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Jacqueline Grima-pettenati - One of the best experts on this subject based on the ideXlab platform.
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Genetic transformation of Eucalyptus globulus using the vascular-specific EgCCR as an alternative to the constitutive CaMV35S promoter
Plant Cell Tissue and Organ Culture (PCTOC), 2014Co-Authors: Francisco De La Torre, Jacqueline Grima-pettenati, Ruth Rodríguez, Gago Jorge, Beatriz Villar, Rosa Álvarez-otero, Pedro Pablo GallegoAbstract:Strong constitutive promoters, such as CaMV35S, are widely used for plant transformation, but undesirable phenotypic changes have been reported when used to drive biotic stress tolerance and/or for modifying lignin content. The promoter of the eucalyptus cinnamoyl CoA Reductase (CCR), a key enzyme of the lignin biosynthetic pathway, was shown to be preferentially expressed in vascular tissues both in herbaceous and woody transgenic plants but not eucalyptus. In this work, we transformed Eucalyptus globulus with the EgCCR promoter governing both β-glucuronidase (GUS) and GFP activity patterns. No statistical differences were found between the survival rate and percentage of GUS positive shoots between eucalyptus transformed with either the constitutive CaMV35Sor with the EgCCR promoter. The EgCCR transformed plantlets exhibited high GUS expression levels associated with the vascular tissues opening the possibility of targeting vascular-associated traits such as lignin content or vascular pathogen resistance in adult elite plants of eucalyptus while avoiding the undesirable pleiotropic effects caused by strong constitutive promoters.
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Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA Reductase (CCR).
Transgenic research, 2007Co-Authors: Johan Wadenbäck, Jacqueline Grima-pettenati, Sara Von Arnold, Ulrika Egertsdotter, Michael H. Walter, Deborah Goffner, Göran Gellerstedt, Terry Gullion, David E. ClaphamAbstract:An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA Reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90–99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway.
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Kinetic and inhibition studies of Cinnamoyl-CoA Reductase 1 from Arabidopsis thaliana
Plant physiology and biochemistry : PPB, 2005Co-Authors: Michel Baltas, C. Lapeyre, F. Bedos-belval, M. Maturano, P. Saint-aguet, L. Roussel, H. Duran, Jacqueline Grima-pettenatiAbstract:Cinnamoyl coenzyme A Reductase (CCR, EC 1.2.1.44), one of the key enzymes in the biosynthesis of lignin monomers, catalyzes the NADPH-dependent reduction of Cinnamoyl-CoA esters to their corresponding cinnamaldehydes. AtCCR1, one of the two distinct isoforms isolated from Arabidopsis thaliana, was shown to be involved in lignin biosynthesis during development. Here, we report on the purification of the recombinant AtCCR1 protein expressed in Escherichia coli and the subsequent determination of its kinetic properties (K(m) and k(cat)/K(m) values) towards its main substrates i.e. feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA esters. In addition, the potential inhibitory effect of five substrate-like analogs possessing an N-acetylcysteamine thioester group was tested on CCR activity using either feruloyl-CoA or sinapoyl-CoA as substrates. The K(i) values were in the range of 4.4-502 microM and the type of inhibition was found to be either uncompetitive or noncompetitive. Interestingly, for compounds 3 and 5, the type of inhibition was found to be different depending on the substrate used to monitor the enzyme activity. The best inhibitors were those possessing the feruloyl (compound 3) and sinapoyl (compound 5) aromatic moiety (4.1 and 7.1 microM) while the enzyme activity was monitored using the corresponding substrates.
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Two Cinnamoyl-CoA Reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria.
Phytochemistry, 2001Co-Authors: Virginie Lauvergeat, Christophe Lacomme, Eric Lacombe, Eric Lasserre, Dominique Roby, Jacqueline Grima-pettenatiAbstract:Abstract Cinnamoyl-CoA Reductase (CCR ; EC 1.2.1.44) catalyses the conversion of Cinnamoyl-CoAs into their corresponding cinnamaldehydes, i.e. the first step of the phenylpropanoid pathway specifically dedicated to the monolignol biosynthetic branch. In previous work, we described the isolation and characterisation of the first cDNA encoding CCR in Eucalyptus (Lacombe, E., Hawkins, S., Van Dorsselaere, J., Piquemal, J., Goffner, D., Poeydomenge, O., Boudet, A.M., Grima-Pettenati, J., 1997. Cinnamoyl CoA Reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant Journal 11, 429–441) and shown the role of this enzyme in controlling the carbon flux into lignins (Piquemal, J., Lapierre, C., Myton, K., O’Connell, A., Schuch, W., Grima-Pettenati, J., Boudet, A.M., 1998. Down-regulation of Cinnamoyl-CoA Reductase induces significant changes of lignin profiles in transgenic tobacco plants. Plant Journal 13, 71–83). Here, we report the characterisation of two functionally and structurally distinct cDNA clones, AtCCR1 and AtCCR2 (81.6% protein sequence identity) in Arabidopsis thaliana. The two recombinant proteins expressed in Escherichia coli are able to use the three Cinnamoyl-CoAs tested but with different levels of efficiency. AtCCR1 is five times more efficient with feruloyl-CoA and sinapoyl-CoA than AtCCR2. In addition, the two genes are differentially expressed during development and in response to infection. AtCCR1 is preferentially expressed in tissues undergoing lignification. In contrast, AtCCR2, which is poorly expressed during development, is strongly and transiently induced during the incompatible interaction with Xanthomonas campestris pv. campestris leading to a hypersensitive response. Altogether, these data suggest that AtCCR1 is involved in constitutive lignification whereas AtCCR2 is involved in the biosynthesis of phenolics whose accumulation may lead to resistance.
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Mapping candidate genes in Eucalyptus with emphasis on lignification genes
Molecular Breeding, 2000Co-Authors: Jean-marc Gion, Philippe Rech, Daniel Verhaegen, Jacqueline Grima-pettenati, Christophe PlomionAbstract:We used the single-strand conformation polymorphism (SSCP) technique to map eight genes on Eucalyptus urophylla and Eucalyptus grandis linkage maps. These included four genes involved in the common phenylpropanoid pathway (caffeic acid 3- 0 -methyltransferase, caffeoyl CoA 3- O -methyltransferase, 4-coumarate CoA ligase and phenylalanine ammonia-lyase), two genes involved in the `lignin specific' pathway (cinnamoyl CoA Reductase and cinnamyl alcohol dehydrogenase), and two symbiosis regulated genes (EgHypar and EgTubA1). A novel source of variation which affects the SSCP pattern, i.e. the presence or absence of electrophoresis buffer upon loading the samples into the polyacrylamide gel, was found. The placement of these genes on the Eucalyptus maps was carried out using an interspecific hybrid mapping population. This will further facilitate the identification or exclusion of `positional' candidate genes for characterizing quantitative trait loci (QTL) for wood quality and vegetative propagation related traits.
Joseph P Noel - One of the best experts on this subject based on the ideXlab platform.
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structural studies of cinnamoyl coa Reductase and cinnamyl alcohol dehydrogenase key enzymes of monolignol biosynthesis
The Plant Cell, 2014Co-Authors: Rui Zhou, Gordo V Louie, Joelle K Muhlema, Eri K Omati, Marianne E Owma, Natalia Dudareva, Richard A Dixo, Joseph P NoelAbstract:The enzymes Cinnamoyl-CoA Reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/Reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates.
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Functional analyses of caffeic acid O-methyltransferase and Cinnamoyl-CoA-Reductase genes from perennial ryegrass (Lolium perenne).
The Plant cell, 2010Co-Authors: Simone Rochfort, Zhiqian Liu, Yidong Ran, Megan Griffith, Pieter Badenhorst, Gordon V. Louie, Marianne E. Bowman, Kevin F. Smith, Joseph P NoelAbstract:Cinnamoyl CoA-Reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.
Bashir M. Khan - One of the best experts on this subject based on the ideXlab platform.
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Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala
Physiology and Molecular Biology of Plants, 2015Co-Authors: Sameer Srivastava, Rishi K. Vishwakarma, Yasir Ali Arafat, Sushim K. Gupta, Bashir M. KhanAbstract:Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.
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Steady State Fluorescence Studies of Wild Type Recombinant Cinnamoyl CoA Reductase (Ll-CCRH1) and its Active Site Mutants
Journal of Fluorescence, 2014Co-Authors: Prashant Sonawane, Somesh Singh, Rishi Kishore Vishwakarma, Sushama Gaikwad, Bashir M. KhanAbstract:Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA Reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA ( K _ a = 3.72 × 10^5 M^−1) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy. Quenching of the fluorescence by acrylamide for wild type and active site mutants was collisional with almost 100 % of the tryptophan fluorescence accessible under native condition and remained same after denaturation of protein with 6 M GdnHCl. In wild type Ll-CCRH1, the extent of quenching achieved with iodide ( f _a = 1.0) was significantly higher than cesium ions ( f _a = 0.33) suggesting more density of positive charge around surface of trp conformers under native conditions. Denaturation of wild type protein with 6 M GdnHCl led to significant increase in the quenching with cesium ( f _a = 0.54), whereas quenching with iodide ion was decreased ( f _a = 0.78), indicating reorientation of charge density around trp from positive to negative and heterogeneity in trp environment. The Stern-Volmer plots for wild type and mutants Ll-CCRH1 under native and denatured conditions, with cesium ion yielded biphasic quenching profiles. The extent of quenching for cesium and iodide ions under native and denatured conditions observed in active site mutants was significantly different from wild type Ll-CCRH1 under the same conditions. Thus, single substitution type mutations of active site residues showed heterogeneity in tryptophan microenvironment and differential degree of conformation of protein under native or denatured conditions.
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Conformational transitions of cinnamoyl CoA Reductase 1 from Leucaena leucocephala.
International journal of biological macromolecules, 2013Co-Authors: Prashant Sonawane, Bashir M. Khan, Sushama M. GaikwadAbstract:Abstract Conformational transitions of cinnamoyl CoA Reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (Ll-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 °C and starts forming aggregates at 55 °C. Ll-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of L1-CCRH1 at pH 2.0 was stable up to 90 °C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCl), it got transformed into different structure which was stable in the vicinity of 2 M GdnHCl (as compared to drastic loss of native structure in 2 M GdnHCl) as seen in far UV-CD spectra. The structural transition of Ll-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein.
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Probing the active site of cinnamoyl CoA Reductase 1 (Ll-CCRH1) from Leucaena leucocephala.
International journal of biological macromolecules, 2013Co-Authors: Prashant Sonawane, Krunal Patel, Rishi K. Vishwakarma, Somesh Singh, Sushama M. Gaikwad, Sameer Srivastava, Bashir M. KhanAbstract:Lack of three dimensional crystal structure of cinnamoyl CoA Reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1.
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Biochemical characterization of recombinant cinnamoyl CoA Reductase 1 (Ll-CCRH1) from Leucaena leucocephala.
International journal of biological macromolecules, 2013Co-Authors: Prashant Sonawane, Rishi K. Vishwakarma, Bashir M. KhanAbstract:Abstract Recombinant cinnamoyl CoA Reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25 °C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×106 M−1 s−1), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20–50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm3 with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme.
Zhongfeng Zhang - One of the best experts on this subject based on the ideXlab platform.
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Dynamics of antioxidant activities, metabolites, phenolic acids, flavonoids, and phenolic biosynthetic genes in germinating Chinese wild rice (Zizania latifolia).
Food chemistry, 2020Co-Authors: Cheng Chu, Zhongfeng Zhang, Hongbo Zhang, John Shi, Xiao-long Yuan, Xinmin Liu, Yanhua Liu, Ning YanAbstract:Abstract In this study, the antioxidant activity of germinating Chinese wild rice was found to decline initially, after which it increased. The largest difference in antioxidant activity was observed between the 36-h (G36) and the 120-h germination (G120) stage. We further assessed the dynamic changes in metabolites, phenolic acids, flavonoids, and phenolic biosynthetic genes in germinating Chinese wild rice. Ultra-high performance liquid chromatography–triple quadrupole mass spectrometry revealed that 315 metabolites were up-regulated and 28 were down-regulated between G36 and G120. Levels of p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillin, p-coumaric acid, ferulic acid, and epigallocatechin increased significantly during germination. Gene expression of four phenylalanine ammonia-lyases, one 4-coumarate-CoA ligase, one Cinnamoyl-CoA Reductase, two cinnamyl alcohol dehydrogenases, one chalcone synthase, and one chalcone isomerase was significantly higher at G120 than at G36 and promoted phenolics accumulation. This study elucidated the biochemical mechanisms involved in antioxidant activity and phenolic profile changes during Chinese wild rice germination.
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iTRAQ-based proteomic analysis reveals the accumulation of bioactive compounds in Chinese wild rice (Zizania latifolia) during germination
Food Chemistry, 2019Co-Authors: Yongmei Du, Hongbo Zhang, Zhongfeng ZhangAbstract:Abstract Polyphenols and γ-aminobutyric acid (GABA) accumulate during seed germination, but the mechanisms involved are poorly understood. The objective of this study was to elucidate the accumulation of these bioactive compounds in Chinese wild rice during germination. The greatest differences in the phenolic content were at 36-h (G36) and 120-h germination (G120) stages. An iTRAQ-based proteomic analysis revealed 7031 proteins, and a comparison of the G120 and G36 stages revealed 956 upregulated and 188 downregulated proteins. The KEGG analysis revealed significant protein enrichment in the “metabolic pathways”, “biosynthesis of secondary metabolites” and “phenylpropanoid biosynthesis”. Four phenylalanine ammonia-lyases, one 4-coumarate-CoA ligase, one Cinnamoyl-CoA Reductase, two cinnamyl alcohol dehydrogenases, and four glutamate decarboxylases exhibited higher expression at the G120 than at the G36 stage and promoted phenolics and GABA accumulation. This study revealed bioactive compound accumulation in germinating Chinese wild rice, and the finding may help develop functional foods derived from this cereal.
David W. Hopkins - One of the best experts on this subject based on the ideXlab platform.
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Simultaneous down-regulation of enzymes in the phenylpropanoid pathway of plants has aggregated effects on rhizosphere microbial communities
Biology and Fertility of Soils, 2013Co-Authors: E. L. Tilston, Claire Halpin, David W. HopkinsAbstract:We have investigated the effects of genetic modifications to lignin biosynthesis in tobacco on the structural and functional characteristics of the microbial community in the rhizospheres of plants with down-regulations to cinnamyl alcohol dehydrogenase (CAD), caffeic acid O-methyltransferase (COMT) and Cinnamoyl-CoA Reductase (CCR) singly and in all the double and triple combinations, and corresponding unmodified plants. The mass of root produced was reduced by the down-regulation of enzymes in the phenylpropanoid pathway, but there were no differences in the microbial biomass in the rhizosphere per mass of roots. The composition of the rhizosphere microbial communities differed between modified and unmodified plants, with the modified plants having smaller proportions of fungal biomass as indicated by phospholipid fatty acid. Subtle changes to both root chemistry and rhizosphere microbial community function associated with down-regulation of CAD have the potential to modify carbon cycling processes.
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Tissue culture propagation alters plant-microbe interactions in tobacco rhizosphere
Biology and Fertility of Soils, 2008Co-Authors: E. L. Tilston, Claire Halpin, David W. HopkinsAbstract:We have compared properties of roots from different lines (genotypes) of tobacco raised either in tissue culture or grown from seed. The different lines included unmodified plants and plants modified to express reduced activity of the enzyme Cinnamoyl-CoA Reductase, which has a pivotal role in lignin biosynthesis. The size and structure of the rhizosphere microbial community, characterized by adenosine triphosphate and phospholipid fatty acid analyses, were related to root chemistry (specifically the soluble carbohydrate concentration) and decomposition rate of the roots. The root material from unmodified plants decomposed faster following tissue culture compared with seed culture, and the faster decomposing material had significantly higher soluble carbohydrate concentrations. These observations are linked to the larger microbial biomass and greater diversity of the rhizosphere communities of tissue culture propagated plants.