3.4 Proteins involved in flavonoid biosynthesis
Proteins related to flavonoid biosynthesis were clustered to better
explore the molecular mechanisms underlying the changes in the
accumulation of secondary metabolites. Figure 4 and Figure S5 showed the
clustering results of the proteins involved in the above pathway, and
the specific positions were mapped, respectively. A total of 51 proteins
were identified, of which 32 were up-regulated and 19 were
down-regulated in TH from FXS (Table S7).
PAL (EC: 4.3.1.24) and 4CL (EC: 6.2.1.12 ) are common precursors in the
flavonoid biosynthsis pathway. PAL functions as a key rate-limiting
enzyme that catalyzes the phenylalanine to trans-cinnamic acid, which
was up regulated in TH. 4CL is the third key enzyme in dihydroflavone
biosynthesis as well. Four 4CLs (TRINITY_DN29174_c0_g1_i1_orf1,
TRINITY_DN3204_c0_g1_i2_orf1, TRINITY_DN5631_c0_g1_i2_orf1,
TRINITY_DN6526_c0_g1_i1_orf1) were identified in TH, where two and
two were up-regulated and down-regulated, respectively. CHI (EC:
5.5.1.6) is the second key enzyme in this pathway, which is important
for the regulation of the synthesis of the entire flavonoid secondary
metabolites, and the expression of CHI gene can provide sufficient
precursors for the synthesis of flavonoids [25]. 3 CHIs
(TRINITY_DN5111_c0_g1_i4_orf1, TRINITY_DN4314_c0_g2_i2_orf1,
TRINITY_DN4314_c0_g2_i1_orf1) were identified, while the expression
trend was not consistent. The midstream enzymes were annotated to FLS
(EC: 1.14.20.6), HCT (EC: 2.3.1.133), F3H (EC: 1.14.11.9), CYP75B1 (EC:
1.14.14.82), and C3’H (EC: 1.14.14.96, CYP98A). FLS directly determines
the content of flavonols in plant tissues and serves as a bridge between
the flavonoid synthesis pathway and the catechin synthesis pathway as
well [26]. The downstream enzymes exhibited not only increasing
trends but also decreasing trends. Other enzymes along this pathway have
been annotated, including ANS (EC: 1.14.20.4), ANR (EC: 1.3.1.77), LAR
(EC: 1.17.1.3), DFR (EC: 1.1.1.219), and UGT73C6. The expression of
upstream enzymes in phenylpropanoid biosynthesis was both up and down
regulated, while the expression of enzymes in the downstream pathway was
predominantly down-regulated in TH from SS. The differences in the
expression of proteins annotated to the flavonoid biosynthesis were not
significant, they still deserved more attention because they might act
synergistically with enzymes in the upstream phenylpropanoid
biosynthesis to regulate flavonoid synthesis and accumulation.