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Summary In rice (Oryza sativa), OsF2H and OsFNSII direct flavanones to independent pathways that form soluble flavone C‐glycosides and tricin‐type metabolites (both soluble and lignin‐bound), respectively. Production of soluble tricin metabolites requires CYP75B4 as a chrysoeriol 5′‐hydroxylase. Meanwhile, the close homologue CYP75B3 is a canonical flavonoid 3′‐hydroxylase (F3′H). However, their precise roles in the biosynthesis of soluble flavone C‐glycosides and tricin–lignins in cell walls remain unknown. We examined CYP75B3 and CYP75B4 expression in vegetative tissues, analyzed extractable flavonoid profiles, cell wall structure and digestibility of their mutants, and investigated catalytic activities of CYP75B4 orthologues in grasses. CYP75B3 and CYP75B4 showed co‐expression patterns with OsF2H and OsFNSII, respectively. CYP75B3 is the sole F3′H in flavone C‐glycosides biosynthesis, whereas CYP75B4 alone provides sufficient 3′,5′‐hydroxylation for tricin–lignin deposition. CYP75B4 mutation results in production of apigenin‐incorporated lignin and enhancement of cell wall digestibility. Moreover, tricin pathway‐specific 3′,5′‐hydroxylation activities are conserved in sorghum CYP75B97 and switchgrass CYP75B11. CYP75B3 and CYP75B4 represent two different pathway‐specific enzymes recruited together with OsF2H and OsFNSII, respectively. Interestingly, the OsF2H‐CYP75B3 and OsFNSII‐CYP75B4 pairs appear to be conserved in grasses. Finally, manipulation of tricin biosynthesis through CYP75B4 orthologues can be a promising strategy to improve digestibility of grass biomass for biofuel and biomaterial production.

Phytoene synthase (PSY) catalyzes the first committed step in the carotenoid biosynthesis pathway and is a major rate-limiting enzyme of carotenogenesis. PSY is highly regulated by various regulators and factors to modulate carotenoid biosynthesis in response to diverse developmental and environmental cues. Because of its critical role in controlling the total amount of synthesized carotenoids, PSY has been extensively investigated and engineered in plant species. However, much remains to be learned on its multifaceted regulatory control and its catalytic efficiency for carotenoid enrichment in crops. Here, we present current knowledge on the basic biology, the functional evolution, the dynamic regulation, and the metabolic engineering of PSY. We also discuss the open questions and gaps to stimulate additional research on this most studied gene/enzyme in the carotenogenic pathway.

Flavonoids are essential for male fertility in some but not all plant species. In rice (Oryza sativa), the chalcone synthase mutant oschs1 produces flavonoid-depleted pollen and is male sterile. The mutant pollen grains are viable with normal structure, but they display reduced germination rate and pollen-tube length. Analysis of oschs1/+ heterozygous lines shows that pollen flavonoid deposition is a paternal effect and fertility is independent of the haploid genotypes (OsCHS1 or oschs1). To understand which classes of flavonoids are involved in male fertility, we conducted detailed analysis of rice mutants for branch-point enzymes of the downstream flavonoid pathways, including flavanone 3-hydroxylase (OsF3H; flavonol pathway entry enzyme), flavone synthase II (CYP93G1; flavone pathway entry enzyme), and flavanone 2-hydroxylase (CYP93G2; flavone C-glycoside pathway entry enzyme). Rice osf3h and cyp93g1 cyp93g2 CRISPR/Cas9 mutants, and cyp93g1 and cyp93g2 T-DNA insertion mutants showed altered flavonoid profiles in anthers, but only the osf3h and cyp93g1 cyp93g2 mutants displayed reduction in seed yield. Our findings indicate that flavonoids are essential for complete male fertility in rice and a combination of different classes (flavanones, flavonols, flavones, and flavone C-glycosides) appears to be important, as opposed to the essential role played primarily by flavonols that has been previously reported in several plant species.