Transcriptional Regulation Of Loquat Fruit Chilling Induced Lignification By EjHAT1 And EjbHLH1

Loquat is one of rosaceae plant,originated from China.It can be divided into red-fleshed and white-fleshed according to the color of the flesh.The red-fleshed loquat‘Luoyangqing'is susceptible to chilling injury during 0~OC storage at postharvest,and results in chilling induced lignification.This appears as flesh becoming juiceless,firmness increased and lignin accumulated,which affects fruit commodity quality.Previous studies confirmed some postharvest treatment,heat shock treatment(HT)and low temperature conditioning treatment(LTC)could reduce such chilling induced lignification,and provided us useful tools to explore the mechanism of lignin biosynthesis and improve fruit quality.In the present study,the red fleshed loquat‘LYQ'was used as experimental material.The mechanism of chilling induced lignin biosynthesis retarded by HT and LTC treatment was explored by real-time fluorescence quantitative PCR,yeast one hybrid assay,bimolecular fluorescence complementarity assay,dual luciferase assay and etc.The main results are as follows:1.EjCAD5 is a member of the CAD family that retards fruit lignification by HT treatment.0~OC low temperature storage induced fruit chilling induced lignification with the increase of CAD enzyme activity while HT treatment retarded the CAD enzyme activities.Four additional CAD genes were isolated according to RNA-seq database,EjCAD3 and EjCAD5 was induced by 0~OC and repressed by HT treatment.Enzyme activity analysis of the two candidate recombinant CAD genes in vitro showed EjCAD5 had high enzyme activities to the three lignin monomer precursors(sinapaldehyde,coniferaldehyde and p-Coumaraldehyde)while EjCAD3 had low enzyme activities to overall substrates.2.EjHAT1 was a transcription factor involved in heat-alleviation of chilling induced lignification.Using EjCAD5 as the target gene,yeast one hybrid screening found a transcription factor EjHAT1 that could bind to the promoter EjCAD5.The expression of EjHAT1 was depressed during 0~OC storage while this repression could be alleviated by HT treatment.Dual luciferase assay showed EjHAT1 inhibited the promoter activity by recognizing the HD-Zip cis-acting element in EjCAD5 promoter.Thus,HT treatment induced the expression of EjHAT1 that repressed by low temperature and increased the repression effect to EjCAD5 promoter to alleviate the chilling induced lignification of loquat fruit.3.EjbHLH1 was a transcription factor involved in LTC-alleviation of chilling induced lignification for loquat fruit.LTC-responsive bHLH transcription factor EjbHLH1 was identified from the RNA-seq database.The expression of EjbHLH1was negatively correlated with the change of fruit lignin content.Dual luciferase assay indicated that it could inhibit the promoter activity of lignin biosynthesis gene Ej4CL1.However,yeast one-hybrid assay showed EjbHLH1 could not bind to the Ej4CL1promoter.Interesting,the results of BIFC and LCI indicated that EjbHLH1 could interaction with EjAP2-1 and EjMYB2 and dual luciferase assay showed that the three transcription factors synergistically regulated the expression of Ej4CL1.Therefore EjbHLH1 indirectly regulated the expression of Ej4CL1 by interacting with EjAP2-1 and EjMYB2 to participate in the chilling induced lignification.In this study,two novel transcription factors EjHAT1,EjbHLH1 and a structural gene EjCAD5 were identified to be involved in chilling induced loquat fruit lignification.EjHAT1 participated in the chilling induced lignification at postharvest by regulating the expression of EjCAD5 in HT treatment.EjbHLH1 participated in chilling induced lignification at postharvest by interacting with EjAP2-1 and EjMYB2proteins,indirectly regulating the expression of Ej4CL1 in LTC treatment.It further enriched the lignin transcription regulatory network and also provided us a theoretical basis for improving fruit quality at postharvest.