Molecular Mechanism Of Calmodulin Binding Transcription Activator Regulating Linalool Synthesis In Peach Fruit

Calmodulin-binding transcription activator(CAMTA)is an important transcription factor family widely present in eukaryotes,which involved in many physiological processes such as plant growth and stress response.However,its function in the formation of fruit flavor quality remains unclear.Aromatic substances are important factors affecting fruit flavor,among which linalool is an important material basis for the formation of aromatic quality of peach and other fruits.In this study,peach fruits of’Prunus persica L.Batsch cv.Hujingmilu’were used as research materials to identify CAMTA members involved in linalool formation and regulation,and to analyze the regulatory mechanism of linalool synthesis from the transcriptional regulation and epigenetic regulation level.The main results are as follows:1.Five Pp CAMTAs were identified in peach.They unevenly distributed on three chromosomes,their gene structures were highly conserved.The proteins encoded by Pp CAMTAs have characteristic domains such as CG-1 and Ca MBD.The promoter regions of Pp CAMTAs contained a large number of cis-acting elements,and about 50%of the elements were able to respond to light signals.Subcellular localization confirmed that peach CAMTA proteins were localized in the nucleus or plasma membrane.Pp CAMTAs had different expression patterns in different organs,developmental stages,and abiotic external stimuli.The conserved function of Pp CAMTA1 involved in low temperature response was verified by allogeneic transgene of Arabidopsis mutants.2.To clarify that transcription factor Pp CAMTA1 was involved in linalool synthesis in peach fruit.Through promoter analysis of volatile aromatic compounds synthesis structural genes in peach,there were a large number of CAMTA binding sites in the promoter regions of two candidate structural genes(Pp AAT1 and Pp TPS3).Using dual-luciferase reporter assay,Pp CAMTA1 had an activation effect on the promoter of terpene synthase Pp TPS3 which responsible for linalool synthesis.EMSA assay and yeast one hybrid assays demonstrated that Pp CAMTA1 directly bound to the promoter of Pp TPS3.The results of homologous transient overexpression in peach leaves showed that increasing the expression of Pp CAMTA1 promoted linalool synthesis.Transgenic material was obtained by using Arabidopsis camta2,3 mutant and driving Pp CAMTA1overexpression with the promoter of At CAMTA3,a homologous gene,which significantly increased linalool content.These results indicated that Pp CAMTA1regulated linalool synthesis in peach fruit by binding to and activating the promoter of the structural gene Pp TPS3.3.To elucidate the relationship between transcription factor Pp CAMTA1 and other transcription factors(Ppb HLH1,Pp ERF61,Pp MADS2)regulating the linalool synthesis in peach fruit.Bimolecular fluorescence complementation assay(Bi FC)found that there was no interaction between Pp CAMTA1 and Ppb HLH1 or Pp ERF61.Combining with firefly luciferase complementation imaging(LCI)and yeast two-hybrid,it was determined that Pp CAMTA1 and Pp MADS2 were bound at the protein level.The dual-luciferase reporter assay showed that there was no upstream or downstream regulatory relationship between Pp CAMTA1 and Ppb HLH1 or Pp ERF61,while Pp MADS2 activated the promoter of Pp CAMTA1.EMSA assay further showed that Pp MADS2 directly bound the Pp CAMTA1 promoter.These results showed that transcription factors Pp MADS2 and Pp CAMTA1 had both protein interaction and transcriptional regulation,Pp MADS2 was involved in regulating the synthesis of linalool by binding and activating Pp CAMTA1 promoter.4.To reveal the molecular mechanism of Pp CAMTA1 involved in UV-B regulation of linalool synthesis.Both RNA-seq and RT-q PCR results showed that UV-B radiation significantly inhibited Pp CAMTA1 expression,and m~6A-seq showed that UV-B radiation increased the m~6A modification level of Pp CAMTA1 transcript.On the one hand,Bi FC and LCI assays showed that Pp CAMTA1 interacted with Pp HY5,a key factor in the light signaling pathway,DLR showed that Pp HY5 inhibited the activity of Pp CAMTA1 promoter,which may be one of the reasons for the decreased expression of Pp CAMTA1 gene under UV-B radiation.On the other hand,Pp ECT5 was identified as an m~6A binding protein containing the characteristic YTH domain.RNA-EMSA analysis showed that Pp ECT5 directly bound the m~6A modification region on Pp CAMTA1 m RNA,further study found that Pp ECT5 promoted the stability of Pp CAMTA1 m RNA.The expression level of Pp ECT5 was significantly inhibited under UV-B irradiation,which affected the expression level of Pp CAMTA1,the synthesis of linalool was further blocked under UV-B radiation.Therefore,Pp CAMTA1 is involved in the synthesis of linalool through transcriptional regulation and epigenetic regulation in peach fruit.