CAD2 deficiency causes both brown midrib and gold hull and internode phenotypes in Oryza sativa L. cv. Nipponbare

Abstract

Several brown midrib (bm) mutants have so far been isolated from the C4 grasses, maize, sorghum and pearl millet, but have not been detected in C3 grasses including rice (Oryza sativa). In the present study we characterized the cad2 (cinnamyl alcohol dehydrogenase 2) null mutant isolated from retrotransposon Tos17 insertion lines of Oryza sativa L. ssp. japonica cv. Nipponbare. This mutant exhibited brown-colored midribs in addition to hulls and internodes, clearly indicating both bm and gold hull and internode (gh) phenotypes. The enzymatic saccharification efficiency in the culm of cad2 null mutant was increased by 16.1% than that of the control plants. The lignin content of the cad2 null mutant was 14.6% lower than that of the control plants. Thioacidolysis of the cad2 null mutant indicated the presence of cinnamaldehyde structures in the lignin. Taken together, our results show that deficiency of OsCAD2 causes the bm phenotype in addition to gh, and that the coloration is probably due to the accumulation of cinnamaldehyde-related structures in the lignin. Additionally, this cad2 null mutant was useful to silage purposes and biofuel production. Lignin is a complex phenylpropanoid polymer, and is biosynthesized via oxidative coupling of phydroxycinnamyl alcohols (monolignols) and related compounds that are formed in the cinnamate/ monolignol pathway (Umezawa 2010). Lignin fills the spaces between cell wall polysaccharides and confers mechanical strength and imperviousness to the cell wall (Boerjan et al. 2003). Therefore, lignin biosynthesis is closely related to the evolution of land plants.Lignin has several properties that present obstacles to chemical pulping, forage digestion, and enzymatic hydrolysis of plant cell wall polysaccharides for biorefining. For these processes, it would be beneficial for plant materials to either have less lignin, or to have lignin that is easier to remove. Mutant plants in which genes encoding lignin biosynthetic enzymes are downregulated are generally expected to have lower lignin content and higher enzymatic saccharification efficiency. For these reasons, lignin biosynthesis is an area of great interest (Chiang 2006;Dixon and Reddy 2003;Vanholme et al. 2008;Weng et al. 2008).Several brown midrib (bm or bmr for sorghum) mutants have been isolated in maize (Zea mays), sorghum (Sorghum bicolor), and pearl millet (Pennisetum glaucum) arising by either spontaneous or chemical mutagenesis (Barrière et al. 2004;Cherney et al. 1991;Sattler et al. 2010). The characteristic reddish-brown to tan colored midribs of the mutant leaf blades contrasts with the pale green midribs of the wild type. In addition, the mutants show similar coloration in stalks and generally have reduced lignin content and higher in vitro digestibility compared with wild-type plants. Hence, the mutants have been receiving a lot of interest in relation not only to silage purposes (Barrière et al. 2004;Cherney et al. 1991;Sattler et al. 2010), but also biofuel production (Sattler et al. 2010).In maize, six...

Disrupting Flavone Synthase II Alters Lignin and Improves Biomass Digestibility

“…As with other putative/known tricin biosynthetic genes, such as OsCHS1 (Shih et al, 2008;Hong et al, 2012), OsCHI (Shih et al, 2008), and OsC59H (CYP75B4; Lam et al, 2015), OsFNSII (CYP93G1; Lam et al, 2014) was expressed most prominently in culm at reproductive and ripening stages, where cell wall lignification typically occurs. We confirmed the concurrent expression of putative/known monolignol biosynthetic genes, including OsCAD2 (Zhang et al, 2006;Koshiba et al, 2013b), OsCAldOMT1 (Koshiba et al, 2013a), and OsPMT (Petrik et al, 2014), as well as the common phenylpropanoid genes OsPAL1/2 (Cass et al, 2015) and Os4CL3 (Gui et al, 2011;Supplemental Fig. S1).…”

MYB-mediated upregulation of lignin biosynthesis in <i>Oryza sativa</i> towards biomass refinery

“…The panicles were removed from the aerial parts, and the remaining plant material was separated into leaves, leaf sheaths, and culms. The separated plant samples were individually cut into pieces with scissors and then pulverized with a TissueLyser (Qiagen, Hilden, Germany), extracted sequentially with methanol, hexane, and distilled water, and then freeze-dried as described previously (Koshiba et al 2013a(Koshiba et al , 2013b.…”

“…Lignin quantitation by the thioglycolic acid method, microscale nitrobenzene oxidation analysis, and thioacidolysis were conducted according to the methods as previously described (Koshiba et al 2013a(Koshiba et al , 2013bSuzuki et al 2009;Yamamura et al 2010Yamamura et al , 2012. Two-dimensional NMR (2D-NMR) analysis was conducted as previously reported (Mansfield et al 2012).…”

“…Quantitative reversetranscription PCR (qRT-PCR) was conducted as previously described (Koshiba et al 2013a(Koshiba et al , 2013b. Each gene was amplified using specific primers: 5′-TTG GTA TGT GGC CAT GTA ACC A-3′ and 5′-GGC CAT ATG CAT GTT GCT GA-3′ for AtMYB55, 5′-AAC ATG GTT GGT TCT GTC CTT CA-3′ and 5′-AAT CGA GGG CTT TAC GCA TAC T-3′ for AtMYB61, 5′-ACA AAC CCG ATC TGC TGG AG-3′ and 5′-TCC AAA TGT CAG GAT CTG AAT CAA-3′ for AtMYB63, respectively.…”

“…japonica cv. Nipponbare) lines with decreased lignin content, which exhibited significantly enhanced enzymatic saccharification efficiency Koshiba et al 2013aKoshiba et al , 2013b.…”