Biosynthesis and antifungal activity of fungus-induced O-methylated flavonoids in maize

Abstract

Fungal infection of grasses, including rice (Oryza sativa), sorghum (Sorghum bicolor), and barley (Hordeum vulgare), induces the formation and accumulation of flavonoid phytoalexins. In maize (Zea mays), however, investigators have emphasized benzoxazinoid and terpenoid phytoalexins, and comparatively little is known about flavonoid induction in response to pathogens. Here, we examined fungus-elicited flavonoid metabolism in maize and identified key biosynthetic enzymes involved in the formation of O-methylflavonoids. The predominant end products were identified as two tautomers of a 2-hydroxynaringenin-derived compound termed xilonenin, which significantly inhibited the growth of two maize pathogens, Fusarium graminearum and Fusarium verticillioides. Among the biosynthetic enzymes identified were two O-methyltransferases (OMTs), flavonoid OMT 2 (FOMT2), and FOMT4, which demonstrated distinct regiospecificity on a broad spectrum of flavonoid classes. In addition, a cytochrome P450 monooxygenase (CYP) in the CYP93G subfamily was found to serve as a flavanone 2-hydroxylase providing the substrate for FOMT2-catalyzed formation of xilonenin. In summary, maize produces a diverse blend of O-methylflavonoids with antifungal activity upon attack by a broad range of fungi.

Evolution of DIMBOA-Glc O-Methyltransferases from Flavonoid O-Methyltransferases in the Grasses

“…However, when the flavonol kaempferol, which, in contrast to the above-mentioned flavonoids, contains an additional hydroxyl group at position 3 of the C ring, was tested as a substrate, ZmBX10-12 and ZnBX10 produced substantial amounts of the 3- O -methylated derivative isokaempferide ( Figure 2 b and Figure S2; Table S1 ), while ZmFOMT2 and SbFOMT2 produced only trace amounts of this compound ( Table S1 ). Notably, the catalytic efficiency of the BX OMTs towards kaempferol followed the same order as shown for DIMBOA-Glc [ 28 , 31 ], with ZmBX12 as the most active OMT, followed by ZmBX10, ZmBX11, and ZmBX14 ( Figure S2 ). The flavonoid 6-OMT ZmFOMT5 [ 28 ] also accepted kaempferol as a substrate and produced minor amounts of isokaempferide ( Figure S2 ).…”

“…Notably, the catalytic efficiency of the BX OMTs towards kaempferol followed the same order as shown for DIMBOA-Glc [ 28 , 31 ], with ZmBX12 as the most active OMT, followed by ZmBX10, ZmBX11, and ZmBX14 ( Figure S2 ). The flavonoid 6-OMT ZmFOMT5 [ 28 ] also accepted kaempferol as a substrate and produced minor amounts of isokaempferide ( Figure S2 ). Untargeted LC–MS measurements with accurate mass determination showed that ZmBX12 and ZnBX10 were also active with quercetin, a flavonol similar to kaempferol, and both the MS/MS fragmentation pattern and the LC elution order indicated methylation at the 3-hydroxyl group of the C ring ( Figure S3 ).…”

“…In a previous study, we showed that ZmBX10-12 and ZmBX14 exhibited nonspecific trace activity with various flavonoids, such as naringenin, apigenin, and scutellarein [ 28 ]. However, when the flavonol kaempferol, which, in contrast to the above-mentioned flavonoids, contains an additional hydroxyl group at position 3 of the C ring, was tested as a substrate, ZmBX10-12 and ZnBX10 produced substantial amounts of the 3- O -methylated derivative isokaempferide ( Figure 2 b and Figure S2; Table S1 ), while ZmFOMT2 and SbFOMT2 produced only trace amounts of this compound ( Table S1 ).…”

“…Similarly, pathogen-induced formation of O -methylflavonoids was also detected in other grasses, such as sorghum ( Sorghum bicolor ), maize ( Zea mays ), and barley ( Hordeum vulgare ) [ 25 , 26 , 27 ]. Recently, we characterized four FOMTs from maize, ZmFOMT2 and ZmFOMT3, ZmFOMT4, and ZmFOMT5, that methylate the hydroxyl groups of various flavonoids regiospecifically at positions 5, 7, and 6 of the A ring, respectively, and are involved in the fungus-induced formation of complex O -methylflavonoid mixtures [ 28 ].…”

“…In this study, we investigated the evolution of DIMBOA-Glc 4-OMTs in maize. Because the recently identified flavonoid 5-OMTs ZmFOMT2 and ZmFOMT3 are closely related to ZmBX10-12 and ZmBX14 [ 28 ], we hypothesized that DIMBOA-Glc 4-OMT activity evolved from FOMT activity in the PACMAD clade of the grasses, one of the two major clades of Poaceae, named after the subfamilies Panicoideae, Aristidoideae, Chloridoideae, Micrairoideae, Arundinoideae, and Danthonioideae [ 41 ]. To investigate this evolutionary scenario, we performed phylogenetic analyses to identify ZmFOMT2 homologs in several Poaceae species, followed by in vitro enzyme characterization.…”