The Brown midrib2 and Brown midrib4 mutants of Maize Link Lignin Biosynthesis to Methylation and Polyglutamylation

Lignin is a heteropolymer component of plant cell walls that provides structural strength and contributes to resistance to some pests and pathogens. The maize brown midrib (bm) mutants, named for their reddish-brown midribs, accumulate less and altered lignin relative to non-mutants. Four of the six currently characterized bm mutants have been cloned and shown to encode enzymes in the lignin biosynthesis pathway. While bm1 and bm3 are involved in the direct synthesis of lignin, bm2 and bm4 reveal a previously uncharacterized link between lignin synthesis and components of the methylation and polyglutamylation pathways. The bm2 gene encodes a functional methylene tetrahydrofolate reductase (MTHFR) involved in the formation of the methyl donor S-adenosyl-L-methionine, which is consumed by lignin pathway enzymes. The bm4 gene encodes a functional folylpolyglutamate synthase (FPGS) that catalyzes the polyglutamlyation of folate compounds upstream of bm2-encoded MTHFR. The bm2 gene was cloned via traditional mapping, RNA-sequencing, and BSR-Seq (Tang et al., Plant J 2014). The bm4 gene was cloned via a combination of traditional mapping, Seq-Walking, and PCR-based sequencing methods (Li et al., accepted pending revisions). Mutants in the bm2 and bm4 genes accumulate 7-14% less lignin in stalks than their wild-type siblings at anthesis. In both cases, however, lignin continues to accumulate until senescence, which can narrow the difference between non-mutant and mutant to as low as 2%.  For both mutants, the ratio of syringyl to guaiacyl lignin is elevated in mutant stalks. Functionality of the bm2 and bm4-encoded enzymes was confirmed via yeast complementation studies. Paralogous genes of bm2 and bm4 have been identified that may explain the viability of these mutants. However, the functional overlap among paralogs is not complete, as evidenced by the alterations in lignin content in both mutants.