| Papaya(Carica papaya L.)fruit exhibits yellowing and rapid softening and has a short-term shelf life due to its climacteric behavior,which significantly limits its market value.Fruit ripening is a complex biological process controlled by various physiological and biochemical factors.Phytohormones and their signaling pathways are considered to be involved in this process.In this study,the roles of and papaya auxin response factor gene CpARF2 underlying mechanisms of CpARF2-mediated auxin signaling involving in fruit postharvest ripening have been extensively explored.The major results are as follows:1.Identification of ripening-induced CpARF2 by RNA-seq analysis.By searching the transcriptomes of papaya fruits at different postharvest stages integrating genome data,27,769 genes were annotated.A large number of auxin signal pathway-related genes were identified and their expression profiles during the fruit ripening process were analyzed.Among the differentially expressed genes,CpARF2 was a fruit ripening-induced gene.A total of 11 ARF family genes were identified,and their evolutionary relationships and many other important parameters,such as protein domain,exon-intron structure,and amino acid content,were also predicted.The differential expression of ARF family genes at different stages of fruit development and ripening of papaya were also analyzed.Of note,CpARF2 expression was significantly induced by fruit development and ripening.2.Confermation of the biological function of CpARF2.In this study,the full length sequence of CpARF2 gene was cloned.Gene expression analysis showed that postharvest ripening and ethylene application could induce the expression of CpARF2 gene.Subcellular localization analysis showed that CpARF2 was localized in the nucleus.The results of transient transformation of papaya fruits showed that over-expression of CpARF2 could accelerate the fruit postharvest ripening and inhibited expression of Cp ARF2 could slow down the fruit postharvest ripening process.Tomato genetic transformation experiment showed that the ecotopic over-expression of CpARF2 in tomato could shorten the color-breaking period,increase ethylene release,and accelerate the senescence of leaves.Ecotopic over-expression of CpARF2 in Arabidopsis could promote the growth and development,and affect the responses of roots to treatments by exogenous auxin and ethylene substances.Furthermore,the affects of over-expression of CpARF2 in tomato and Arabidopsis were analyzed by RNA-seq method.It was found that the expression levels of several cell wall degradation-and ethylene synthesis-related genes were significantly affected by the over-expression of CpARF2 in both transgenic tomato and Arabidopsis lines.These results indicated that CpARF2 plays an important role in the regulation of papaya fruit postharvest ripening.3.Several CpARF2 interacting proteins in papaya were isolated and the interactions between CpARF2 and CpEIL1,CpAP2/ERF were both confirmed in vitro and in vivo.In this study,yeast expression cDNA library of papaya fruit was constructed,and the recombination rate and homogenization efficiency of the library reached a high level.Using CpARF2 as bait protein,15 possible interacting proteins with different biological functions were obtained by yeast two-hybrid method.The results of yeast two-hybrid,GST Pull-down,Bi FC and LCI showed that CpARF2 could not only form homodimers,but also interact with CpEIL1 and CpAP2/ERF in vitro and in vivo.The interactions between CpARF2 and CpEIL1 or CpARF2 and CpAP2/ERF may be the key nodes of signal interaction between auxin and ethylene signaling pathways.4.Clarification of the transcription regulation mechanism of papaya CpARF2 gene involved in the postharvest ripening of papaya.The results of EMSA experiment showed that CpARF2 could bind to AuxRE(TGTCTC)region in the promoters of ethylene synthesis genes(CpACS1,CpACO1)and cell wall metabolism-related genes(CpXTH12,CpPE51).In addition,CpEIL1 can also bind to the promoters of the four above-mentioned genes in vitro,which contain the EIN3/EIL1 binding element(ATGTA).It was further confirmed by EMSA that CpARF2 promoted the binding activities of CpEIL1 to the promoters of CpACS1,CpACO1,CpXTH12 and CpPE51 genes.Double-luciferase reporter assay found that the interaction between CpARF2 and CpEIL1 could significantly induce the activities of CpACS1,CpACO1,CpXTH12 and CpPE51 promoters,thus regulating the postharvest ripening process of papaya fruits.In this study,ARF gene family was used as the starting point to screen the interacting proteins of CpARF2.The molecular mechanism of ARF-mediated auxin signaling pathway in the regulation of postharvest ripening of papaya fruit was revealed by physiological,biochemical,genetic and molecular biological methods.Our results may serve as a comprehensive resource for investigating the regulation mechanism involved in the ripening process of papaya fruit. |
