Comparative phytochemical and genomic characterization of genes associated with gallic acid metabolism in: Juglans australis and J. regia

Abstract

Juglans australis Griseb. is a native South American walnut species poorly phytochemical and molecularly characterized. In this study, the phenolic composition, antioxidant activity, and candidate genes associated with shikimate-derived gallic acid metabolism were investigated through an integrated phytochemical and genomic approach. Green husk (exocarp) extracts were obtained using methanol, ethanol/water (50:50), and aqueous at 25 °C and 120 °C, and were evaluated for extraction yield, total phenolic content, antioxidant activity (DPPH, FRAP, and TEAC), and phenolic composition by UHPLC-UV-MS. The hydroethanolic extract of J. australis showed the highest total phenolic content (42.09 ± 2.68 ug GAE/mg de), whereas hydrothermal extraction exhibited the greatest antioxidant activity (DPPH: 62.32 ± 6.17 mM Trolox/g dry extract d.e., FRAP: 115.54 ± 9.32 mM Trolox/g d.e., TEAC: 196.65 ± 15.89 mM Trolox/g d.e.). Nine phenolic compounds were identified by UHPLC-UV-MS analysis, including gallic acid, ethyl gallate, juglone, and several flavonoid derivatives. Genomic mining of the J. australis draft genome identified candidate genes that may encode enzymes involved in hydrolyzable tannin biosynthesis: a bifunctional dehydroquinate dehydratase/shikimate dehydrogenase (DHQD/SDH), a UDP-glycosyltransferase (UGT84 family), and a BAHD acyltransferase. Structural modeling, conserved catalytic residue analysis, and phylogenetic reconstruction supported the annotation of the predicted DHQD/SDH protein as a conserved plant ortholog closely related to those of other Juglandaceae species. These findings provide integrated phytochemical and genomic evidence supporting the occurrence of a shikimate-derived gallic acid biosynthetic pathway in J. australis and establish a possible molecular framework for future studies on hydrolyzable tannin biosynthesis in this native walnut species.