EjbHLH14-EjHB1-EjPRX12 Regulation Pathway Involved In Methyl Jasmonate Alleviation Of Low Temperature-induced Lignin Deposition In Loquat Fruit

Loquat is a kind of subtropical evergreen fruit tree that is native to china.The thin skin and juicy flesh of loquat fruit result in short shelf life.Low temperature is an effective method to extend storage time,but red-flesh loquat exhibits increased firmness and lignin content during 0℃ storage,which is chilling-lignification,damaging fruit quality and commodity value.In the present study,mature loquat fruit‘Luoyangqing’ was used as experimental material,and Ej PRX12 was identified as a target of Me JA treatment.The alleviation of chilling-lignification by Me JA was through EjbHLH14-Ej HB1-Ej PRX12 pathway.The main conclusions are as follows:1.Ej PRX12 was characterized to be the target of Me JA treatment.Me JA treatment could alleviate loquat fruit chilling-lignification.Based on RNA-seq data,Ej PRX12 showed highest expression and its expression was inhabited by Me JA treatment.Phylogenetic analysis indicated Ej PRX12 and At PRX64 clustered in the same subgroup.Subcellular analysis presented that Ej PRX12 located at cell membrane and nuclear membrane.In vitro enzyme activity analysis indicated Ej PRX12 could catalyze sinapyl alcohol rather than coniferyl alcohol.Overexpression of Ej PRX12 in Arabidopsis induced 27% lignin deposition in stem.2.Ej HB1 was identified as an activator of Ej PRX12 promoter.Luciferase assays showed Ej HB1 significantly activated Ej PRX12 promoter,reaching 2.5-folds of the control.Moreover,Y1 H assay indicated that Ej HB1 could directly bind to Ej PRX12 promoter and EMSA presented that Ej HB1 could recognize CAATAATTG cis-element in Ej HB1 promoter.Meanwhile,expression of Ej HB1 was inhabited by Me JA treatment.Compared with WT,overexpression of Ej HB1 in Arabidopsis promoted 27% increase in lignin content in the stem through inducing expression of lignin-related structure genes,including At4CL1,At CCR1,At COMT,At CAD6 and At PRX64,of that At PRX64 was induced up to14.3-folds compared to WT,and Ej PRX12 belongs to the same subclass in phylogenetic analysis,suggesting the activation of Ej HB1 on PRXs is conserved between species.3.EjbHLH14 was identified as a JA signaling pathway repressor to repress Ej HB1 promoter.Based on RNA-seq data,through analyzing expression of homologous transcriptional factors with Arabidopsis in JA signaling pathway,EjbHLH14 was recognized to be induced most dramatically.Bi FC confirmed that EjbHLH14 combined to Ej JAZ1/3/6/7/8/10/12.Luciferase assays showed EjbHLH14 repressed activity of Ej HB1 promoter.EMSA indicated that EjbHLH14 recognized and bound to E-box in Ej HB1 promoter.The results above revealed that Me JA induced the degradation of JAZ proteins,and EjbHLH14 was released to inhabit Ej HB1 expression,resulting in alleviation of fruit chilling-lignification.Above all,lignin monomer polymerization was inhabited and chillinglignification in loquat was alleviated by Me JA treatment,the underlying molecular mechanism is EjbHLH14-Ej HB1-Ej PRX12 regulation pathway.In this study,the new gene target and new regulation pathway of alleviation of chilling-lignification by Me JA was identified,which enriched the molecular mechanism of lignin accumulation regulated by JA.