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.