The maize brown midrib4 (bm4) gene encodes a functional folylpolyglutamate synthase

Abstract

Mutations in the brown midrib4 (bm4) gene affect the accumulation and composition of lignin in maize. Fine-mapping analysis of bm4 narrowed the candidate region to an approximately 105 kb interval on chromosome 9 containing six genes. Only one of these six genes, GRMZM2G393334, showed decreased expression in mutants. At least four of 10 Mu-induced bm4 mutant alleles contain a Mu insertion in the GRMZM2G393334 gene. Based on these results, we concluded that GRMZM2G393334 is the bm4 gene. GRMZM2G393334 encodes a putative folylpolyglutamate synthase (FPGS), which functions in one-carbon (C1) metabolism to polyglutamylate substrates of folate-dependent enzymes. Yeast complementation experiments demonstrated that expression of the maize bm4 gene in FPGS-deficient met7 yeast is able to rescue the yeast mutant phenotype, thus demonstrating that bm4 encodes a functional FPGS. Consistent with earlier studies, bm4 mutants exhibit a modest decrease in lignin concentration and an overall increase in the S:G lignin ratio relative to wild-type. Orthologs of bm4 include at least one paralogous gene in maize and various homologs in other grasses and dicots. Discovery of the gene underlying the bm4 maize phenotype illustrates a role for FPGS in lignin biosynthesis.

Keywords: Zea mays; brown midrib; folylpolyglutamate synthase; lignin; methylenetetrahydrofolate reductase.

Figure 1

BM4 in connection with lignin biosynthesis. FPGS (BM4) is connected to the lignin biosynthesis pathway through a series of polyglutamylated molecules and BM2. Chemical structures were downloaded from KEGG (http://www.genome.jp/kegg/). ‘…’ indicates a series of steps that converts polyglutamylated THF to polyglutamylated 5,10-methylene THF (Cossins and Chen, 1997).

Figure 2

Phenotypic characterization of bm4 mutant phenotypes. bm4 mutants display reddish coloring of their leaf midribs. Plants shown were grown in the greenhouse under 27°C daytime temperature and 24°C night-time temperature. (a, b) B73 wild-type and bm4-ref; (c, d) B73; (e, f) bm4-ref; (g, h) bm4-Mu 13-7027D; (i, j) bm4-Mu 13-7029B; (k, l) bm4-Mu 13-7033E; (m, n) bm4-Mu 13-7064E; (o, p) bm4-Mu 11-8034B; (q, r) bm4-Mu 11-8050D. Phenotypic appearance of the adaxial (a) and abaxial (b) sides of bm4-ref and B73 leaves. (g, i, k, m, o, q) Phenotypic appearance of the abaxial sides of bm4-Mu mutants. Phloroglucinol-hydrochloric acid staining of the adaxial (c) and abaxial (d) sides of B73 wild-type midrib tissue. Phloroglucinol-HCl staining of the adaxial (e) and abaxial (f, h, j, l, n, p, r) sides of bm4 mutant midrib tissue. Scale bars = 100 μm.

Figure 3

qRT-PCR expression analysis of bm4-Mu alleles. The relative expression of bm4 in bm4-ref mutants and five homozygous bm4-Mu mutants compared with B73 wild-type and the bm2-ref mutant (Tang et al., 2014). Error bars indicate the standard error between technical replicates.

Figure 4

Sequence analysis of bm4-Mu alleles. Mu insertions in bm4 were detected in alleles: (1) bm4-Mu 13-7033E, (2) bm4-Mu 11-8050D, (3) bm4-Mu 13-7029B, and (4) bm4-Mu 13-7064E.

Figure 5

Maize-derived Bm4 complements the yeast met7 mutant phenotype. met7 = untransformed haploid mutant MATα met7 strain; MET7 = untransformed haploid MATα non-mutant parent; pBm4 = MATα met7 haploid mutant transformed with Bm4-containing p413-ADH shuttle vector; p413 = MATα met7 haploid mutant transformed with empty p413-ADH shuttle vector. YPD = YPD media; −Me = SDC −Met medium.

Figure 6

Homolog analysis of BM4. Homolog analysis of BM4 across various plant species. ‘|’ separates the associated organism, Protein ID, and Gene ID of each protein sequence in the tree. The protein sequences used to construct the tree are the same as in Table S5. Brackets indicate either the name of the protein encoded or its subcellular localization. Boxes separate the two observed clades of FPGS proteins.