The Mechanism Of Abscisic Acid-Regulated Strawberry Fruit Ripening And Identification Of Key MiRNAs Involved

The mechanism of fruit ripening is an important scientific issue.An in-depth understanding of the basic molecular mechanism of regulating fruit ripening will not only help improve fruit quality,but also can help directly promote the economic benefits of the fruit industry,as well as enhance the consumer product experience and encourage the development of efficient postharvest preservation technologies.Abscisic acid(ABA)signaling plays an important role in strawberry fruit ripening,but it is unclear how the genes involved in fruit ripening and key regulatory factors are regulated at its downstream.Therefore,the effects of ABA treatment on the metabolic pathways and genes involved during strawberry fruit ripening were analyzed at the physio-biochemical level as well as by applying integrated multiomics technologies.Key regulatory transcription factors and mi RNAs and their possible functions were also studied.The main conclusions are:1.Exogenous ABA treatment accelerated many ripening-related processes,including the formation of red color,fruit development and enlargement,firmness decrease,the accumulation of soluble solid content either in postharvest fruits or on vine fruits.ABA-treated fruits accumulated 17.6% more flavonoids.ABA treatment also induced more accumulation of ascorbic acid and promoted chlorophyll degration.DPPH and FRAP tests showed that ABA enhanced the antioxidant ability of the fruits.It was also found that ABA remarkably induced anthocyanin biosynthesis,by enhancing the activity of anthocyanin biosynthetic enzymes,including PAL,Tyrosine ammonia-lyase(TAL),4-coumarate Co A ligase(4CL)and Dihydroflavonol-4-reductase(DFR).However,the effect of ABA treatment on Cinnamic acid 4-hygroxylase(C4H)was minor.Moreover,it was found that folate biosynthesis was rarely influenced by ABA and ?-carotene was important for ABA biosynthesis.2.Transcriptome analyses found 4164 significantly differentially expressed genes in ABA or NDGA-treated fruits.In particular,genes in the anthocyanin biosynthesispathway were actively regulated by ABA,including PAL?4CL?chalcone synthase?chalcone isomerase?flavanone 3-hydroxylase?DFR and anthocyanidin synthase,with the exception of C4 H.Chlorophyll degradation was accelerated by ABA mainly owing to the higher expression of gene encoding pheide a oxygenase.In addition,evaluation on the folate biosynthetic pathway showed that ABA had minor regulatory function on the expression of genes involved in this nutrient's biosynthesis process,however,the expression of genes responsible for folate metabolism balance was remarkedly affected by ABA,indicating that ABA may be involved in the regulation of folate homeostasis.Moreover,although ABA promoted ascorbic acid accumulation,expressions of genes involved in its biosynthesis were significantly lower in ABA-treated fruit,indicating that the biosynthesis of ascorbic acid in strawberry fruit may be regulated by some unknown ways or ascorbic acid could be transported from some other tissues in the plant.3.KEGG enriched pathways of the significantly differentially expressed genes found that ABA actively regulated genes involved in the pathways of plant hormone signaling transduction and the biosynthesis of secondary metabolites.ABA down-regulated the expression of cytokinin receptor genes AHK2/3/4 and cytokinin downstream gene CYCD3.ABA also inhibited the expression of IAA27 and SAUR in auxin signaling pathway.These results indicate that ABA may play a regulatory role in the transition of fruit development to fruit ripening.In addition,the expression of ethylene biosynthetic gene ACO3 and key signaling component gene CTR1 was affected by ABA,implying that ethylene may be involved in the regulation of strawberry fruit ripening.Moreover,a co-expression analysis between the differentially expressed genes and transcription factors found that WRKY and HSF transcription factors might play key roles in regulating the expression of ABA inducible genes,whereas the KNOX and SBP18 might be responsible for ABA-downregulated genes.4.Forty-seven conserved mi RNAs and 28 novel mi RNAs were identified during strawberry fruit ripening,among them,20 and 6 were significantly differentially expressed in response to ABA.Targets of the differentially expressed mi RNAs werefound to be involved in processes of phytohormonal metabolism balance,formation of fruit color and cell wall degradation.In addition,degradome analysis showed that one novel mi RNA,Fa_novel6,could degrade its target gene HERCULES1(HERK1),which likely contributed to fruit size determination during strawberry ripening.5.A novel mi RNA,Fan_mi R73 was found to target ABI5 downstream in the ABA signaling pathway.Exogenous ABA treatment accelerated fruit ripening through differentially regulating the expression of ABA metabolism and signal transduction related genes,including NCED1,PYR1,ABI1,and Sn RK2.2.Expression of Fan-mi R73 was down-regulated in response to exogenous ABA treatment in a dosage-dependent manner,which resulted in an accumulation of ABI5 transcripts in the ripening-accelerated fruits.In addition,both UV-B radiation and salinity stress reduced the transcript levels of Fan-mi R73,whereas promoted ABI5 expression.Furthermore,the high negative correlations between the transcriptional abundance of Fan-mi R73 and ABI5 indicate that Fan_mi R73-ABI5 pair may play a role in strawberry fruit ripening and abiotic stress response.