Metabolomic and Transcriptomic Analyses of Anthocyanin Biosynthesis Mechanisms in the Color Mutant <i>Ziziphus jujuba</i> cv. Tailihong

Abstract

The purplish-red color of "Tailihong" jujube fruit skins is caused primarily by anthocyanin accumulation, but the mechanisms that underlie anthocyanin biosynthesis in jujube fruit have rarely been studied. We performed metabolomic and transcriptomic analyses of jujube fruit skins at different developmental stages and identified a total of 158 flavonoids, among which cyanidin-3-<i>O</i>-rutinoside and peonidin-3,5-<i>O</i>-diglucoside were the primary anthocyanins. During fruit development and maturation, the anthocyanin content was strongly correlated with the expression of <i>ZjANS</i> and <i>ZjUGT79B1</i>, suggesting that these are key genes in the anthocyanin biosynthesis process. Transcriptomic analysis indicated that the transcription factors ZjMYB5, ZjTT8, and ZjWDR3 regulated anthocyanin biosynthesis in jujube fruit skins. Subcellular localization experiments confirmed that ZjANS and ZjUGT79B1 were localized to the nucleus and the endoplasmic reticulum. ZjMYB5 and ZjTT8 were found only in the nucleus, whereas strong fluorescence signals from ZjWDR3 were observed in the nucleus and cytoplasm. Prokaryotic expression and <i>in vitro</i> enzyme activity assays showed that the recombinant ZjANS protein catalyzed the formation of cyanidin from (+)-catechin. Secondary glycosylation by ZjUFGT79B1 modified cyanidin-3-<i>O</i>-glucoside to produce cyanidin-3-<i>O</i>-rutinoside, and ZjCCoAOMT readily catalyzed the production of the methylated anthocyanin peonidin-3,5-<i>O</i>-diglucoside from cyanidin 3,5-<i>O</i>-glucoside. Dual-Luciferase and GUS activity assays showed that the <i>ZjANS</i> and <i>ZjUGT79B1</i> promoters were activated by ZjMYB5, ZjTT8, and ZjWDR3. All data indicated that these three transcription factors played important roles in anthocyanin biosynthesis in the color mutant <i>Ziziphus jujuba</i> cv. Tailihong, contributing to anthocyanin accumulation by enhancing the expression of <i>ZjANS</i> and <i>ZjUGT79B1</i>.