Transcriptional Regulation Of Loquat Fruit Postharvest Chilling Injury And Lignification By EjAP2-1and EjHSF3

Loquat (Eriobortya japonica Lindl.) is a subtropical fruit native to China. It belongs to non-climacteric fruit and can be divided into two groups according to the flesh color, i.e. white-fleshed and red-fleshed. Red-fleshed loquat fruit are susceptive to low temperature (0℃) and exhibited lignification during low temperature storage. The present study investigated the transcriptional regulatory mechanisms of EjAP2/ERF, EjHSF and their transcription complex on loquat fruit lignification. The main results are as followed:1. Isolation and characterization of lignification-related EjAP2/ERF. Eighteen EjAP2/ERF genes were isolated based on RNA-Seq database, and they responded differentially to low temperature. Among them, EjERF8and EjERF16(putative activators) were inhibited by low temperature conditioning (LTC) treatment and positively correlated with fruit lignification, while EjERF7and EjAP2-1(putative repressors) which contain EAR motifs were activated by LTC treatment. However, dual-luciferase assay results showed that only EjAP2-1had significant in vivo repressive effects on the Ej4CL1promoter.2. EjAP2-1participated in the transcriptional regulation of fruit lignification. Yeast-two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that EjAP2-1interacted with both EjMYB1and EjMYB2. The biological functions of protein-protein interaction of EjAP2-1and EjMYBs were further tested using the dual-luciferase assay. EjAP2-1counteracted trans-activation activity of EjMYB1on the Ej4CL1promoter and the combination of EjAP2-1and EjMYB2resulted in a stronger repressive effect. After double mutated the two EAR motifs within EjAP2-1deduced amino acid sequence, mEjAP2-1lost the synergistic effect with EjMYBs. Moreover, protein-protein interactions still existed between mEjAP2-1and EjMYBs. Thus, these results indicated that the repression effect of EjAP2-1on lignin biosynthesis genes originated from EAR motifs, whose functions are manifested via EjAP2-1/EjMYBs protein-protein complexes.3. EjHSF3participated in the transcriptional regulation of fruit lignification, at least via protein-protein interaction with EjAP2-1. Based on the loquat RNA-Seq database, eight heat shock protein (EjHsp70-1/2, EjsHspl/2/3/4/5/6) genes and three heat shock factor (EjHSF1, EjHSF2and EjHSF3) genes were isolated. Heat treatment (HT) alleviated lignification and induced EjHSFl and EjHsp gene expression, meanwhile, EjHSFl trans-activated promoters of EjHsps, which further supported the regulatory cascade of EjHSF1-EjHsp-chilling tolerance. EjHSF3was suppressed by LTC treatment, which was positively correlated with fruit lignification. The role of EjHSF3in fruit lignification regulation via both transactive lignin biosynthetic gene Ej4CL1promoter, and protein-protein interaction with lignin regulator EjAP2-1transcription factor.In conclusion, the present study indicated two novel transcription factors, i.e. EjAP2-1and EjHSF3, regulated loquat fruit chilling injury and lignification via different mechanisms. Moreover, these two transcription factors also had linkage with protein-protein interaction.