Transcriptome Analysis Identifies Two Ethylene Response Factors That Regulate Proanthocyanidin Biosynthesis During Crabapple Fruit Development.

Proanthocyanidins (PAs) are a class of flavonoid compounds in plants that play many important roles in pest and disease resistance and are beneficial components of the human diet. The crabapple () provides an excellent model to study PA biosynthesis and metabolism; therefore, to gain insights into the PA regulatory network in plants, we performed RNA-seq profiling of fruits of the 'Flame' cultivar at five sequential developmental stages. KEGG () enrichment analysis showed that differentially expressed genes (DEGs) related to the functional category 'plant hormone signal transduction' were significantly enriched during fruit development. Further analysis showed that ethylene signal transduction pathway genes or response genes, such as ERS (ethylene response sensor), EIN3 (ETHYLENE INSENSITIVE 3) and ERFs (ethylene response factors), may play an important role in the regulatory network of PA biosynthesis. Additionally, 12 DEGs, including 10 ERFs, 1 MYB, and 1 bHLH transcription factor, associated with PA biosynthesis were identified using WGCNA. The expression patterns of these genes correlated with PA accumulation trends and transcriptome data from qRT-PCR analysis. The expression of (RELATED TO APETALA 2-4) and (related to ABI3/VP1), which belong to the ERF transcription factor family, showed the greatest correlations with PAs accumulation among the 12 identified TFs. mediated-transient overexpression of the led to an increase in PA abundance in crabapple leaves and apple fruits, and the opposite results were observed in -overexpressed crabapple leaves and apple fruits. Moreover, a yeast one-hybrid assay showed that RAP2-4 and RAV1 specifically bound the promoters of the PA biosynthetic genes and , respectively. These results indicate that RAP2-4 act as an inducer and RAV1 act as a repressor of PA biosynthesis by regulating the expression of the PA biosynthetic genes and . Taken together, we identified two potential regulators of PA biosynthesis and provide new insights into the ethylene-PA regulatory network.