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

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-O-rutinoside and peonidin-3,5-O-diglucoside were
the primary anthocyanins. During fruit development and maturation,
the anthocyanin content was strongly correlated with the expression
of ZjANS and ZjUGT79B1, 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 in vitro enzyme activity assays showed
that the recombinant ZjANS protein catalyzed the formation of cyanidin
from (+)-catechin. Secondary glycosylation by ZjUFGT79B1 modified
cyanidin-3-O-glucoside to produce cyanidin-3-O-rutinoside, and ZjCCoAOMT readily catalyzed the production
of the methylated anthocyanin peonidin-3,5-O-diglucoside
from cyanidin 3,5-O-glucoside. Dual-Luciferase and
GUS activity assays showed that the ZjANS and ZjUGT79B1 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 Ziziphus jujuba cv. Tailihong, contributing to anthocyanin
accumulation by enhancing the expression of ZjANS and ZjUGT79B1.