Overexpression of PgbHLH94‐Like Increases Shikimate, Hydrolyzable Tannin, and Lignin Levels in Pomegranate ( Punica granatum L.) Hairy Roots

ABSTRACT Pomegranate fruit and juice products are rich in shikimate pathway‐derived metabolites, such as hydrolyzable tannins (HTs) and flavonoids, which contribute to plant fitness and human health. The fruit peel, in particular, is highly enriched in these metabolites and thus provides an excellent model for studying the regulation of their accumulation. To investigate the transcriptional control of shikimate pathway‐related metabolites and identify transcription factors (TFs) regulating HT biosynthesis in pomegranate, we searched for TFs whose expression correlated with gallic acid, the precursor of HT biosynthesis, using transcriptome data from pomegranate fruit peels and hairy roots. This screen identified a candidate TF, Pg bHLH94‐like. Overexpression of PgbHLH94‐like in pomegranate hairy roots significantly increased the accumulation of gallic acid and its downstream products, including galloylglucose esters and several HTs. PgbHLH94‐like overexpression also increased shikimate and lignin levels while decreasing flavonoid levels. Interestingly, transcriptome analysis of PgbHLH94‐like ‐overexpressing hairy roots revealed reprogramming of cell wall‐related genes, as well as upregulation of genes (potentially) involved in galloylglucose ester and HT metabolism. To the best of our knowledge, this represents the first TF shown to regulate gallic acid‐derived metabolites. The identification of Pg bHLH94‐like also provides insights into the regulation of shikimate‐related pathways and a foundation for defining the biosynthetic enzymes of HTs, many of which remain unknown. Moreover, it can serve as a molecular marker for breeding pomegranate fruits with enhanced HT content and improved health benefits.