Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula.
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ABSTRACT:
SUBMITTER: Zhao Q
PROVIDER: S-EPMC3746925 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step in the biosynthesis of monolignols. In the present study, a cDNA encoding a CAD was isolated from wheat, designated as TaCAD1. A genome-wide data mining in the wheat EST database revealed another 10 CAD-like homologues, namely TaCAD2 to TaCAD11. A phylogenetic analysis showed that TaCAD1 belonged to the bona fide CAD group involved in lignin synthesis. Two other putative CADs from the wheat genome (TaCAD2 and TaCAD4) also belonged to this group and were very close to TaCAD1, but lacked C-terminal domain, suggesting that they are pseudogenes. DNA gel blot analysis for the wheat genome showed two to three copies of CAD related to TaCAD1, but RNA gel blot analysis revealed only single band for TaCAD1, which was highly expressed in stem, with quite low expression in leaf and undetectable expression in root. The predicted three-dimension structure of TaCAD1 resembled that of AtCAD5, but two amino acid substitutions were identified in the substrate binding region. Recombinant TaCAD1 protein used coniferyl aldehyde as the most favoured substrate, also showed high efficiencies toward sinapyl and p-coumaryl aldehydes. TaCAD1 was an enzyme being pH-dependent and temperature-sensitive, and showing a typical random catalysing mechanism. At the milky stage of wheat, TaCAD1 mRNA abundance, protein level and enzyme activity in stem tissues were higher in a lodging-resistant cultivar (H4546) than in lodging-sensitive cultivar (C6001). These properties were correlated to the lignin contents and lodging indices of the two cultivars. These data suggest that TaCAD1 is the predominant CAD in wheat stem for lignin biosynthesis and is critical for lodging resistance.
| S-EPMC2882267 | biostudies-literature
Project description:Artemisia annua is the only medicinal crop that produces artemisinin for malarial treatment. Herein, we describe the cloning of a cinnamyl alcohol dehydrogenase (AaCAD) from an inbred self-pollinating (SP) A. annua cultivar and its effects on lignin and artemisinin production. A recombinant AaCAD was purified via heterogeneous expression. Enzyme assays showed that the recombinant AaCAD converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates involved in the biosynthesis of lignin. Km, Vmax, and Vmax/Km values were calculated for all three substrates. To characterize its function in planta, AaCAD was overexpressed in SP plants. Quantification using acetyl bromide (AcBr) showed significantly higher lignin contents in transgenics compared with wild-type (WT) plants. Moreover, GC-MS-based profiling revealed a significant increase in coumarin contents in transgenic plants. By contrast, HPLC-MS analysis showed significantly reduced artemisinin contents in transgenics compared with WT plants. Furthermore, GC-MS analysis revealed a decrease in the contents of arteannuin B and six other sesquiterpenes in transgenic plants. Confocal microscopy analysis showed the cytosolic localization of AaCAD. These data demonstrate that AaCAD plays a dual pathway function in the cytosol, in which it positively enhances lignin formation but negatively controls artemisinin formation. Based on these data, crosstalk between these two pathways mediated by AaCAD catalysis is discussed to understand the metabolic control of artemisinin biosynthesis in plants for high production.
| S-EPMC6018409 | biostudies-literature
Project description:Cinnamoyl CoA reductases (CCR) convert hydroxycinnamoyl CoA esters to their corresponding cinnamyl aldehydes in monolignol biosynthesis. We identified two CCR genes in the model legume Medicago truncatula. CCR1 exhibits preference for feruloyl CoA, but CCR2 prefers caffeoyl and 4-coumaroyl CoAs, exhibits sigmoidal kinetics with these substrates, and is substrate-inhibited by feruloyl and sinapoyl CoAs. M. truncatula lines harboring transposon insertions in CCR1 exhibit drastically reduced growth and lignin content, whereas CCR2 knockouts grow normally with moderate reduction in lignin levels. CCR1 fully and CCR2 partially complement the irregular xylem gene 4 CCR mutation of Arabidopsis. The expression of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) is up-regulated in CCR2 knockout lines; conversely, knockout of CCoAOMT up-regulates CCR2. These observations suggest that CCR2 is involved in a route to monolignols in Medicago whereby coniferaldehyde is formed via caffeyl aldehyde which then is 3-O-methylated by caffeic acid O-methyltransferase.
| S-EPMC2955080 | biostudies-other
Project description:l-Tyrosine (Tyr) is an aromatic amino acid synthesized de novo in plants and microbes downstream of the shikimate pathway. In plants, Tyr and a Tyr pathway intermediate, 4-hydroxyphenylpyruvate (HPP), are precursors to numerous specialized metabolites, which are crucial for plant and human health. Tyr is synthesized in the plastids by a TyrA family enzyme, arogenate dehydrogenase (ADH/TyrAa), which is feedback inhibited by Tyr. Additionally, many legumes possess prephenate dehydrogenases (PDH/TyrAp), which are insensitive to Tyr and localized to the cytosol. Yet the role of PDH enzymes in legumes is currently unknown. This study isolated and characterized Tnt1-transposon mutants of MtPDH1 (pdh1) in Medicago truncatula to investigate PDH function. The pdh1 mutants lacked PDH transcript and PDH activity, and displayed little aberrant morphological phenotypes under standard growth conditions, providing genetic evidence that MtPDH1 is responsible for the PDH activity detected in M. truncatula. Though plant PDH enzymes and activity have been specifically found in legumes, nodule number and nitrogenase activity of pdh1 mutants were not significantly reduced compared with wild-type (Wt) during symbiosis with nitrogen-fixing bacteria. Although Tyr levels were not significantly different between Wt and mutants under standard conditions, when carbon flux was increased by shikimate precursor feeding, mutants accumulated significantly less Tyr than Wt. These data suggest that MtPDH1 is involved in Tyr biosynthesis when the shikimate pathway is stimulated and possibly linked to unidentified legume-specific specialized metabolism.
| S-EPMC7196213 | biostudies-literature
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.
| S-EPMC6222663 | biostudies-literature
Project description: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.
| S-EPMC341741 | biostudies-literature
Project description:The experimental results on detection and identification of intermediate radicals and molecular products from gas-phase pyrolysis of cinnamyl alcohol (CnA), the simplest non-phenolic lignin model compound, over the temperature range of 400-800 °C are reported. The low temperature matrix isolation - electron paramagnetic resonance (LTMI-EPR) experiments along with the theoretical calculations, provided evidences on the generation of the intermediate carbon and oxygen centered as well as oxygen-linked, conjugated radicals. A mechanistic analysis is performed based on density functional theory to explain formation of the major products from CnA pyrolysis; cinnamaldehyde, indene, styrene, benzaldehyde, 1-propynyl benzene, and 2-propenyl benzene. The evaluated bond dissociation patterns and unimolecular decomposition pathways involve dehydrogenation, dehydration, 1,3-sigmatropic H-migration, 1,2-hydrogen shift, C-O and C-C bond cleavage processes.
| S-EPMC5363761 | biostudies-literature
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.
| S-EPMC3945063 | biostudies-literature
Project description:Lignin is a complex phenylpropanoid polymer deposited in plant cell walls. Lignin has long been recognized as an important limiting factor for the polysaccharide-oriented biomass utilizations. To mitigate lignin-associated biomass recalcitrance, numerous mutants and transgenic plants that produce lignocellulose with reduced lignin contents and/or lignins with altered chemical structures have been produced and characterised. However, it is not fully understood how altered lignin chemistry affects the supramolecular structure of lignocellulose, and consequently, its utilization properties. Herein, we conducted comprehensive chemical and supramolecular structural analyses of lignocellulose produced by a rice cad2 mutant deficient in CINNAMYL ALCOHOL DEHYDROGENASE (CAD), which encodes a key enzyme in lignin biosynthesis. By using a solution-state two-dimensional NMR approach and complementary chemical methods, we elucidated the structural details of the altered lignins enriched with unusual hydroxycinnamaldehyde-derived substructures produced by the cad2 mutant. In parallel, polysaccharide assembly and the molecular mobility of lignocellulose were investigated by solid-state 13C MAS NMR, nuclear magnetic relaxation, X-ray diffraction, and Simon's staining analyses. Possible links between CAD-associated lignin modifications (in terms of total content and chemical structures) and changes to the lignocellulose supramolecular structure are discussed in the context of the improved biomass saccharification efficiency of the cad2 rice mutant.
| S-EPMC6868246 | biostudies-literature
Project description:Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step of the monolignol biosynthesis, the conversion of cinnamyl aldehydes to alcohols, using NADPH as a cofactor. Seven members of the CAD gene family were identified in the genome of Brachypodium distachyon and five of these were isolated and cloned from genomic DNA. Semi-quantitative reverse-transcription PCR revealed differential expression of the cloned genes, with BdCAD5 being expressed in all tissues and highest in root and stem while BdCAD3 was only expressed in stem and spikes. A phylogenetic analysis of CAD-like proteins placed BdCAD5 on the same branch as bona fide CAD proteins from maize (ZmCAD2), rice (OsCAD2), sorghum (SbCAD2) and Arabidopsis (AtCAD4, 5). The predicted three-dimensional structures of both BdCAD3 and BdCAD5 resemble that of AtCAD5. However, the amino-acid residues in the substrate-binding domains of BdCAD3 and BdCAD5 are distributed symmetrically and BdCAD3 is similar to that of poplar sinapyl alcohol dehydrogenase (PotSAD). BdCAD3 and BdCAD5 expressed and purified from Escherichia coli both showed a temperature optimum of about 50 °C and molar weight of 49 kDa. The optimal pH for the reduction of coniferyl aldehyde were pH 5.2 and 6.2 and the pH for the oxidation of coniferyl alcohol were pH 8 and 9.5, for BdCAD3 and BdCAD5 respectively. Kinetic parameters for conversion of coniferyl aldehyde and coniferyl alcohol showed that BdCAD5 was clearly the most efficient enzyme of the two. These data suggest that BdCAD5 is the main CAD enzyme for lignin biosynthesis and that BdCAD3 has a different role in Brachypodium. All CAD enzymes are cytosolic except for BdCAD4, which has a putative chloroplast signal peptide adding to the diversity of CAD functions.
| S-EPMC3481213 | biostudies-literature