The Mechanism Of Internal Browning In Harvested Pineapple Fruit Based On Metabolomics

’Comte de Paris’ pineapple is the major variety in China,accounting for more than 80% of the growing area,but this variety of pineapple is highly susceptible to internal browning,which seriously affects its commercial value,results in great economy loss and has a negative effect on the pineapple industry.Research of the machanism can help to develop new methods to control pineapple blackheart.This study,based on metabolomics research methods,combined with gene expression analysis,was conducted to identify differential metabolites and metabolic pathways during blackheart development between green-ripe pineapple and full-ripe pineapple,aiming to unravel the relationship between key metabolic pathways and pineapple blackheart,hoping to provide new perspective for understanding the mechanism of pineapple blackheart.The main results are as follows:1.The differential metabolites in the development of postharvest blackheart of greenripe pineapple and full-ripe pineapple fruit were detected by non-targeted LC-MS metabolomics.The results showed that,when comparing the critical period of browning of post-harvest pineapple with the day of harvest(S2/S1),nearly 95.5% of significant differential metabolites common for the two different harvest maturity were opposite in trend of change,while comparing the browning of the tissue with the critical period(S3/S2),92% of the significant differential metabolites displayed the same trend.Among those that showed the same trends of changes,15 differential metabolites displayed fold change over 10,were closely related to aging and browning.Of these,the metabolites of increasing trends included one organic acid,two lipids,three phenylpropanoids and two unknown metabolites,those of decreasing trend included five amino acids and derivatives,two terpenoid,and one steroid.Pathway enrichment analysis showed that L-glutamine and isocitrate were enriched in more metabolic pathways than other differential metabolites.In addition,in both green-ripe fruit and full-ripe fruit,citric acid content decreased,while isocitrate content increase during the development of blackheart,and the content of phenolic caffeic acid and epicatechin were significantly increased,indicating that the citric acid degradation pathway is further addressed.2.The intermediate metabolites for the citric acid degradation pathway were identified using HPLC,amino acid analyzer and enzyme-linked immunosorbent assay,and the expression of genes in the citrate degradation pathway analyzed by fluorescence quantitative PCR.During the development of blackheart of green-ripe pineapple,the relative content of citric acid was significantly reduced,and the relative contents of isocitrate,α-ketoglutaric acid,L-glutamate and L-glutamine increased.The expression levels of AcAco1,AcAco3,AcNAD-IDH1,AcNAD-IDH4,AcNADP-IDHs and AcGS1-4 were up-regulated.Therefore,glutamine pathway is suggested to play a major role in citric acid degradation pathway in green-ripe pineapple fruit.Although the difference of GABA content was not significant,the expression of AcGAD3,AcGAD4 and AcGABA-T were upregulated,so the GABA shunt may also be involved in citric acid degradation.3.During development of blackheart of full-ripe pineapple,among the intermediate metabolites of citric acid degradation pathway,the content of citric acid,L-glutamate and L-glutamine decreased,while the relative content of isocitrate and α-ketoglutaric acid increased.In addition,AcAco3,AcNAD-IDH3,AcNADP-IDH1,AcGDH3,AcGAD2,AcGAD3 were up-regulated,while AcGSs and AcGABA-T were down-regulated,implying that the glutamine pathway is not the main pathway for citric acid degradation in full-ripe pineapple fruit.As the GABA shunt was blocked downstream,that upstream may participate in the citric acid degradation in full-ripe fruit.In conclusion,citric acid degradation of postharvest pineapple fruit is closely related to the occurrence of harvested pineapple blackheart,but there are some differences between green-ripe fruit and full-ripe fruit in citric acid degradation.It could be speculated that in the green-ripe fruit glutamine pathway and and the GABA shunt are mainly responsible for the citric acid degradation.However,in full ripe fruit,the upstream part of GABA shunt may be involved in the citric acid degradation,while glutamine pathway may not be play a major role.