The Mechanism Of Exogenous Acetylsalicylic Acid In Regulating Ethylene Biosynthesis In Kiwifruit

Levels of ethylene,implicated in a diverse array of plants for inducing fruit ripening,is influenced by genetic factors and other plant hormones.Among these,salicylic acid(SA)and its derivative,acetylsalicylic acid(ASA)have been demonstrated to inhibit ethylene biosynthesis and improve quality properties in various fruit,yet the underlying regulatory mechanisms remain elusive.In this study,‘Hayward’ kiwifruit and research methods such as RNA-sequencing,dual-luciferase assay,electrophoretic mobility shift assay(EMSA),kiwifruit stable genetic transformation,co-immunoprecipitation(Co IP)and firefly luciferase complementation imaging assay(LCI),were used to clarify the transcriptional and post-transcriptional mechanisms by which exogenous ASA inhibits ethylene biosynthesis.The main results are as follows:1.Kiwifruit flesh was processed into discs and treated with 0.5 m M ASA.Physiological and biochemical results showed that ASA treatment dramatically reduced ethylene production and the content of 1-aminocyclopropane-1-carboxilic acid(ACC),as well as activities of ACC synthase(ACS)and ACC oxidase(ACO).While the content of methionine,the precursor of ethylene,showed no significant difference between the ASA-treated and control discs.Based on physiological data and transcriptome analysis,ACS and ACO were assumed to be the key target genes of ASA-mediated ethylene decrease,and three key ACS members as well as two ACO members were identified.2.RNA sequencing was performed among kiwifruit discs treated with/without exogenous ASA,and a total of 201 differentially expressed genes including 15 transcription factors were isolated.Dual-luciferase assay was performed to investigate the effects of these transcription factors on the promoters of Ad ACS1/2 and Ad ACO5.The results showed that Ad ERF105 L is a transcriptional repressor of Ad ACS1/2,and Ad WRKY29 is a transcriptional activator of Ad ACO5.EMSA results indicated that Ad ERF105 L protein and Ad WRKY29 protein had direct binding effects with Ad ACS1/2 promoters and Ad ACO5 promoter,respectively.Moreover,transgenic kiwifruit plants confirmed the regulatory effects of these two ASA-responsive transcription factors on expression of downstream target genes.3.In response to exogenous ASA treatment,Ad ACS3 protein levels decreased while Ad ACO3 protein levels were maintained.Phylogenetic tree analysis showed that Ad ACS3 belongs to Type 1 ACS subfamily,which contains target sites for mitogen-activated protein kinase(MAPK).Through luciferase imaging assay,we identified a protein kinase Ad MPK16 that can phosphorylate and stabilize Ad ACS3 protein.Co IP and LCI also confirmed the protein-protein interaction between Ad ACS3 and Ad MPK16.Meanwhile,combining co-immunoprecipitation and mass spectroscopy(Co IP-MS)and LCI results,we identified an aspartic peptidase(AP),which enhanced the protein activity of Ad ACO3 without altering its protein abundance.RT-q PCR results indicated that exogenous ASA treatment down-regulated the expression of Ad MPK16 and Ad AP,which reduced the protein stability of Ad ACS3 and inhibited the protein activity of Ad ACO3,ultimately influencing ethylene biosynthesis.4.ASA treatment was performed on different climacteric fruit discs.In addition to kiwifruit,ethylene biosynthesis in mango and tomato discs were inhibited.By analyzing expression pattern of several pivotal ACS and ACO genes,the mechanism of exogenous ASA inhibiting ethylene biosynthesis among different species was revealed.Moreover,treatment with ASA was performed on fresh-cut kiwifruit and the results showed that ASA could effectively delay fruit ripening,maintain fruit quality and extend shelf-life during low-temperature storage,indicating the potential value of ASA in postharvest storage and preservation of fruits and vegetables.These results proposed a multidimensional system,involving the regulation of Ad ERF105 L and Ad WRKY29 on expression of target genes,Ad MAPK on protein abundance and Ad AP on protein activity,which enriched the regulatory network of ethylene biosynthesis and clarified the molecular basis of ASA-inhibition on ethylene.