Preliminary Functional Study And Application Of FaPKc2.2 In Regulating Strawberry Fruit Ripening And Quality

As one of the key rate-limiting enzymes in glycolysis,pyruvate kinase is closely linked to the tricarboxylic acid cycle and is a potential candidate involved in the regulation of fruit ripening.This experiment mainly changed the expression level of FaPKc2.2 gene in strawberry fruit by transient overexpression,supplemented by gene phylogenetic analysis,subcellular localization,determination of maturity-related physiological indicators and combined analysis of transcriptomics and metabolomics,aimed to preliminarily elucidate the function of FaPKc2.2 in the regulation of strawberry fruit ripening and quality.The main findings were as follows:1.A total of 53 candidate pyruvate kinase genes were identified through a genome-wide search of the octoploid strawberry.Phylogenetic analysis showed that,similar to the grouping of pyruvate kinases in Arabidopsis thaliana and potato,strawberry pyruvate kinases were mainly divided into two isozyme groups,including plastidial and cytosolic groups,and each group contained two subgroups: plastidial subgroup α and β,and cytosolic subgroup 1 and 2 respectively.At the same time,according to the previous transcriptome data,it was found that the expression levels of two cytosolic pyruvate kinase genes(Fxa C＿14g00330,FaPKc2.1 and Fxa C＿15g00080,FaPKc2.2)showed a highly similar trend in the four fruit development stages.Based on the previous transcriptome data of our group,the transcription level of FaPKc2.2 was significantly inhibited by exogenous ABA,sucrose and their mixed treament,while FaPKc2.1 was not.At the same time,through the re-validation of ABA and sucrose exogenous treatment experiments,it was consistent with the transcriptome results.Based on this result,we selected FaPKc2.2 as a candidate gene for subsequent study on the regulation of strawberry ripening.2.Using the cultivated strawberry ‘Benihoppe’ as a template,the FaPKc2.2 gene was cloned,which full-length of the coding region was 1533 bp.The spatiotemporal expression pattern analysis showed that FaPKc2.2 was widely expressed and significantly different in roots,crowns,leaves,flowers and five fruit developmental stages.In addition,subcellular localization showed that the fluorescence of FaPKc2.2 fusion protein was detected in the cytoplasm,which was consistent with the phylogenetic analysis results.3.By transiently overexpressing the FaPKc2.2 gene in de-greening stage strawberry fruits,it was found that the transcriptional abundance of the gene was significantly increased.Comparing with the empty-infected fruit,the anthocyanin content and the color difference a* value of the fruits were significantly reduced.Meanwhile,transient overexpression of FaPKc2.2 also significantly inhibited the increase of fruit firmness and endogenous ABA content.Furthermore,transient overexpression of FaPKc2.2 had no effect on the levels of several soluble sugars(sucrose,fructose,glucose),but significantly inhibited the accumulation of titratable acid,citric acid and malic acid in strawberry fruit.In conclusion,the results indicated that FaPKc2.2 was a negative regulator which was involved in the regulation of strawberry fruit ripening and quality.4.Through the metabolomics detection and analysis of FaPKc2.2 transiently overexpressed strawberry fruits,a total of 150 differential metabolites were detected.The maturation-related metabolism processes of anthocyanins,organic acids and amino acids were analyzed,and there were one anthocyanin,four organic acids and one amino acid significantly down-regulated,and three organic acid derivatives and five amino acids were significantly up-regulated.However,sucrose,fructose and glucose were not identified as differential metabolites,and the relative content changes of these metabolites were basically consistent with the previous physiological measurements.This indicated that the transient overexpression of FaPKc2.2 may affect the ripening and quality formation of strawberry fruit by reorganizing the biosynthesis of secondary,amino acids and organic acids metabolism in strawberry fruit.5.Transcriptomics analysis of FaPKc2.2 transiently overexpressed fruits showed that a total of 1406 differential genes(DEGs)were detected,which were annotated into 15 KEGG classification pathways.Among them,the proportion of DEGs enriched in general metabolic pathways was the largest,followed by secondary metabolites biosynthesis pathways.Meanwhile,some DEGs enriched in carbohydrate metabolic pathways were specifically inhibited,including glycolysis/gluconeogenesis,tricarboxylic acid cycle(TCA cycle),pyruvate metabolism,and glyoxylate and dicarboxylate metabolism.In addition,Gene Ontology(GO)enrichment analysis also showed that there were significant differential gene enrichment in two carbohydrate metabolism-related pathways(glyoxylate cycle and glyoxylate metabolic process).6.Combining the experimental results of the metabolome and transcriptome,the combined analysis of metabolomics and transcriptomics were focused on glycolysis,TCA cycle and amino acid metabolism pathways.The results showed that a significant change in related intermediates and gene was not detected in glycolysis pathway,but two encoding fructose-1,6-bisphosphatase(FBP)genes in the gluconeogenesis pathway and one encoding 6-phosphogluconate dehydrogenase(6PGD)gene in the pentose phosphate pathway were detected and significantly up-regulated.In the TCA cycle,although the contents of citric acid,malic acid and α-ketoglutarate were significantly decreased at the metabolic level.But at the transcription level,except for two encoding succinate dehydrogenase(SDH)genes,and four encoding isocitrate lyase(ICL)genes and one malate synthase(MS)gene in the glyoxylate cycle were significantly changed,there were no significant differences in other genes related to the TCA cycle.In addition,amino acids and their derivatives were also affected among the metabolites.There were six amino acids were detected significant changes at the metabolic level,but only one related enzyme 3-isopropylmalate dehydrogenase(IPMDH),was significantly up-regulated at the transcription level.Therefore,transient overexpression of FaPKc2.2affected the flux between glycolysis,TCA cycle and amino acid metabolism.7.Similar to the physiological changes caused by transient overexpression of FaPKc2.2,exogenous arginine spraying also resulted in a significant inhibition of strawberry fruit coloration,as well as a significant decrease in anthocyanin,chromatic aberration a* value and organic acid content.In addition,exogenous arginine increased the expression level of FaPKc2.2 to a certain extent.In conclusion,it is considered that exogenous arginine can be used as a potential foliar fertilizer for regulating strawberry fruit ripening and quality.