Transcriptomic and metabolomic analysis reveals a protein module involved in preharvest apple peel browning.

Peel browning is a natural phenomenon that adversely affects the appearance of fruits. Research on the regulation of browning in apples (Malus × domestica Borkh.) has mainly focused on postharvest storage, while studies at the preharvest stage are relatively rare. Apple is an economically important horticultural crop prone to peel browning during growth, especially when the fruits are bagged (dark conditions). The present study's integrated transcriptomics and metabolomics analysis revealed that preharvest apple peel browning was primarily due to changes in phenolics and flavonoids. The detailed analysis identified MdLAC7's (laccase 7) role in the preharvest apple peel browning process. Transient injection, overexpression, and CRISPR/Cas9 knockout of the MdLAC7 gene in apple fruit and calli identified vallinic acid, anthocyanidin, tannic acid, sinapic acid, and catechinic acid as its catalytic substrates. In addition, yeast one-hybrid assay, electrophoretic mobility shift assay, luciferase reporter assay, and ChIP-PCR analysis revealed that MdWRKY31 binds to the promoter of MdLAC7 and positively regulates its activity to promote peel browning of bagged fruits (dark conditions). Interestingly, upon light exposure, the light-responsive transcription factor MdHY5 (ELONGATED HYPOCOTYL 5) bound to the promoter of MdWRKY31 and inhibited the gene's expression, thereby indirectly inhibiting the function of MdLAC7. Subsequent analysis showed that MdHY5 binds to the MdLAC7 promoter at the G-box1/2 site and directly inhibits its expression in vivo. Thus, the study revealed the MdLAC7-mediated mechanism regulating preharvest apple peel browning and demonstrated the role of light in inhibiting MdLAC7 activity and subsequently reducing peel browning. These results provide theoretical guidance for producing high-quality apple fruits.

Metabolomic insights into the browning of the peel of bagging 'Rui Xue' apple fruit.

BACKGROUND: Bagging is one of the most important techniques for producting high-quality fruits. In the actual of cultivating, we found a new kind of browning in peel of apple fruit that occurs before harvest and worsen during storage period. There are many studies on metabonomic analysis of browning about storage fruits, but few studies on the mechanism of browning before harvest. RESULTS: In this study, five-year-old trees of 'Rui Xue' (CNA20151469.1) were used as materials. Bagging fruits without browning (BFW) and bagging fruits with browning (BFB) were set as the experimental groups, non-bagging fruits (NBF) were set as control. After partial least squares discriminant analysis (PLS-DA), 50 kinds of metabolites were important with predictive VIP > 1 and p-value < 0.05. The most important differential metabolites include flavonoids and lipids molecules, 11 flavonoids and 6 lipids molecules were significantly decreased in the BFW compared with NBF. After browning, 11 flavonoids and 7 lipids were further decreased in BFB compared with BFW. Meanwhile, the significantly enriched metabolic pathways include galactose metabolism, ABC membrane transporter protein, flavonoid biosynthesis and linoleic acid metabolism pathways et al. Physiological indicators show that, compared with NBF, the content of malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide anion (O2-) in peel of BFW and BFB were significantly increased, and the difference of BFB was more significant. Meanwhile, the antioxidant enzyme activities of BFW and BFB were inhibited, which accelerated the destruction of cell structure. In addition, the metabolome and physiological data showed that the significantly decrease of flavonoid was positively correlated with peel browning. So, we analyzed the expression of flavonoid related genes and found that, compared with NBF, the flavonoid synthesis genes MdLAR and MdANR were significantly up-regulated in BFW and BFB, but, the downstream flavonoids-related polymeric genes MdLAC7 and MdLAC14 were also significantly expressed. CONCLUSIONS: Our findings demonstrated that the microenvironment of fruit was changed by bagging, the destruction of cell structure, the decrease of flavonoids and the increase of triterpenoids were the main reasons for the browning of peel.