Regulation Of MicroRNA162 On Stomatal Clsoure Of Tomato Leaves Under Low Night Temperature

MicroRNA(miRNA)are 22 nucleotide(nt)endogenous non-coding RNAs,and miRNA can respond to a variety of abiotic stress.Previous studies had discovered that mi R162 was the differentially expressed miRNA and could be induced by abscisic acid under low night temperature,miR162 responded to low night temperature by DCL1-dependent ABA signaling pathway.In this study,we constructed mi R162 overexpressing and silencing vectors to obtain transgenic plants.The phenotype and stomatal ultrastructure of transgenic plants were obtained,the Fv/Fm and ABA content of transgenic plants under low night temperature were measured,the genes related to ABA synthetic,decomposition and signaling pathways were identified.We also analyzed the expression level of cold-stress related genes and DCL1-cleaved mi RNAs.The role of miR162 in regulating the stomatal opening and closing under low night temperature and the specific function of miR162 in tomato under low night temperature stress was clarified.The main results are as follows:1.miR162 affects the opening state of stomata.Compared with the wild-type plants,the aperture of stomatal in miR162 overexpressing plants was reduced,while the aperture of stomatal in miR162 silencing plants was increased.Under low night temperature(6?),the stomatal aperture gradually decreased in wild type plants,mi R162 overexpressing plants and silencing plants,but the stomatal aperture in mi R162 silencing plants was the highest among them,indicated that silencing of miR162 could alleviate stomata closure under low night temperature.This study also found that the miR162 silencing plants can alleviate the decrease of Fv/Fm to resist cold stress.2.miR162 affects the change of ABA content.Compared with the wild-type plants,the ABA content in miR162 overexpressing plants was increased and was decreased in miR162 silencing plants.Under low night temperature(6?),the ABA content was increased on the ninth day in overexpressing plants,and was decreased after 6 days in silencing plants.It was speculated that the silencing of miR162 could alleviate the increase of ABA content to reduce the stomatal closure to resist low night temperature stress.3.miR162 regulates the expression of ABA synthesis,catabolism,signaling pathway and cold-stress related genes.Compared with the wild type plants,NCRD1,ZEP1,ABA2,and AAO in the ABA synthesis pathway and ABI3,ABF4,AREB and MYB1 in ABA signaling pathway were up-regulated in mi R162 overexpressing plants,the CYP707A1 and CYP707A2 in ABA decomposition pathway were down-regulated.The opposite trend appeared in the silencing plants.Under low night temperature(6?),the expression of ABA synthesis-related genes,ABA decomposition-related genes,and ABA signaling pathway-related genes changed significantly,suggested that miR162 regulated the expression of ABA synthesis,decomposition,and signaling pathway related genes under low night temperature to affect ABA content.Analysis of cold stress genes CBF1,CBF2 and CBF3 revealed that low night temperature could induce the expression of CBF1,CBF2 and CBF3 and demonstrated thatmiR162 may regulate ABA content and the expression of cold stress related genes.4.mi R160,miR164,and miR171 a were up-regulated in miR162 overexpressing plants by analyzing the expression level of the DCL1-cleaved mi RNAs and their target genes;the results showed that miR160,miR164,and miR171 a were down-regulated in silencing plants,and target genes changes in the opposite trend.The results speculated that miR162 may play a role through DCL1 to regulate the expression of miR160,miR164 and miR171 a in response to low night temperature.