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Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

ABSTRACT: BACKGROUND: Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production. RESULTS: We developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants. CONCLUSION: These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency.

SUBMITTER: Trabucco GM

PROVIDER: S-EPMC3734214 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

Trabucco Gina M GM Matos Dominick A DA Lee Scott J SJ Saathoff Aaron J AJ Priest Henry D HD Mockler Todd C TC Sarath Gautam G Hazen Samuel P SP

BMC biotechnology 20130731

<h4>Background</h4>Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized c ...[more]

PMID: 23902793

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Project description:Cinnamyl alcohol dehydrogenase (CAD) is involved in the final step of the phenylpropanod pathway, catalyzing the NADPH-dependent reduction of hydroxy-cinnamaldehydes into the corresponding alcohols. The rice genome contains twelve CAD and CAD-like genes, collectively called OsCADs. To elucidate the biochemical function of the OsCADs, OsCAD1, 2, 6, and 7, which are highly expressed in rice, were cloned from rice tissues. The cloned OsCADs were heterologously expressed in Escherichia coli as His-tag fusion proteins. The activity assay of the recombinant OsCADs showed that OsCAD2, 6, and 7 have CAD activity toward hydroxycinnamaldehydes, but OsCAD1 has no detectable catalytic activity. The kinetic parameters of the enzyme reactions demonstrated that OsCAD2 has the highest catalytic activity among the examined enzymes. This result agrees well with the finding that the Zn binding and NADPH binding motifs and the residues constituting the substrate binding pocket in bona fide plant CADs were fully conserved in OsCAD2. Although they have large variations in the residue for the substrate binding pocket, OsCAD6 and 7 catalyzed the reduction of hydroxycinnamaldehydes with a similar efficiency. Alignment of amino acid sequences showed that OsCAD1 lacks the GxxxxP motif for NADPH binding and has mismatches in residues important in the reduction process, which could be responsible for the loss of catalytic activity. OsCAD2 belongs to CAD Class I with bona fide CADs from other plant species and is constitutively expressed throughout the developmental stages of rice, with preferential expression in actively lignifying tissues such as the root, stem, and panicle, suggesting that it is mainly involved in developmental lignification in rice. The expression of OsCAD2 was also induced by biotic and abiotic stresses such as Xanthomonas oryzae pv. oryzae (Xoo) infection and UV-irradiation, suggesting that it plays a role in the defense response of rice, in addition to a bona fide role in developmental lignification. OsCAD6 and 7 belong in CAD Class II. Their expression is relatively lower than that of OsCAD2 and is confined to certain tissues, such as the leaf sheath, stem, and panicle. The expression of OsCAD6 was stimulated by Xoo infection and UV-irradiation. Thus OsCAD6 appears to be an inducible OsCAD that is likely involved in the defense response of rice against biotic and abiotic stresses.

Project description:BACKGROUND: Adverse environmental conditions severely influence various aspects of plant growth and developmental processes, causing worldwide reduction of crop yields. The C-repeat binding factors (CBFs) are critical transcription factors constituting the gene regulatory network that mediates the acclimation process to low temperatures. They regulate a large number of cold-responsive genes, including COLD-REGULATED (COR) genes, via the CBF-COR regulon. Recent studies have shown that the CBF transcription factors also play a role in plant responses to drought and salt stresses. Putative CBF gene homologues and their downstream genes are also present in the genome of Brachypodium distachyon, which is perceived as a monocot model in recent years. However, they have not been functionally characterized at the molecular level. RESULTS: Three CBF genes that are responsive to cold were identified from Brachypodium, designated BdCBF1, BdCBF2, and BdCBF3, and they were functionally characterized by molecular biological and transgenic approaches in Brachypodium and Arabidopsis thaliana. Our results demonstrate that the BdCBF genes contribute to the tolerance response of Brachypodium to cold, drought, and salt stresses by regulating downstream targets, such as DEHYDRIN5.1 (Dhn5.1) and COR genes. The BdCBF genes are induced under the environmental stress conditions. The BdCBF proteins possess transcriptional activation activity and bind directly to the promoters of the target genes. Transgenic Brachypodium plants overexpressing the BdCBF genes exhibited enhanced resistance to drought and salt stresses as well as low temperatures, and accordingly endogenous contents of proline and soluble sugars were significantly elevated in the transgenic plants. The BdCBF transcription factors are also functional in the heterologous system Arabidopsis. Transgenic Arabidopsis plants overexpressing the BdCBF genes were also tolerant to freezing, drought, and salt stresses, and a set of stress-responsive genes was upregulated in the transgenic Arabidopsis plants. CONCLUSIONS: Taken together, our results strongly support that the BdCBF transcription factors are key regulators of cold stress responses in Brachypodium and the CBF-mediated cold stress signaling pathway is conserved in this plant species. We believe that this study would confer great impact on stress biology in monocot species and could be applied to engineer abiotic stress tolerance of bioenergy grass species.

Dataset Information

Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

Publications

Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon.

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