Regulation Mechanisms Of Transcription Factors Cs PIF8 And CsAGL6 On Citrus Fruit Metabolism And Cold Tolerance

Citrus is one of the most important fruit.Improving the quality of citrus fruit has always been one of the most vital goals of citrus breeding work.Citrus fruit quality is closely related to the composition of primary metabolites and secondary metabolites.Studies on the biosynthesis and regulation mechanism of citrus fruit quality-related metabolism will provide a theoretical basis for molecular breeding of citrus.Transcription factors(TFs)are important regulators in different stages of plant metabolism.In this study,transcription factor gene Cs AGL6 responding to IAA and transcription factor gene Cs PIF8 responding to cold stress were screened.Subsequently,the function and regulatory mechanism of the two transcription factors were analyzed in depth.Detailed results are showed as follows:1.Effects of IAA on citrus fruit and callus metabolism.In this study,the fruit of citrus sinensis and the callus of citrus paradis were treated with IAA for metabolomics and transcriptome analysis.The results indicated that the expression levels of genes and the content involved in carotenoid pathway were up-regulated in fruit and callus.Besides,it was found that the contents of soluble sugar,d-limonene and lignin in callus were all decreased,while the content of citric acid was increased.In addition,the gene expression level of an IAA-responsive transcription factor gene,Cs AGL6,was upregulated about 100 folds.2.Regulatory mechanism of IAA response transcription factor Cs AGL6 in metabolic pathway.In this study,we detected the contents of metabolites and related gene expression levels of overexpressed Cs AGL6 in sweet orange fruits and grapefruit callus.The results showed that the contents of citrus carotenoids and ABA were significantly increased in overexpressed lines,and the expression levels of BCH,ZEP and NCED6,involved in downstream carotenoid biosynthesis pathway were significantly up-regulated.In addition,the expression levels of soluble sugar,d-limonene and flavonoids biosynthetic genes were significantly down-regulated in grapefruit callus,while the expression levels of lignin and organic acid biosynthetic genes exhibited the opposite effects.Besides,the promoters of ten primary and secondary metabolic pathway genes(SS,PAL,F3H/FLS,F3'H,ZEP,NCED6,RHM1,CHS,LS and CCo AOMT)were found to be bound by Cs AGL6 transcription factors by Ch IP-seq,Ch IP-PCR,Ch IP-q PCR and EMSA analysis.Furthermore,it was found that 9 interacted proteins of Cs AGL6 were identified by Co IP-MS and Y2 H analysis,including heat Shock protein HSP70-2/5,CLPB1/3/4,Phosphorylated protein kinase(GAPDH,NDPK3),E3 ubiquitin Ligases Slr P and proteolytic enzyme CLPX.3.Identification and molecular functional analysis of the cold stress response transcription factor Cs PIF8.In this study,a linear up-regulation expression of a b HLH transcription factor(Cs PIF8)gene was identified in sweet orange leaf transcriptomic data treated at low temperature for 0-72 h.The relative electrolyte leakage and the content of MDA of the overexpressed Cs PIF8 tomato plants were significantly lower than those of the wild-type,while virus-induced gene silencing-mediated suppression of PIF8 increased the relative conductivity and the content of MDA sensitivity in seedlings of Poncirus trifoliata.Futhermore,the results of Ch IP-seq,Ch IP-PCR,Ch IP-q PCR and EMSA experiments showed that Cs PIF8 directly bound the promoter of Cs SOD and activated the expression of Cs SOD.Meanwhile,the expression level of Cs SOD and the activity of SOD enzyme were significantly increased,whereas the level of O2.-was significantly reduced in the transgenic lines.The Poncirus trifoliata seedlings with VIGS-mediated suppression of PIF8 exhibited the opposite effects.These results have shown that Cs PIF8 improved cold tolerance in citrus through regulating the expression level of SOD and SOD activity.It showed that PIF8 enhanced the quality of resistance to low temperature stress.In conclusion,this study build foundamental bases for in-depth analysis of the biological nature of plant multiple metabolic networks,and also provides important theoretical basis for industrial practice to improve the comprehensive quality of citrus.