Dataset Information

Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis.

ABSTRACT: Of 17 genes annotated in the Arabidopsis genome database as cinnamyl alcohol dehydrogenase (CAD) homologues, an in silico analysis revealed that 8 genes were misannotated. Of the remaining nine, six were catalytically competent for NADPH-dependent reduction of p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes, whereas three displayed very low activity and only at very high substrate concentrations. Of the nine putative CADs, two (AtCAD5 and AtCAD4) had the highest activity and homology (approximately 83% similarity) relative to bona fide CADs from other species. AtCAD5 used all five substrates effectively, whereas AtCAD4 (of lower overall catalytic capacity) poorly used sinapyl aldehyde; the corresponding 270-fold decrease in k(enz) resulted from higher K(m) and lower k(cat) values, respectively. No CAD homologue displayed a specific requirement for sinapyl aldehyde, which was in direct contrast with unfounded claims for a so-called sinapyl alcohol dehydrogenase in angiosperms. AtCAD2, 3, as well as AtCAD7 and 8 (highest homology to sinapyl alcohol dehydrogenase) were catalytically less active overall by at least an order of magnitude, due to increased K(m) and lower k(cat) values. Accordingly, alternative and/or bifunctional metabolic roles of these proteins in plant defense cannot be ruled out. Comprehensive analyses of lignified tissues of various Arabidopsis knockout mutants (for AtCAD5, 6, and 9) at different stages of growth/development indicated the presence of functionally redundant CAD metabolic networks. Moreover, disruption of AtCAD5 expression had only a small effect on either overall lignin amounts deposited, or on syringyl-guaiacyl compositions, despite being the most catalytically active form in vitro.

SUBMITTER: Kim SJ

PROVIDER: S-EPMC341741 | biostudies-literature | 2004 Feb

REPOSITORIES: biostudies-literature

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Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis.

Kim Sung-Jin SJ Kim Mi-Ran MR Bedgar Diana L DL Moinuddin Syed G A SG Cardenas Claudia L CL Davin Laurence B LB Kang ChulHee C Lewis Norman G NG

Proceedings of the National Academy of Sciences of the United States of America 20040126 6

Of 17 genes annotated in the Arabidopsis genome database as cinnamyl alcohol dehydrogenase (CAD) homologues, an in silico analysis revealed that 8 genes were misannotated. Of the remaining nine, six were catalytically competent for NADPH-dependent reduction of p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes, whereas three displayed very low activity and only at very high substrate concentrations. Of the nine putative CADs, two (AtCAD5 and AtCAD4) had the highest activit ...[more]

PMID: 14745009

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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: Lignin is a phenolic heteropolymer in secondary cell walls that plays a major role in the development of plants and their defense against pathogens. The biosynthesis of monolignols, which represent the main component of lignin involves many enzymes. The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. The CAD gene family has been studied in Arabidopsis thaliana, Oryza sativa and partially in Populus. This is the first comprehensive study on the CAD gene family in woody plants including genome organization, gene structure, phylogeny across land plant lineages, and expression profiling in Populus. RESULTS: The phylogenetic analyses showed that CAD genes fall into three main classes (clades), one of which is represented by CAD sequences from gymnosperms and angiosperms. The other two clades are represented by sequences only from angiosperms. All Populus CAD genes, except PoptrCAD 4 are distributed in Class II and Class III. CAD genes associated with xylem development (PoptrCAD 4 and PoptrCAD 10) belong to Class I and Class II. Most of the CAD genes are physically distributed on duplicated blocks and are still in conserved locations on the homeologous duplicated blocks. Promoter analysis of CAD genes revealed several motifs involved in gene expression modulation under various biological and physiological processes. The CAD genes showed different expression patterns in poplar with only two genes preferentially expressed in xylem tissues during lignin biosynthesis. CONCLUSION: The phylogeny of CAD genes suggests that the radiation of this gene family may have occurred in the early ancestry of angiosperms. Gene distribution on the chromosomes of Populus showed that both large scale and tandem duplications contributed significantly to the CAD gene family expansion. The duplication of several CAD genes seems to be associated with a genome duplication event that happened in the ancestor of Salicaceae. Phylogenetic analyses associated with expression profiling and results from previous studies suggest that CAD genes involved in wood development belong to Class I and Class II. The other CAD genes from Class II and Class III may function in plant tissues under biotic stresses. The conservation of most duplicated CAD genes, the differential distribution of motifs in their promoter regions, and the divergence of their expression profiles in various tissues of Populus plants indicate that genes in the CAD family have evolved tissue-specialized expression profiles and may have divergent functions.

Project description:BACKGROUND: In recent decades cultivation of flax and its application have dramatically decreased. One of the reasons for this is unpredictable quality and properties of flax fibre, because they depend on environmental factors, retting duration and growing conditions. These factors have contribution to the fibre composition, which consists of cellulose, hemicelluloses, lignin and pectin. By far, it is largely established that in flax, lignin reduces an accessibility of enzymes either to pectin, hemicelluloses or cellulose (during retting or in biofuel synthesis and paper production).Therefore, in this study we evaluated composition and properties of flax fibre from plants with silenced CAD (cinnamyl alcohol dehydrogenase) gene, which is key in the lignin biosynthesis. There is evidence that CAD is a useful tool to improve lignin digestibility and/or to lower the lignin levels in plants. RESULTS: Two studied lines responded differentially to the introduced modification due to the efficiency of the CAD silencing. Phylogenetic analysis revealed that flax CAD belongs to the "bona-fide" CAD family. CAD down-regulation had an effect in the reduced lignin amount in the flax fibre cell wall and as FT-IR results suggests, disturbed lignin composition and structure. Moreover introduced modification activated a compensatory mechanism which was manifested in the accumulation of cellulose and/or pectin. These changes had putative correlation with observed improved fiber's tensile strength. Moreover, CAD down-regulation did not disturb at all or has only slight effect on flax plants' development in vivo, however, the resistance against flax major pathogen Fusarium oxysporum decreased slightly. The modification positively affected fibre possessing; it resulted in more uniform retting. CONCLUSION: The major finding of our paper is that the modification targeted directly to block lignin synthesis caused not only reduced lignin level in fibre, but also affected amount and organization of cellulose and pectin. However, to conclude that all observed changes are trustworthy and correlated exclusively to CAD repression, further analysis of the modified plants genome is necessary. Secondly, this is one of the first studies on the crop from the low-lignin plants from the field trail which demonstrates that such plants could be successfully cultivated in a field.

Dataset Information

Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis.

Publications

Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis.

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