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Biomedical subjects

A M Boudet

Publications and source records attributed to A M Boudet.

At least 19 recordsLinked to original sources
PubMed2001

Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants.

Different transgenic tobacco lines down-regulated for either one or two enzymes of the monolignol pathway were compared for their lignin content and composition, and developmental patterns. The comparison concerned CCR and CAD down-regulated lines (homozygous or heterozygous for the transgene) and the hybrids resulting from the crossing of transgenic lines individually altered for CCR or CAD activities. Surprisingly, the crosses containing only one allele of each antisense transgene, exhibit a dramatic reduction of lignin content similar to the CCR down-regulated parent but, in contrast to this transgenic line, display a normal phenotype and only slight alterations of the shape of the vessels. Qualitatively the lignin of the double transformant displays characteristics more like the wild type control than either of the other transgenics. In the transgenics with a low lignin content, the transformations induced other biochemical changes involving polysaccharides, phenolic components of the cell wall and also soluble phenolics. These results show that the ectopic expression of a specific transgene may have a different impact depending on the genetic background and suggest that the two transgenes present in the crosses may operate synergistically to reduce the lignin content. In addition, these data confirm that plants with a severe reduction in lignin content may undergo normal development at least in controlled conditions.

Alcohol Oxidoreductases↗
PubMed2001

In situ analysis of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and subcellular levels.

Using tobacco transgenic lines altered in the monolignol biosynthetic pathway and which differ in their lignin profiles we have evaluated lignin deposition at the cellular and subcellular levels using several microanalytical techniques. Surprisingly, whereas a Cinnamoyl CoA reductase (CCR) down-regulated line with a strong decrease in lignin content exhibited an overall reduction in lignin deposition in the walls of the different xylem cell types, this reduction was selectively targeted to the fibers in a double transformant (down-regulated for both CCR and Cinnamyl alcohol dehydrogenase (CAD)) displaying a similar degree of global lignin content decrease. Fiber and vessel secondary walls of the transgenic tobacco line homozygous for the ccr antisense gene (CCR.H) down-regulated plants were dramatically destructured, particularly in the S2 sublayer, whereas the deposition of lignins in the S1 sublayer was not significantly modified. In contrast, cell wall organization was slightly altered in xylem cells of the double transformant. The relative distribution of non-condensed and condensed units in lignin, evaluated microscopically with specific antibodies, was differentially affected in the transgenics studied and, in a general way, a drop in non-condensed lignin units (beta- 0-4 interunit linkages) was associated with a loss of cohesion and extensive disorganization of the secondary wall. These results demonstrate that lignification is tightly and independently regulated in individual cell types and cell wall sublayers. They also show that down-regulation of specific genes may induce targeted changes in lignin structure and in spatial deposition patterns of the polymer.

Aldehyde Oxidoreductases↗
PubMed2001

Simultaneous down-regulation of caffeic/5-hydroxy ferulic acid-O-methyltransferase I and cinnamoyl-coenzyme A reductase in the progeny from a cross between tobacco lines homozygous for each transgene. Consequences for plant development and lignin synthesis.

Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants.

Aldehyde Oxidoreductases↗
PubMed2000

Cross-coupling of hydroxycinnamyl aldehydes into lignins.

Pathways for hydroxycinnamyl aldehyde incorporation into lignins are revealed by examining transgenic plants deficient in cinnamyl alcohol dehydrogenase, the enzyme that converts hydroxycinnamyl aldehydes to the hydroxycinnamyl alcohol lignin monomers. In such plants the aldehydes incorporate into lignins via radical coupling reactions. As diagnostically revealed by long-range (13)C-(1)H correlative NMR, sinapyl aldehyde (3, 5-dimethoxy-4-hydroxy-cinnamaldehyde) 8-O-4-cross-couples with both guaiacyl (3-methoxy-4-hydroxyphenyl-propanoid) and syringyl (3, 5-dimethoxy-4-hydroxyphenyl-propanoid) units, whereas coniferyl aldehyde cross-couples only with syringyl units.

Alcohol Oxidoreductases↗
PubMed2000

Characterization of cis-elements required for vascular expression of the cinnamoyl CoA reductase gene and for protein-DNA complex formation.

Cinnamoyl-CoA reductase (CCR) catalyses the first specific step in the biosynthesis of monolignols, the monomeric units of lignins. We examined the developmental regulation of the Eucalyptus gunnii EgCCR promoter by analysing the expression of EgCCR-GUS fusions in tobacco. EgCCR promoter activity was strongest in lignified organs (stems and roots) consistent with the EgCCR mRNA level in these organs. Histochemical analysis showed expression in vascular tissues (cambium, young differentiating xylem, ray cells, internal and external phloem) of stems and roots in agreement with in situ hybridization data. Promoter deletion analysis and gain-of-function experiments identified the sequences between positions -119 and -77 as necessary and sufficient for expression in vascular tissues of stems. Electrophoretic mobility-shift assays showed that this region is specifically recognized by nuclear proteins present in tobacco stems, giving rise to two retarded complexes, LMC1 and LMC2. Using overlapping EgCCR fragments and mutated oligonucleotides as competitors in gel-shift assays, two DNA-protein interaction sites were mapped. Finally, the role of protein-protein interactions in the formation of the LMC1 and LMC2 complexes was investigated using the detergent sodium deoxycholate, and protein fractionation onto a heparin Sepharose column.

Aldehyde Oxidoreductases↗
PubMed1999

Biochemical characterization, molecular cloning and expression of laccases - a divergent gene family - in poplar.

The nature of the enzyme(s) involved in the dehydrogenative polymerization of lignin monomers is still a matter of debate. Potential candidates include laccases which have recently received attention due to their capacity to oxidize lignin monomers and close spatial and temporal correlation with lignin deposition. We have characterized two H2O2-independent phenoloxidases with approximate molecular masses of 90 kDa and 110 kDa from cell walls of Populus euramericana xylem that are capable of oxidizing coniferyl alcohol. The 90-kDa protein was purified to apparent homogeneity and extensively characterized at the biochemical and structural levels. To our knowledge, this is the first report of a plant laccase purified to homogeneity from a lignifying tissue of an angiosperm. The cDNA clones corresponding to the 90-kDa and 110-kDa proteins, lac90 and lac110, were obtained by a PCR-based approach using specific oligonucleotides derived from peptide sequences. Sequence analysis indicated that lac90 and lac110 encoded two distinct laccases. In addition, heterologous screening using an Acer pseudoplatanus laccase cDNA enabled us to obtain three additional cDNAs (lac1, lac2, lac3) that did not correspond to lac90 and lac110. The five laccase cDNAs correspond to a highly divergent multigene family but Northern analysis with gene-specific probes indicated that all of the genes are exclusively and abundantly expressed in stems. These results highlight the polymorphism of plant laccases by an integrated biochemical and molecular approach, and provide the tools that will enable us to clearly determine the function of these enzymes in plants by molecular and genetic approaches.

Acrolein↗
PubMed1998

Cloning and characterization of two maize cDNAs encoding cinnamoyl-CoA reductase (CCR) and differential expression of the corresponding genes.

Cinnamoyl-CoA Reductase (CCR, EC 1.2.1.44) catalyses the first step of the lignin pathway. Two full-length cDNAs identified by sequence analysis as CCR-encoding cDNAs were isolated from a maize root cDNA library. These two cDNAs designated ZmCCR1 and ZmCCR2 exhibit 73% sequence conservation at the nucleotide level for their coding regions and are relatively divergent at their 5'- and 3'-untranslated regions. They both contain a common signature which is thought to be involved in the catalytic site of CCR. Northern blot analysis indicated that ZmCCR2 was expressed at very low levels in roots whereas ZmCCR1 was widely expressed in different organs. The high level of ZmCCR1 gene expression along the stalk suggests that the corresponding enzyme is probably involved in constitutive lignification.

Aldehyde Oxidoreductases↗
PubMed1998

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase.

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl-SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl-SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Journal Article↗
PubMed1998

A comparative study of two isoforms of laccase secreted by the "white-rot" fungus Rigidoporus lignosus, exhibiting significant structural and functional differences.

Two isoforms of laccase were obtained as the predominant phenol-oxidases in defined medium liquid cultures of the "white-rot" fungus Rigidoporus lignosus (R. lignosus). A characterization of the two laccases was made in terms of molecular mass, isoelectric point, metal content and N-terminal sequence. Furthermore, in order to gain information on the structural features related to the metal centers, a study of their geometric arrangement and their redox ability was made. It turned out that the two isoenzymes greatly differed with regard to pH stability, catalytic and copper centers features. It is proposed that all such differences are dependent on the amino acid sequences, which cause a distortion of the copper sites, thus accounting for the redox potential values and kinetic properties.

Amino Acid Sequence↗
PubMed1998

A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression.

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, an atypical form of CAD (CAD 1) was identified in Eucalyptus gunnii [12]. We report here the molecular cloning and characterization of the corresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol dehydrogenase. The identity of CAD 1-5 was unambiguously confirmed by sequence comparison of the cDNA with peptide sequences derived from purified CAD 1 protein and by functional expression of CAD 1 recombinant protein in Escherichia coli. Both native and recombinant CAD 1 exhibit high affinity towards lignin precursors including 4-coumaraldehyde and coniferaldehyde, but they do not accept sinapaldehyde. Moreover, recombinant CAD 1 can also utilize a wide range of aromatic substrates including unsubstituted and substituted benzaldehydes. The open reading frame of CAD 1-5 encodes a protein with a calculated molecular mass of 35,790 Da and an isoelectric point of 8.1. Although sequence comparisons with proteins in databases revealed significant similarities with dihydroflavonol-4-reductases (DFR; EC 1.1.1.219) from a wide range of plant species, the most striking similarity was found with cinnamoyl-CoA reductase (CCR; EC 1.2.1.44), the enzyme which directly precedes CAD in the lignin biosynthetic pathway. RNA blot analysis and immunolocalization experiments indicated that CAD 1 is expressed in both lignified and unlignified tissues/cells. Based on the catalytic activity of CAD 1 in vitro and its localization in planta, CAD 1 may function as an 'alternative' enzyme in the lignin biosynthetic pathway. However, additional roles in phenolic metabolism are not excluded.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships.

Cinnamoyl CoA:NADP oxidoreductase (CCR, EC 1.2.1.44) catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes, i.e. the first specific step in the synthesis of the lignin monomers. The cloning of a cDNA encoding CCR in Eucalyptus gunnii (EUCCR) is reported here. The identity of the EUCCR cDNA was demonstrated by comparison with peptide sequence data from purified CCR and functional expression of the recombinant enzyme in Escherichia coli. Sequence analysis revealed remarkable homologies with dihydroflavonol-4-reductase (DFR), the first enzyme of the anthocyanin biosynthetic pathway. Moreover, significant similarities were found with mammalian 3 beta-hydroxysteroid dehydrogenase and bacterial UDP-galactose-4-epimerase, suggesting that CCR shared a common ancestor with these enzymes and can therefore be considered as a new member of the mammalian 3 beta-hydroxysteroid dehydrogenase/ plant dihydroflavonol reductase superfamily. In Eucalyptus gunnii, CCR is encoded by one gene containing four introns whose positions are similar to those of introns I, II, III and V in DFR genes from dicots. In agreement with the involvement of CCR in lignification, the CCR transcript was shown to be expressed in lignified organs, i.e. root and stem tissues, and was localized mainly in young differentiating xylem. On the other hand, its abundance in Eucalyptus leaves suggests that monolignols may be precursors of end products other than lignins. This first characterization of a gene corresponding to CCR opens new possibilities to genetically engineer plants with lower lignin content. This is particularly important for woody plants such as Eucalyptus which are used for pulp making.

Aldehyde Oxidoreductases↗
PubMed1995

Site-directed mutagenesis of a serine residue in cinnamyl alcohol dehydrogenase, a plant NADPH-dependent dehydrogenase, affects the specificity for the coenzyme.

Using recombinant cinnamyl alcohol dehydrogenase isoform 2 (CAD2, EC 1.1.1.195), an NADPH-dependent aromatic alcohol dehydrogenase involved in lignification in vascular plants, we have investigated the detailed steady-state kinetic mechanism of CAD2 and the role of a serine residue in determining the cofactor specificity of CAD2. Site-directed mutagenesis (S212D) and overexpression of the WT and mutant S212D forms of CAD2 in Escherichia coli, followed by kinetic studies on the purified WT and mutant proteins, confirmed the involvement of S212D in recognizing the phosphate group of NADPH and provided information on the structural requirements for NADPH specificity. From substrate kinetic patterns and product inhibition studies both WT and S212D mutant forms of CAD2 have been shown to follow rapid equilibrium random bireactant kinetics with the value of the interaction factor (alpha) for WT (0.25) being significantly less than that for S212D CAD2 (0.45). The changes in binding energy arising from the mutation on the binding of the 2'-phosphate site of the coenzyme were assessed. A marked degree of physical interaction was detected between the enzymatic binding sites of the coniferyl alcohol substrate and the 2'-phosphate binding region, which are quite distant in the three-dimensional structure. The inhibition by 2',5'-ADP and 5'-AMP was found to be weak for both WT and S212D CAD2. Strong substrate inhibition was detected for CAD2, and its implications for plant physiological studies were assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗
PubMed1995

Introduction of specific carbohydrates into Eucalyptus gunnii cells increases their freezing tolerance.

The comparison of soluble sugar content in various cell lines of Eucalyptus gunnii exhibiting different freezing resistances revealed that the most resistant cell line contained the highest soluble sugar content. It was possible to increase the freezing resistance of the sensitive cell line by progressive exposure to low temperatures (acclimation). During the early stage of cold acclimation, an increase of soluble sugar concentration was observed in the cells confirming the correlation between freezing resistance and soluble carbohydrate content in this species. In addition, feeding experiments on the sensitive cell line were performed to introduce specific sugars into the cells. Both electropulsation and long-term incubation in the presence of fructose and raffinose led to an increase in the tolerance of the cells during a freezing programme. Using radioactive fructose, the uptake of the sugar into cells and protoplasts was checked. In the light of these results, hypotheses are presented concerning the possible role of intracellular sugars in cryoprotection.

Adaptation, Physiological↗
PubMed1994

Purification and characterization of cinnamyl alcohol dehydrogenase isoforms from Phaseolus vulgaris.

Cinnamyl alcohol dehydrogenase (CAD) catalyses the reduction of hydroxycinnamaldehydes (p-coumaryl, coniferyl, sinapyl) to the corresponding alcohols which are the monomeric precursors of lignins. We have demonstrated the occurrence of two isoforms of CAD (CAD1 and CAD2) in bean which differ in terms of subunit Mr, specific activity, substrate affinity and antigenicity. The most abundant polypeptide in bean pods, organs with very limited lignification, is a low affinity CAD isoform (CAD1). This enzyme which is distinct from a benzyl alcohol dehydrogenase with broad substrate specificity, was purified to apparent homogeneity and partial amino acid sequencing was carried out using internal peptides obtained by trypsin cleavage.

Alcohol Oxidoreductases↗
PubMed1993

A molecular model for cinnamyl alcohol dehydrogenase, a plant aromatic alcohol dehydrogenase involved in lignification.

The plant aromatic alcohol dehydrogenase, cinnamyl alcohol dehydrogenase (CAD2 from Eucalyptus) was found by sequence analysis of its cloned gene to be homologous to a range of dehydrogenases including alcohol dehydrogenases, L-threonine-3-dehydrogenase, D-xylose reductase and sorbitol dehydrogenase. A homology model of CAD2 was built using the X-ray crystallographic coordinates of horse-liver alcohol dehydrogenase to provide the template, with additional modelling input from other analogous regions of structure from similar enzymes where necessary. The structural model thus produced rationalised the Zn-binding properties of CAD2, indicated the possession of a Rossmann fold (GXGXXG motif), and explained the class A stereospecificity (pro-R hydrogen removal from substrate alcohol) and aromatic substrate specificity of the enzyme. A range of potential ligands was designed based on the homology model and tested as inhibitors of CAD2 and horse liver alcohol dehydrogenase.

Alcohol Dehydrogenase↗