACS1 B.Furthermore,the transcriptional activation effect of Sl NAM3 on Sl ACO1,Sl ACO4,Sl ACS1 A and Sl ACS1 B genes was confirmed by gel electrophoresis migration assay,chromatin immunoprecipitation and dual luciferase assay.4.The mechanism of ethylene involved in regulating the cold tolerance in tomato was explored.Using nor mutant and WT plants with exogenous ACC treatment,the Sl JAZ8 gene was screened.Besides,through exogenous ACC treatment on Sl JAZ8-silenced plants and silencing of Sl JAZ8 gene in nor mutants,it was confirmed that ethylene regulated cold resistance in a manner dependent on Sl JAZ8 gene.The inhibitory effect of ethylene-induced Sl-miR169e-5p on the accumulation of Sl JAZ8 transcript and protein content was confirmed by dual-luciferase assay and western blot.Further,by luciferase complementary imaging technology(LCI),and bimolecular fluorescence complementary experiments(Bi Fc),it was proved that exogenous ethylene treatment weakened the interaction signal between Sl JAZ8 and Sl MYC2 dependent on Sl-miR169e-5p in vivo.In summary,we confirmed that the miR164/Sl NAM3 module relied on ethylene synthesis pathway to improve cold resistance in tomato plants.In addition,we explored the mechanism of ethylene regulated cold resistance.We found that ethylene induced the inhibition of Sl-miR169e-5p on the accumulation of Sl JAZ8 transcript and protein content and weakened the interaction signal of Sl JAZ8-Sl MYC2.Our results enriched signal transduction network of cold in tomato and provided theoretical guidance and scientific basis for the reasonable use of gene editing,plant growth regulators and other means to induce cold resistance in tomato.