ssociation mapping using the common linear model (GLM) and 80,440 single-nucleotide polymorphisms (SNPs) that identified

ssociation mapping using the common linear model (GLM) and 80,440 single-nucleotide polymorphisms (SNPs) that identified very considerable SNPs on chromosome 9 (B73 RefGen_v2) (Figure 2A; Supplemental Figure S2). The corresponding chromosomal area contained two putative OMT genes named FOMT2 (Zm00001d047192) and FOMT3 (Zm00001d047194). For clarity, unless otherwise noted, gene and protein abbreviations refer to line B73 (RefGen_v4) reference sequences. Additionally, a genomewide association study (GWAS) making use of the Goodman association panel (mixed linear model (Multilevel marketing), 25,457,708 SNPs; Flint-Garcia et al., 2005) was performed working with genkwanin or the apigenin/genkwanin ratio as traits (Figure 2B; Supplemental Figure S3), which revealed a second genomic area on chromosome 9 containing a third putative OMT gene named FOMT4 (Zm00001d048087). Initial sequence analyses from the identified OMT genes in diverse maize inbred lines revealed that W22 includes a second copy of FOMT2 (Zm00004b033403 and Zm00004b033399, W22 RefGen_v2) on chromosome 9, differing only in a single synonymous nucleotide (Supplemental Figures S4 and S5). In addition, FOMT2 and FOMT3 are closely associated and encode proteins with 79 amino acid sequence identity. RNA sequencing (RNA-seq) of W22 leaves, damaged and treated with either water (handle) or B. maydis hyphae for 4 d, showed significantly increased accumulation of transcripts encoding both copies of FOMT2 and FOMT4 as predicted for their involvement in flavonoid O-methylation (Figure 2C; Supplemental Table S2; Supplemental Information Set S1). In contrast, FOMT3 displayed substantially decrease expression levels that did not show statistically considerable variations between the treatments. Phylogenetic analyses demonstrated that FOMT2/3 are closely related to maize BX10/11/12/14, which catalyze various O-methylations of benzoxazinoid (BX) defensecompounds (Meihls et al., 2013), and to an uncharacterized maize OMT named FOMT5 (Zm00001d051934) (Figure 2D; Supplemental Figure S6; Supplemental Table S3). Notably, BX10/11/14 and FOMT5 transcripts also elevated immediately after fungal elicitation in our experiments (Figure 2C; Supplemental Figure S7; Supplemental Table S2). In contrast to FOMT2/3, FOMT4 showed the closest relation to OsNOMT, accountable for production from the COX-2 Modulator Accession phytoalexin sakuranetin in rice, and also other Poaceae FOMTs, including maize OMT1 (FOMT1), which has been described to O-methylate the B-ring of different flavonoids (Figure 2D; Supplemental Figure S6).FOMT2/3, FOMT4, and FOMT5 catalyze the regiospecific O-methylation of diverse flavonoids in vitroTo characterize the enzymatic activity of FOMT2, FOMT3, FOMT4, and FOMT5, we expressed the comprehensive open reading frames in Escherichia coli and tested the purified recombinant proteins in enzyme assays with potential flavonoid substrates in the presence on the cofactor SAM. Applying scutellarein as a substrate, LC S/MS analysis revealed that FOMT2, FOMT4, and FOMT5 every single made a various single item peak that was not present within the empty vector (EV) control (Figure 2E). Solution purification followed by NMR structure elucidation (Supplemental Table S4; Supplemental Data Set S2) or comparison with commercially out there requirements confirmed regiospecific O-methylation on positions 5, 7, and six of the flavonoid A-ring catalyzed by FOMT2, FOMT4, and FOMT5, respectively. In an enzyme assay containing both FOMT2 and FOMT4, a 5,7-O-dimethylated product was detected (Figure 2E). HDAC6 Inhibitor manufacturer Interestingly,