Characterization of tricin biosynthesis pathway and lignin composition in rice

Abstract

Tricin, a 3’,5’-dimethoxylated flavone, is an ubiquitous secondary metabolite in a number of monocots including sedges, palms and grasses. In plants, tricin exists in the forms of aglycone, O-glycosides and O-flavanolignols, and is also covalently linked to cell wall lignin polymers. Soluble tricin O-conjugates functions as allelochemicals and insect deterrents, while tricin in lignin polymers acts as a nucleation site for lignification. To humans, consumption of tricin is beneficial to health due to its anti-oxidant, anti-cancer and anti-inflammatory properties. However, not much is known about how tricin is synthesized in plants. In this study, key enzymes involved in the biosynthesis tricin in rice were identified and characterized.

This study has provided in vitro and in vivo evidence to show CYP93G1 as a flavone synthase II (FNSII) that channels flavanones to the formation of flavones, including tricin. In vitro enzyme assays have shown that recombinant CYP93G1 can convert naringenin and eriodictyol into apigenin and luteolin, respectively. Transgenic Arabidopsis over-expressing CYP93G1 accumulated flavone O-glycosides, and a rice CYP93G1 mutant was depleted in the accumulation of all flavone O-conjugates. The acquisition of FNSIIs in monocots was expected to be independent from the dicot FNSIIs of the CYP93B subfamily, leading to the wide spread occurrence of tricin type flavone derivatives in monocots.

The enzyme responsible for the 5’-hydroxylation of tricin was also isolated from characterization of rice mutants. A rice CYP75B4 mutant was depleted in the accumulation of selgin and tricin, which are 5’-hydroxylated and 5’-methoxylated flavones, respectively. Co-expressing CYP75B4 and CYP93G1 in Arabidopsis also resulted in the generation of selgin and tricin. In vitro enzyme assays confirmed that CYP75B4 catalyzed 3’-hydroxylation of most classes of flavonoids, while the 5’-hydroxylation activity was specific to chrysoeriol (a 3’-methoxylated flavone). The identification of CYP75B4 in tricin biosynthesis might indicate that grass species acquired chrysoeriol 5’-hydroxylase from gene duplication and function diversification of flavonoid 3’-hydroxylases, and is distinct from the flavonoid 3’,5’-hydroxylases in the CYP75A subfamily that catalyze a broad range of flavonoid substrates by sequential 3’ and 5’-hydroxylation. Moreover, this study also showed that CAldOMT1/ROMT9 is an O-methyltransferase involved in tricin biosynthesis in rice by metabolite analysis of a rice CAldOMT1/ROMT9 knockout mutant.

Finally, the roles of tricin in lignification were investigated by cell wall characterizations of the rice CYP93G1, CYP75B4 and CAldOMT1/ROMT9 mutants. Histochemical staining and thioglycolic acid lignin analysis showed that flavonoid and lignin contents were largely reduced in these mutants. Thioacidolysis indicated that the knocking out CYP93G1 or CYP75B4 did not change the core lignin composition. 2D-HSQC-NMR analysis confirmed the depletion of tricin in these two mutants while naringenin was incorporated in the cell wall lignin of the CYP93G1 mutant. On the other hand, 2D-NMR analysis also confirmed the roles of CAldOMT1/ROMT9 as a bifunctional enzyme involved in both S-lignin and tricin biosynthesis in rice. Collectively, this study has shown that tricin depletion in cell wall lignin in rice reduced lignin content but did not alter lignin and other cell wall components compositions, nor exerted significant impact on plant growth.