GOLD HULL AND INTERNODE2 Encodes a Primarily Multifunctional Cinnamyl-Alcohol Dehydrogenase in Rice

Abstract

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis.

FLEXIBLE CULM 1 encoding a cinnamyl-alcohol dehydrogenase controls culm mechanical strength in rice

“…The reddish coloration of CAD-deficient plants has been attributed to the incorporation of cinnamaldehydes in polymer. Especially dark red coloration occurred after staining with phloroglucinol reagent, and the color intensity was stronger in the gh2 and bm1 mutants than in their respective wild-types (Halpin et al 1998;Zhang et al 2006). However, an opposite phloroglucinol staining pattern existed between fc1 and its wild-type.…”

“…Plant protein extraction and enzyme assay Plant total proteins were extracted using the method described by Zhang et al (2006). Fresh tissues (about 500 mg) at the heading stage were separately milled to a fine powder in liquid nitrogen, then extracted with 1,000 ll extraction buffer (100 mM Tris-HCl, pH 7.5; 2% polyethylene glycol 6000, 5 mM dithiothreitol, 2% polyvinylpolypyrrolidone) for 2.5 h at 4°C.…”

“…For histochemical localization of the accumulated lignin, phloroglucinol staining was performed according to the method described by Zhang et al (2006). Fresh hand-cut sections from the second internode of rice plants at the heading stage were incubated for 10 min in phloroglucinol solution (1% in 70% ethanol), the phloroglucinol was removed and treated with 18% HCl for 5 min, then photographed under a light microscope (model DM4000B; Leica, Germany).…”

“…Many CAD homologs have been isolated from various plant species, and they have been well studied using traditional mutants and transgenic approaches Halpin et al 1998;Sibout et al 2005;Zhang et al 2006). For example, Halpin et al (1998) reported that the natural CADdeficient mutant brown midrib1 (bm1) of maize (Zea mays) showed a reddish brown pigment in leaf midribs and stem sclerenchyma; bm1 had a 20% decrease in lignin content and better digestibility.…”

Free Phenols in Maize Pith and Their Relationship with Resistance to <I>Sesamia nonagrioides</I> (Lepidoptera: Noctuidae) Attack

“…Previous studies have indicated impairing different steps in monolignol biosynthesis results in reddish brown to tan stem and stalk pigmentation, including the CAD and COMT mutants examined in this study (Mackay et al 1997, Tsai et al 1998, Sibout et al 2005, Zhang et al 2006). There are differences in pith and midrib (leaf vein) coloration between bmr6 and bmr12 (Porter et al 1978, Saballos et al 2009), probably because bmr6 and bmr12 block different steps of the monolignol biosynthetic pathway.…”