The role of pgbhlh94-like in the synthesis of hydrolysable tannins and lignins in outer peels of pomegranate fruits

The outer peels of pomegranate (Punica granatum) fruit are highly enriched in metabolites produced from the shikimate pathway and its products, the aromatic amino acids. These include flavonoids, monomers of lignins (monolignols), hydrolysable tannin [HTs; including gallotannins (GTs) and ellagitannins (ETs)]. These metabolites benefit human health and protect the fruit from environmental stresses. To understand the transcriptional control of shikimate pathway-related metabolites in pomegranate, we searched for transcription factors that positively correlated to the levels of gallic acid, the first substrate of the HT biosynthetic pathway. Our previous analysis showed that a bHLH family transcription factor, PgbHLH94-like, exhibited a strong correlation with the gallic acid content during three stages of fruit development in two different pomegranate accessions [1]. In this study, we overexpressed PgbHLH94-like in pomegranate hairy roots, which were subjected to LC-MS/MS analysis. A total of 166 metabolites related to phenols, and the shikimate pathway were detected. The transgenic hairy roots showed significantly higher levels of shikimate, gallic acid, monolignols, and all the detected metabolites that belong to GTs, relative to the control lines. However, the level of ETs did not significantly change compared to the control hairy roots expressing an empty vector. Notably, there was a significant increase in total lignins in PgbHLH94-like-overexpressing hairy roots. RNAseq analysis of the transgenic hairy roots showed that expression of the gene gallate 1-beta-glucosyltransferase-like was upregulated, supported by the LC-MS/MS data of high producing 1-galloyl-beta-glucose, the product of gallate 1-beta-glucosyltransferase using gallic acid as substrate. The transcriptome analysis of PgbHLH94-like-overexpressing hairy roots also revealed reprogramming of defense response and cell wall-related genes. These results together suggested that PgbHLH94-like activates genes in lignin biosynthesis and the GT biosynthetic pathway, but acts as a repressor of genes related to flavonoid biosynthesis. The results also suggest that a high level of GTs does not support ETs formation.

[1] Harel-Beja, R., Tian, L., Freilich, S., Habashi, R., Borochov-Neori, H., Lahav, T., et al. (2019). Gene expression and metabolite profiling analyses of developing pomegranate fruit peel reveal interactions between anthocyanin and punicalagin production. Tree Genet. Genomes 15. doi: 10.1007/s11295-019-1329-6.