Expression of flavonoid 3’-hydroxylase is controlled by P1, the regulator of 3-deoxyflavonoid biosynthesis in maize

BMC Plant Biology Expression of flavonoid 3'-hydroxylase is controlled by P1, the regulator of 3-deoxyflavonoid biosynthesis in maize Mandeep Sharma 0 Chenglin Chai 2 Kengo Morohashi 2 Erich Grotewold 2 Maurice E Snook 1 Surinder Chopra 0 0 Department of Plant Science, Pennsylvania State University , University Park, Pennsylvania, PA16802 , USA 1 USDA-ARS, Russell Research Center , 950 College Station Road, Athens, GA 30605 , USA 2 Center for Applied Plant Sciences and Department of Molecular Genetics, Ohio State University , Columbus, OH 43210 , USA Background: The maize (Zea mays) red aleurone1 (pr1) encodes a CYP450-dependent flavonoid 3'-hydroxylase (ZmF3'H1) required for the biosynthesis of purple and red anthocyanin pigments. We previously showed that Zmf3'h1 is regulated by C1 (Colorless1) and R1 (Red1) transcription factors. The current study demonstrates that, in addition to its role in anthocyanin biosynthesis, the Zmf3'h1 gene also participates in the biosynthesis of 3-deoxyflavonoids and phlobaphenes that accumulate in maize pericarps, cob glumes, and silks. Biosynthesis of 3-deoxyflavonoids is regulated by P1 (Pericarp color1) and is independent from the action of C1 and R1 transcription factors. Results: In maize, apiforol and luteoforol are the precursors of condensed phlobaphenes. Maize lines with functional alleles of pr1 and p1 (Pr1;P1) accumulate luteoforol, while null pr1 lines with a functional or non-functional p1 allele (pr1;P1 or pr1;p1) accumulate apiforol. Apiforol lacks a hydroxyl group at the 3'-position of the flavylium B-ring, while luteoforol has this hydroxyl group. Our biochemical analysis of accumulated compounds in different pr1 genotypes showed that the pr1 encoded ZmF3'H1 has a role in the conversion of mono-hydroxylated to bi-hydroxylated compounds in the B-ring. Steady state RNA analyses demonstrated that Zmf3'h1 mRNA accumulation requires a functional p1 allele. Using a combination of EMSA and ChIP experiments, we established that the Zmf3'h1 gene is a direct target of P1. Highlighting the significance of the Zmf3'h1 gene for resistance against biotic stress, we also show here that the p1 controlled 3-deoxyanthocyanidin and C-glycosyl flavone (maysin) defence compounds accumulate at significantly higher levels in Pr1 silks as compared to pr1 silks. By virtue of increased maysin synthesis in Pr1 plants, corn ear worm larvae fed on Pr1; P1 silks showed slower growth as compared to pr1; P1 silks. Conclusions: Our results show that the Zmf3'h1 gene participates in the biosynthesis of phlobaphenes and agronomically important 3-deoxyflavonoid compounds under the regulatory control of P1. Anthocyanins; Flavones; Flavonoids; F3'H; Maysin; Phlobaphenes - Background The maize (Zea mays) flavonoid biosynthesis provides an excellent system to study gene interaction in plants because of its extensive characterization at genetic, biochemical, and molecular levels [1]. Different flavonoid compounds share the same basic skeleton of the flavannucleus consisting of two aromatic rings with six carbon atoms (ring A and B) which are interconnected by a hetero-cyclic ring with three carbon atoms (ring C). Maize produces 3-hydroxyflavonoids (anthocyanidins) and 3-deoxyflavonoids which include phlobaphenes, 3deoxyanthocyanidins, and C-glycosyl flavones. These compounds are synthesized in different tissues and this spatial distribution depends on the genetic constitution of the plant. Anthocyanins can accumulate in most plant parts whereas phlobaphenes are predominantly found in kernel pericarp (outer layer of ovary wall), cob-glumes (palea and lemma), tassel glumes, and husk [2]. The 3deoxyanthocyanins and C-glycosyl flavones primarily accumulate in silks [3-5]. However, in some high altitude 4-Coumaroyl-CoA + 3-Malonyl-CoA 2 OH-eriodictyol Dehydration GT RT Apimaysin R1 = H Maysin R1 = OH Pelargonidin R1 = H Cyanidin R1 = OH Apigeninidin R1 = H Luteolinidin R1 = OH Apiferol R1 = H Luteoforol R1 = OH Figure 1 Flavonoids biosynthetic pathway in maize. Biosynthetic genes (enzymes) in the pathway are: c2 (CHS), chalcone synthase; chi1 (CHI), chalcone isomerase; f3h (F3H), flavanone 3-hydroxylase; pr1 (F3H), flavonoid 3-hydroxylase; a1 (DFR), dihydroflavonol 4-reductase; a2 (AS), anthocyanidin synthase; bz1 (UFGT), UDP-glucose flavonoid 3-O-glucosyltransferase; and bz2 (GST), glutathione S-transferase; f2h1 (F2H), flavanone-2-hydroxylase; GT, C-glycosyl transferase; sm2 (RT), rhamnosyl-transferase; salmon silk1 (sm1) [28]; and brown pericarp1 (bp1) [29,30]; Pathway modeled after [31]; [27] (3-deoxyanthocyanidins); and [21] (C-glycosyl flavones). Putative steps in the pathway are shown as broken arrows with or without probable gene/enzymes involved. maize lines C-glycosyl flavones can also accumulate in leaves [6] indicating genetic diversity for developmental accumulation of flavonoid metabolites. The 3-hydroxy- and 3-deoxy-flavonoids in maize are regulated by independent sets of transcription factors. Accumulation of 3-hydroxyflavonoids (anthocyanins) is controlled by two sets of duplicated genes: colorless1 (c1)/purple leaf1 (pl1) are members of the R2R3-MYB family of transcription factors [7], and booster1 (b1)/red1 (r1) are members of the basic helix-loop-helix (bHLH) family [8,9]. Studies have shown that C1 or PL1 proteins interact directly with R1 or B1 to activate transcription of anthocyanin biosynthetic genes in seed and plant body, respectively [10,11]. In contrast, 3-deoxyflavonoid pathway genes are regulated by pericarp color1 (p1), which encodes an R2R3-MYB transcription factor [12]. The p locus is a complex of duplicated MYB-homologous genes p1 and p2 on chromosome 1 [13]. The p locus is a major QTL for the biosynthesis of C-glycosyl flavones [14,15] and 3-deoxyanthocyanidins in silks [16]. Three flavonoid biosynthetic genes; colorless2 (c2), chalcone isomerase1 (chi1), and anthocyaninless1 (a1) encode chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol 4-reductase (DFR), respectively. These three genes are common to the anthocyanin and phlobaphene pathways, but are independently Figure 2 Luteoforol accumulates in Pr1; P1 cob glumes. (A) Ear phenotypes showing cob glumes pigmentation. Cob glumes of pr1 are light red while Pr1 has dark red cob glumes in the presence of P1-wr or P1-rr alleles. Pr1 and pr1 ears carrying p-del2 do not show any pigmentation. (B) Absorption spectra of cob glume extracts. Methanolic extracts from cob glumes of Pr1 ears in genetic background of P1-wr and P1-rr alleles gave maximum absorption at 498 nm. Cob glumes from pr1 ears in P1-wr and P1-rr genetic background gave maximum absorption at 482 nm. These spectra at 498 and 482 correspond to peaks for standard luteolinidin and apigeninidin, respectively (see inset). No absorption peak for flavan 4-ols was observed in p-del2 cob glumes. regulated by the corresponding sets of transcription factors [10,17,18]. In vitro and in vivo studi (...truncated)