Academic literature on the topic 'Cinnamyl alcohol dehydrogenase'

Abstract Alcohol oxidases and dehydrogenases are poorly studied in the Mollusca, the second largest phylum of metazoans. In order to obtain an overview of the distribution of aromatic alcohols and ethanol-oxidizing enzymes in the gastropod phylogenetic tree, we investigated the activity of these enzymes in the digestive gland of 26 gastropod species in the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, as well as herbivores and carnivores, were sampled so that gastropods varying widely in habitat and diet were included in the study. An aromatic alcohol oxidase, which was previously reported in herbivorous terrestrial gastropods, was detected in 25 of the studied species. The activity of a cinnamyl alcohol dehydrogenase was detected for the first time in gastropods and this enzyme was found to be present in all the species that were studied. Our study, thus, demonstrates that alcohol oxidases and dehydrogenases are ubiquitous enzymes among gastropods; these enzymes are found across the gastropod phylogenetic tree and across species varying widely in habitat and diet. The enzymes that catalyze the oxidation or dehydrogenation of cinnamyl alcohol must be involved in the metabolism of aromatic alcohols of very different dietary origins and conceivably have a detoxification function. Oxidase or dehydrogenase activities involving ethanol as a substrate were detected only in a few species, mostly those belonging to the Panpulmonata. This suggests that for many gastropods ethanol may not be metabolically relevant.

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.

京都大学
0048
新制・課程博士
博士(農学)
甲第22502号
農博第2406号
新制||農||1077(附属図書館)
学位論文||R2||N5282(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 梅澤 俊明, 教授 矢﨑 一史, 教授 渡邊 隆司
学位規則第4条第1項該当
Doctor of Agricultural Science
Kyoto University
DGAM

Cost effective production of biofuel from plant biomass (second generation biofuels) is currently a key challenge. To achieve this, accessibility of plant cell wall polysaccharides to chemical, enzymatic and microbial digestion could be improved by altering lignin structure and composition or by reducing lignin content, as lignin is one cell wall component that has already been shown to contribute to biomass recalcitrance. Therefore, this thesis reports the genetic manipulation of lignin biosynthesis through down-regulation of cinnamyl alcohol dehydrogenase (CAD) genes in barley (Hordeum vulgare L.). Barley has been chosen as the target plant for lignin manipulation for a few reasons: it is a major cereal crop that produces large amounts of lignocellulosic plant biomass that can potentially be used as animal feed or to produce second generation biofuels and also because it is a model grass for other bioenergy crops. CAD, as the final enzyme in the lignin pathway, is a perfect target for lignin manipulation. Characterised CAD mutants and transgenics have shown that down-regulation of CAD improves digestibility and does not influence plant growth and fertility. Due to the difficulty and complexity of transformation of monocot species, there are only a few reports describing down-regulation of CAD in monocots, and none in barley. Here, in this thesis, lignin was altered by down-regulating CAD genes using an RNAi construct with part of the HvCAD2 gene, the gene which has the highest expression level of all CAD genes. Transgenic barley plants showed reduced enzyme activity in the T0 generation (31% compared to EV plants) and enzyme activity was reduced even more in the T1 (to 3%) and T2 (to 2%) generations. The HvCAD2 RNAi barley lines had similar or slightly reduced Klason total lignin contents relative to control plants, but lignin structure and composition were altered. The RNAi plants had lower thioacidolysis yields, S/G ratio was reduced (1.59 in the empty vector controls versus 0.96–1.21 in the transgenic barley plants), the relative frequency of S units was reduced by 11–20%, the proportion of G units was increased by 17–32%, there was increased sinapaldehyde accumulation in lignin and ferulic acid abundance was reduced relative to control plants. Analysed transgenic barley plants had an orange stem phenotype. Growth season and conditions hugely affected the intensity of the phenotype. Because lignin plays a major role in culm strength and pathogen resistance, the influence of down-regulation of CAD on these features was characterised. The changed physicochemical nature of cell walls in HvCAD2 RNAi lines does not decrease the strength of the straw and does not decrease the resistance to the biotrophic Blumeria graminis and to the hemibiotrophic Rhynchosporium commune pathogens. The modified cell walls in the HvCAD2 RNAi lines had moderately improved sugar release for biofuel production. This study proves that it is possible to down-regulate CAD in cereal crops in order to change lignin structure and composition in plants without a negative impact on plant growth, fertility or pathogen resistance.

La récente définition de Brachypodium distachyon comme modèle des graminées en fait un organisme de choix pour l'étude de leur paroi cellulaire, en particulier dans le cadre de leur utilisation comme matière première renouvelable pour le bioéthanol de seconde génération. Les lignines, dont les trois unités (H, G et S) proviennent de la polymérisation des monolignols, sont associées aux acides hydroxycinnamiques dans la paroi des céréales et représentent l'obstacle majeur à l'exploitation industrielle de la biomasse lignocellulosique. L'acquisition de connaissances sur les mécanismes dirigeant leur mise en place et leur organisation permettrait d'identifier des facteurs modulant les rendements de production qui y sont associés. Quatre familles de gènes ont été étudiées et l'implication dans la voie de biosynthèse des monolignols de trois gènes a été montrée : BdF5H2 possède une activité férulate-5-hydroxylase permettant la synthèse des précurseurs des unités S des lignines, BdCOMT3 est l'isoforme principale des acide cafféique O-Méthyltransférases et sa perte partielle de fonction cause une diminution de la quantité de lignine, la modification du rapport S/G et une baisse de quantité d'acide p-coumarique dans deux lignées mutantes indépendantes. Enfin, BdCAD1 est l'isoforme principale des alcools cinnamylique déshydrogénases : sa perte de fonction dans deux lignées indépendantes cause la diminution de la quantité globale de lignine et d'acide p-coumarique, une baisse du rapport S/G ainsi que l'accumulation de sinapaldéhyde. Par ailleurs ces deux lignées présentent des rendements de saccharification augmentés de plus d'un quart par rapport au sauvage.

碩士
國立中興大學
基因體暨生物資訊學研究所
99
A cDNA encoding a cinnamyl alcohol dehydrogenase (CAD) gene with 1155 mucleotides was isolated from Chlorella sorokiniana T89. Recombinant CAD protein was expressed in Escherichia coli as a C-terminal histidin-tagged fusion protein and purified by DEAE-Sephacel ion exchange chromatography and TALON affinity chromatography. By these steps, the CAD was purified 1250 fold over the crude protein, and the total recovery was 3%. The optimal pH for the recombinant CAD protein was 6.5, and the optimal temperature was 33 °C. 1 mM Co2+, 1 mM Ni2+ and 1 mM Cu2+ significantly inhibited the activity of the enzyme. 1% 2-Mercaptoethanol, 1 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfateand 2 M urea significantly inhibited the activity of th enzyme. The recombinant CAD protein sequence analysis and prediction three-dimensional structure. There were some features of recombinant CAD protein. First, it may be combination of two zinc ions which related to the structural zinc ion and catalytic zinc ion. Second, the [GX(X)GXXG] motif may be the NADP+-binding domain. Finally, two pairs of disulfide bonds may form between C131 and C134, C75 and C191, respectively.

Research Doctorate - Doctor of Philosophy (PhD)
Energy is the lifeblood of human society with over 82% of demand derived from the combustion of fossil fuels. However, fossil fuels are non-renewable sources that will be depleted within the next 100 years. Furthermore, combustion of fossil fuels emits large amounts of greenhouse gases, and this is a primary contributor to global warming. Finding renewable energy sources is a major target to reduce modern society’s current dependence on fossil fuel and to secure industrially and ecologically sustainable development. Lignocellulosic biomass derived from C₄ crops and energy feedstocks including maize, sugarcane, sorghum, switchgrass and Miscanthus x giganteus offers a great potential alternative energy source. However, production of lignocellulose-derived bioethanol is a time- and energy-consuming procedure impeded by recalcitrant properties of lignified plant cell walls when subjected to enzymatic hydrolysis. Identification of more efficient cell wall hydrolysing enzymes and/or the genetic modification of lignocellulosic biomass composition and structure are necessary to increase bioethanol production efficiency and reduce production cost. Setaria has been identified as a promising model system for genetic studies of C₄ monocot growth and development. Setaria viridis (green foxtail) and its domesticated relative Setaria italica (foxtail millet) are phylogenetically related to many economically important C₄ crops in the Panicoideae subfamily, a subfamily that also includes maize, sorghum, sugarcane and potential bioenergy crops such as Miscanthus x giganteus and switchgrass. Setaria viridis (Accession 10 (A10)) possesses many desirable traits of a model plant, including a small stature (10-15 cm in height), short life cycle (6-9 weeks from seed to seed), prolific seed production (13,000 seeds per plant), and a small sized, sequenced and reasonably well annotated genome. The potential for using S. viridis as a C₄ genetic model has led to an increased demand for the development of efficient and reliable molecular tools to enable the investigation of complex gene networks to assign biological function to such genetic pathways. Therefore, the main aims of this thesis was to (1) establish an Agrobacterium tumefaciens-mediated transformation system for the genetic manipulation of S. viridis, and (2) identify stably expressed reference genes for quantitative real time PCR (RT-qPCR) analysis to confirm the continued use of S. viridis as an attractive genetic model to molecularly characterise the C₄ grasses. The established system would facilitate the identification and evaluation of potential target genes for manipulation of lignin composition and cell wall anatomy in S. viridis transformants. Firstly, an Agrobacterium-mediated transformation system for S. viridis was established. This was achieved via evaluation of several key factors, including the; (i) use of mature seeds as the plant material for callus induction; (ii) age of the seed used for callus induction; (iii) composition of the callus induction media; (iv) co-cultivation approach, and; (v) concentration of selective agent used at the plant regeneration stage. Together, the Agrobacterium-mediated transformation system established here significantly enhanced the frequency of callus induction from mature S. viridis seeds (~76%) and the overall efficiency of transformation (~6%). Further, this system only required a relatively short period of approximately 4 months to generate S. viridis transformant lines from mature seed-derived callus. Secondly, the potential S. viridis candidate genes, including 7 (out of 11 genes identified) CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes and 7 (out of 18 genes identified) LACCASE (LAC) genes, were identified for further molecular assessment of their respective, putative roles in lignin biosynthesis. In addition, a putative regulator, the microRNA397 (miR397) small RNA (sRNA) with the potential to regulate the S. viridis LAC gene expression at the posttranscriptional level was also identified. To facilitate expression profiling of the selected CAD and LAC genes, 3 reference gene candidates, including ADENYLYLSULFATE REDUCTASE 6 (ASPR6), DUAL SPECIFICITY PROTEIN (DUSP) and SERINE/THREONINE PROTEIN PHOSPHATASE 2A (PP2A), were identified by the 2 most frequently used algorithms, NormFinder and geNorm, as the optimal reference gene set for gene expression normalisation across S. viridis tissues. Gene expression analyses identified CAD2 and 7 LAC genes as displaying the most functionally relevant expression profiles, that is; high expression in tissues undergoing lignification. In addition, the miR397 sRNA was putatively demonstrated to be a potential posttranscriptional regulator of LAC gene expression. Thirdly, one of the initial aims of this thesis was to produce transformed S. viridis lines with modulated expression levels of the identified target gene(s), and to subsequently study the effect of these molecular manipulations on lignin biosynthesis. However, my research suffered from several major incidents and as a consequence of these combined hurdles, my original research plan was significantly delayed. Therefore, an alternative strategy was employed and involved the utilisation of Arabidopsis thaliana, a genetic model plant species with exceptionally well established transformation protocols. Based on the interesting regulatory role that the miR397 sRNA appeared to direct control of LAC gene expression at the posttranscriptional level in S. viridis, this sRNA was constitutively and ubiquitously overexpressed in Arabidopsis tissues to examine the effects of modified LAC gene expression on lignin biosynthesis. The Arabidopsis system clearly showed that the miR397 sRNA is indeed a key posttranscriptional regulator of the expression of LAC genes central to lignin biosynthesis. In summary, the knowledge derived from the Arabidopsis system can be readily transferred to the S. viridis system in the future to genetically engineer S. viridis plants with altered lignin content. In conclusion, the results presented in this thesis have further confirmed the suitability of S. viridis for use as a model system to genetically characterise the C₄ monocot grasses. Furthermore, the Arabidopsis work has demonstrated that manipulation of the miR397 abundance allowed for the altered expression regulation of LAC gene expression, and in turn, modification of the lignin content of the Arabidopsis inflorescence stem. Identification of the same miR397/LAC gene expression module in S. viridis allows for a similar Agrobacterium-mediated approach to be utilised in this species to generate S. viridis plant lines with modified lignin contents desirable for different industrial purposes, namely the production of biofuel.

Lignin is a significant recalcitrant 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. We identified candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon and developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and 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-silenced and wildtype 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 decrease in total lignin content, a significant reduction of guaiacyl lignin, and a modest reduction of syringyl lignin. Ethanol yield by microbial fermentation was enhanced in both BdCAD1- and BdCOMT4-downregulated plants. These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater biomass yield and bioconversion efficiency.