Functional characterization of flavonoid biosynthesis in rice and sorghum

Abstract

Flavonoids are one of the most abundantly accumulated secondary metabolites in plants with multiple biological activities. The biosynthetic pathways and functions of flavonoids in plant have been studied for decades. However, more in-depth analyses are required to further understand the precise roles of different flavonoid subclasses in monocots.

Recently, tricin, a 3', 5'-dimethylated flavone, was reported to be incorporated into cell wall lignin in the grass family. Here, I investigated the roles of early flavonoid biosynthetic enzymes, CHALCONE SYNTHASE (OsCHS1), CHALCONE ISOMERASE (OsCHI), and non-catalytic enhancer protein, CHALCONE ISOMERASE-LIKE (OsCHIL1 and OsCHIL2), on tricin-lignin biosynthesis, and the effects of deficiency of these proteins on cell wall properties and biomass digestibility. Tricin-lignin and the soluble flavonoid metabolites were completely or moderately depleted in these mutants. Total lignin content was reduced in OsCHS1 and OsCHI mutant, while increased in OsCHIL mutants compared with wild-type. However, monolignol-derived lignin composition and inter-monomeric linkages of lignin polymers were not largely altered in these mutants. Significant alteration in biomass digestibility was also not found. Together with the results of the other recently characterized grass tricin depleted mutants, results here suggest that tricin-lignin content alone does not directly impact total lignin content and biomass digestibility in grasses.

Rice OsCHS1 homozygous mutants are devoid of flavonoids (flavonol, flavone, flavone C-glycosides and flavanone) in anthers, while their pollens are viable with normal structure but displayed reduction in germination rate and pollen tube length, leading to complete sterility. Analysis of pollens of OsCHS1 heterologous lines further showed that flavonoid deposition in pollen is paternal effect and male fertility is independent from the OsCHS1 genotypes of microgametophytes and megagametophytes. Rice mutants deficient in branch point enzymes of the downstream flavonoid pathways, including FLAVANONE 3-HYDROXYLASE (OsF3H), FLAVONE SYNTHASE II (CYP93G1) and FLAVANONE 2-HYDROXYLASE (CYP93G2) were used to determine which subclass of flavonoids are essential for male fertility. These mutants displayed shifts in flavonoid profiles but only osf3h and cyp93g1 cyp93g2 double mutants displayed slight and moderate reduction in fertility rate, respectively. Results here indicated that not a specific flavonoid subclass is indispensable for male fertility and a balance of flavonoid composition might be required for full fertility in rice, as opposed to the essential role of flavonols reported previously in other plant species.

F3H is a key enzyme that channels carbon flow towards the production of 3-hydroxylated flavonoids, including flavonols and anthocyanidins. However, F3H-encoding genes are generally inactive in vegetative tissues of grass species such as sorghum which accumulates flavone derivatives predominantly. Microprojectile bombardment method was applied to generated SbF3H1 overexpression sorghum lines. Flavonol O-glycosides were detected amongst all transgenic tissues, and anthocyanidins were also detected in transgenic grains. By the contrary, only flavonol O-glycosides were detected with constitutive SbF3H1 expression in wild-type spikelet and pollen. Furthermore, SbF3H1 transgenic seedlings showed reduced susceptibility to Colletotrichum sublineola, the cause agent of sorghum anthracnose. Results here suggested that the enriched flavonoid profile as a result of SbF3H1 overexpression may improve defense responses to fungal infection and serve as an excellent source of nutraceuticals.