Transciptional Regulation Of NAC And MADS On Loquat Fruit Lignification Induced By Postharvest Chilling Injury

Loquat(Eriobotrya japonica)is subtropical fruit which originates from China,since its harvest time coincides with high temperature,it is not easy to store the fruit.Low temperature storage is a predominant approach for delaying decrease of fruit quality.However,for 'Luoyangqing',a typical red fleshed loquat fruit,various physiological injury symptoms will occur when subjected to inappropriately low temperature storage,inducing accumulation of lignin in flesh which leads to decrease of commercial quality.The present study applied heat treatment(HT)and low temperature conditioning(LTC)to alleviate chilling induced lignification of loquat fruit during 0? storage.In addition,multiple methods including bioinformatics,gene quantification,dual luciferase assay,yeast one hybrid assay,EMSA and transgene were applied to identify potential NAC and MADS family members which response to temperature,and to reveal their molecular mechanism of regulating chilling induced fruit lignification.The research results enrich the transcriptional regulation network of lignin biosynthesis.Principals are as follows:1.Characterization of fruit lignification related NAC and MADS genes.Six NAC genes and a MADS gene were identified via RNA-seq or yeast one hybrid library screening.Among them,EjNAC3 and EjNAC4 showed the positive relationship with fruit lignification,as their expression were significantly suppressed by HT and LTC;while EjMADS1 was induced by LTC,negatively related to fruit lignification procedure.2.EjNAC3 was involved in fruit lignification via direct regulation to EjCAD-like.Dual-luciferase assay indicated that EjNAC3 could transactivate promoter of EjCAD-like(?6 fold),while EjNAC4 had very limited effects on all examinated promoters.Analysis on EjCAD-like promoter revealed multiple binding elements for NAC proteins.EMSA indicated EjNAC3 could bind to-502 bp to-454 bp region and-142 bp to-103 bp region.In vivo confirmation by yeast one hybrid assay proved the physical binding between EjNAC3 and EjCAD-like promoter.These evidences indicate a distinct mechanism of lignification regulation of EjNAC3 compared with those of secondary cell wall related NAC genes.3.EjMADSl was involved in fruit lignification via transcriptional regulation to EjMYB8.Yeast one hybrid indicated EjMADS1 could physically bind to promoter of EjMYB8,which is an activator of Ej4CL1,Dual-luciferase assay indicates that EjMDS1 acted as a repressor,which significantly suppressed the activity of the EjMYB8 promoter.Taken together the expression pattern of EjMADS1,it could be proposed that EjMADSl is the secondary transcriptional regulator of loquat fruit lignification,which is different with the direct regulator EjNAC3.Meanwhile,EjMADSl is the first MIKC*subgroup member being involved in regulation of lignin biosynthesis.In summary,this work reveals the fact that EjNAC3 takes part in fruit lignification through directly binding and activting promoter of EjCAD-like.Meanwhile,EjMADS1 is the first MIKC*subgroup member that regulates lignification other than development of pollen by targeting at EjMYB8.All these findings are beneficial to the better understanding on the mechanisms of cascade transcriptional regulation in loquat fruit lignification.