Identification of flavone phytoalexins and a pathogen-inducible flavone synthase II gene (SbFNSII) in sorghum

Abstract

Following inoculation with the anthracnose pathogen Colletotrichum sublineolum, seedlings of the sorghum resistant cultivar SC748-5 showed more rapid and elevated accumulation of luteolin than the susceptible cultivar BTx623. On the other hand, apigenin was the major flavone detected in infected BTx623 seedlings. Luteolin was demonstrated to show stronger inhibition of spore germination of C. sublineolum than apigenin. Because of their pathogen-inducible and antifungal nature, both flavone aglycones are considered sorghum phytoalexins. The key enzyme responsible for flavone biosynthesis has not been characterized in monocots. A sorghum pathogen-inducible gene encoding a cytochrome P450 protein (CYP93G3) in the uncharacterized CYP93G subfamily was identified. Transgenic expression of the P450 gene in Arabidopsis demonstrated that the encoded protein is a functional flavone synthase (FNS) II in planta. The sorghum gene was then termed SbFNSII. It is a single-copy gene located on chromosome 2 and the first FNSII gene characterized in a monocot. Metabolite analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in precursor ion scan mode revealed the accumulation of 2-hydroxynaringenin and 2-hydroxyeriodictyol hexosides in the transgenic Arabidopsis plants. Hence, SbFNSII appears to share a similar catalytic mechanism with the licorice and Medicago truncatula FNSIIs (CYP93B subfamily) by converting flavanones to flavone through the formation of 2-hydroxyflavanones.

CYP93G2 Is a Flavanone 2-Hydroxylase Required for C-Glycosylflavone Biosynthesis in Rice

“…MS/MS of the mass-to-charge ratio (m/z) 289 ion generated the characteristic fragmentation pattern (Fig. 2C), which was reported previously for a [2-hydroxynaringenin + H] + ion (Zhang et al, 2007;Du et al, 2010). Upon acid treatment of the reaction mixture, 2-hydroxynaringenin was dehydrated to apigenin ( Fig.…”

“…C, The single peak detected in the CYP93G2 reaction generated a major [M + H] + ion at m/z 289 on MS analysis. The MS/MS spectrum revealed the fragmentation pattern consistent with that reported for 2-hydroxynarigenin (Zhang et al, 2007;Du et al, 2010). D, Upon acid treatment, the reaction product was converted to apigenin (API), which was confirmed by an authentic standard.…”

“…The foreign flavonoid metabolites in transgenic Arabidopsis have been modified by endogenous flavonoid O-glycosyltransferases, which generally show a broad substrate range. Previously, we also detected the presence of 2-hydroxyflavanone O-glycosides in Arabidopsis overexpressing CYP93G3 (Du et al, 2010), which is the closest homolog of CYP93G2. In major cereal crops, flavones occur predominately as C-glycosides that accumulate constitutively in different tissues.…”

“…The P450 CYP93G subfamily was first described in rice with two sequences (http://drnelson.uthsc.edu/ rice.html), and a sorghum protein was subsequently assigned as a new member (Du et al, 2010). Phylogenetic analysis revealed that CYP93G2 is closely related to CYP93G3 (Fig.…”

“…A recombinant rice DOX protein, designated as OsFNS1-1, was found to convert naringenin directly to apigenin, and it represented the first FNSI reported outside Apiaceae (Lee et al, 2007). On the other hand, we recently described a sorghum (Sorghum bicolor) pathogen-inducible P450 gene that resulted in the accumulation of 2-hydroxyflavanone O-hexosides in transgenic Arabidopsis (Arabidopsis thaliana; Du et al, 2010). The encoded protein is a new member (CYP93G3) belonging to the CYP93G subfamily, which was first annotated in the rice genome (Nelson et al, 2004).…”