The Mechanisms Of PIF4-Mediated Light Quality-Regulated Cold Tolerance In Tomato

Tomato(Solanum lycopersicum)plays an important role in vegetable cultivation in protected areas of our country with high economic efficiency.However,tomato is a kind of warm-loving vegetable,which can't tolerate low temperature and freezing damage.Low temperature and chilling injury often results in the growth and development of tomato is hindered,and the quality and yield of tomato are decreased.Therefore,it is of great practical significance to take some measures to improve the low temperature tolerance of tomato.Phytochrome-interacting factor PIFs are involved in many plant growth and development processes,which not only play a key role in photothermal signal transduction,but also regulate the stress response of plants by participating in the synthesis of various hormones and signal transduction.In this paper,tomato was selected as the experimental material,and molecular biology,plant physiology as well as other technologies were used to study the role of phytochrome-interacting factor PIF4 in the regulation of low temperature resistance of tomato mediated by light quality.It was clear that PIF4 could improve the cold tolerance of plants by regulating CBFs pathway related genes and phytohormones.The mechanism of PIF4 transcriptional regulation of CBF1 and GAI4 was investigated.The main contents are as follows:1.The role of phytochrome-interacting factor PIF4 in low temperature resistance of tomato was studied.Low temperature inhibited and induced the expression of PHYB and PHYA genes of phytochrome PHYB and PHYA respectively,which are sensitive to red light(R)and far red light(FR).Cold and FR not only induced the gene expression of PIF4,but also enhanced the stability of PIF4 protein.At low temperature,PIF4 expression decreased in phyA and phyAB1B2 mutants,while PIF4 transcription increased in phyB1B2 mutants.The low temperature resistance of pif4 mutant plants decreased while the PIF4 over-expressed plants increased significantly,which suggested that PIF4 positively regulated the low-temperature resistance of tomato.Further studies showed that PIF4 promoted the transcription of CBF1 by directly binding to the promoter of CBF1 at low temperature to enhance the cold resistance of tomato.On the other hand,PIF4 induces the accumulation of phytohormones ABA and JA by promoting the expression of ABA and JA biosynthesis genes,and meanwhile inhibits the expression of GAs biosynthesis genes and reduces GAs content,thus regulating the low temperature resistance of tomato.The above results indicated that FR promoted the accumulation of PIF4 through phyA under low temperature,and the transcription factor PIF4 enhanced the cold resistance of tomato plants by transcriptional regulating CBF1,inducing the synthesis of ABA and JA and inhibiting the synthesis of GAs.2.The interaction of PIF4 and GAI4 in the low temperature resistance of tomato controlled by light quality was clarified.The phytochrome-interacting factor PIF4 is the key junction connecting the light signal and hormone signal,while the interaction between PIF4 and the negative regulation gene GAI4 in the GAs signal pathway at low temperature is not clear.It was found that the content of GAs increased and the expression of GAI4 decreased significantly in pif4 mutants under low temperature.Using gel electrophoresis transfer assay(EMSA),double luciferase reporter gene system and chromatin immunoprecipitation assay(ChIP),we found that PIF4 binds to promoter of GAI4 gene to promote its expression at low temperature.This indicated that PIF4 positively regulates GAI4 expression under cold stress.3.The effect of GAI4 on low temperature tolerance of tomato induced by far-red light was clarified.The results showed that under low temperature,the maximum photochemical efficiency(Fv/Fm)of tomato photosystem II was significantly decreased,the relative electrolyte leakage(REL)was significantly increased,and the expression of cold-resistant CBF1 gene was significantly down-regulated after silencing GAI4 gene in tomato.However,after overexpression of GAI4 gene,Fv/Fm increased,REL decreased significantly,and CBF1 gene expression increased,which indicated that GAI4 regulated low-temperature resistance of tomato.To further clarify the mechanism of GAI4 in FR induced low temperature resistance in tomato,we detected the phytohormones ABA and JA in GAI4 silencing and overexpression materials.The research showed that overexpression of GAI4 significantly induced the synthesis of ABA and JA and the expression of signaling genes,and promoted the accumulation of ABA and JA.GAI4 gene silencing significantly weakens this induction.This suggest that GAI4 regulates ABA and JA signaling pathways.Exogenously spraying GA3 and its inhibitor PAC on WT and ABA-deficient mutant not plants showed that ABA signal acted on the downstream of GAs signal.Taken together,GAI4 gene positively regulates FR induced low temperature resistance of tomato through CBFs and ABA,JA hormone pathways.4.It is clear that GAI4 feedback supresses PIF4 at low temperature.Exogenous spraying of GA3 and its inhibitor PAC on WT,pif4 mutants and PIF4 overexpressed plants showed that GA3 significantly inhibited while PAC promoted the low temperature resistance of tomato.In addition,the accumulation of PIF4 protein was significantly inhibited by exogenous PAC spraying at low temperature,and PIF4 protein was also decreased after overexpression of GAI4 gene.On the contrary,PIF4 protein significantly accumulates after silencing GAI4 gene.This indicates that GAI4 feedback inhibits PIF4 at low temperature.PIF4-GAI4 forms a feedback loop to finely regulates the cold resistance of tomato.