Structure and expression profile of the cinnamyl alcohol dehydrogenase gene and its association with lignification in the sorghum (Sorghum bicolor (L.) Moench) bmr-6 mutant

Abstract

Lignin, one of the major components of plant cell walls, has negative impacts on forage digestibility and bioethanol production through fermentation. Understanding the regulatory mechanism of lignin biosynthesis is essential to developing plants with altered lignin contents. The sorghum (Sorghum bicolor [L.] Moench) brown-midrib-6 mutant (bmr-6) has reduced levels of several key enzymes in the lignin biosynthesis pathway. Among the genes encoding these enzymes, the gene for cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) contains a nonsense mutation (responsible for bmr-6) that results in reduced CAD gene transcript levels. We found that the CAD coding region was 4225 bp and consisted of four exons separated by three introns. Bioinformatic analysis of the 5′-flanking region identified several putative binding sites for transcription factors. Moreover, a cross-species comparison of CAD genomic DNA sequences revealed evolutionary and structural variation among plant taxa. The expression level of the CAD gene was much lower in bmr-6 than in normal isoline (N-6) in root, stem, leaf, and midrib. Expression levels of other lignin biosynthesis genes were comparable in bmr-6 and N-6. These results suggest that the depression of CAD enzymatic activity in bmr-6 could affect enzymatic activity and post-translational regulation of other enzymes related to lignin biosynthesis.

Dynamic expression of the nutritive values in forage sorghum populations associated with white, green and brown midrid genotypes

“…A number of the BMR mutants of sorghum have been identified and characterized since the late 1970s, two of which mutants, BMR-6 and BMR-12, appeared to be the most promising for improvement of forage quality (Porter et al, 1978;Tsuruta et al, 2010). Previous studies on ADL content of BMR-6 and -12 genes to each other were very limited except for Oliver's research (Oliver et al, 2005) which reported that the BMR-12 had significantly (P < 0.05) less ADL content than BMR-6 counterparts at dough stage, and the similar results were confirmed in our research that BMR-12 populations had significantly (P < 0.05) lower ADL content than that of BMR-6 at the later growing stage (milk and Dough) ( Fig.…”

“…Previous research suggested two enzymes exhibit reduced activity as the result of BMR mutations, the activity of cinnamyl alcohol dehydrogenase (CAD) was decreased in bmr-6 sorghum (Tsuruta et al, 2010) while the activity of caffeic acid O-methyltransferase (COMT) was reduced in the bmr-12 mutants (Bout and Vermerris, 2003). Therefore, it can be deduced that there were not significant (P > 0.05) differences in relative expression level of CAD and COMT genes from joint to head stage ( Fig.…”

The Present Status of C₄ Tropical Grasses Breeding and Molecular Approaches

“…The brown midrib mutants in sorghum were chemically induced via the mutagen diethyl sulfate (DES) (Porter et al 1978), a monofunctional alkylating agent that acts primarily as a base-substitution mutagen and has specificity for G/C-to-A/ T transitions (Hoffman 1980). In previous reports, base substitutions resulting in premature stop codons were identified in the coding regions of the genes encoding COMT (Bout & Vermerris 2003) and CAD (Saballos et al 2009, Sattler et al 2009, Tsuruta et al 2010) in sorghum brown midrib mutants (Table 5). In these mutants, the binding site of the enzyme has been changed, resulting in a loss of enzymatic activity.…”

“…Bout & Vermerris (2003) have developed cleaved amplified polymorphic sequence (CAPS) markers to identify the bmr-12 and bmr-18 genotypes, while we have developed single-nucleotide polymorphism (SNP) markers to identify the bmr-6 genotype. A comparison of the sequences derived from N-6 and bmr-6 revealed a C-to-T transition in the third exon of bmr-6, leading to a single amino acid alteration (glutamine to a termination codon; Tsuruta et al 2010). To verify the single base-pair change observed in bmr-6, SNP markers were developed to distinguish the wild-type and mutant CAD alleles (Fig.…”

“…The improved digestibility is one of key areas of focus in guineagrass, while in brachiariagrass, the marker-assisted selection (MAS) method used for guineagrass was applied (Ebina et al 2005), spawning faster breeding. In sorghum, the lignification mechanisms have been revealed by molecular techniques (Tsuruta et al 2010) and used to produce higher-quality forage cultivars. In zoysiagrass, a domestic forage grass in Japan, molecular techniques also provide a number of advantages.…”