Isolation and characterisation of a cDNA clone encoding cinnamyl alcohol dehydrogenase in Eucalyptus globulus Labill (original) (raw)
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Molecular Breeding, 2003
A procedure for A. tumefaciens-mediated genetic transformation of a juvenile E. camaldulensis clone is presented. CAD antisense full-length cDNAs from Eucalyptus gunnii or Nicotiana tabacum was introduced under the control of the CaMV 35S DE promoter. From 44 individual transgenic shoots selected by PCR analysis, 32% exhibited a significant reduction of CAD activity, up to 83%. The use of the heterologous tobacco CAD cDNA construct was less efficient (up to 65% reduction). Transcript levels in 3 lines obtained using the homologous eucalyptus cDNA confirmed the under-expression of the CAD gene, and Southern blot data indicated a low transgene copy number ranging between 1 and 3. The most down-regulated plant contained a single transgene copy. Therefore, for the first time in eucalyptus, genetically modified plantlets exhibiting a strong inhibition of CAD activity associated with decreased transcription were recovered. Five transgenic lines, transferred to the greenhouse for 10 months, went through a wood chemical analysis that showed no differences in lignin quantity (through Fourier transform infrared spectroscopy), composition (through analytical pyrolysis) or pulp yield (through Kraft pulping) compared to control trees. Despite the down-regulation of the CAD gene in this Eucalyptus species of economic interest, the lack of significant changes in lignin profiles indicates that probably the trees were not sufficiently suppressed in CAD throughout development to exhibit obvious modifications in lignin and pulping. This raises the problem of the requirements for an efficient modulation of lignification in trees such as eucalyptus.
Plant Science, 2003
The developmental tissue-and cell-specific expression pattern of two 'lignification' genes, caffeic acid O -methyltransferase (C-OMT) and cinnamyl alcohol dehydrogenase (CAD), was analysed by in situ hybridisation in leaf and stem samples of Eucalyptus plantlets. Both genes are expressed, in a coordinated, developmental fashion, in the same cell types */especially developing vessels */ of differentiating stem xylem tissue confirming their role in lignification and demonstrating that this process is under strict developmental control. C-OMT, but not CAD, transcripts were also localised to developing xylem vessels in the midribs of leaves. Histochemical analyses revealed that, in stem xylem tissues, C-OMT and CAD are expressed in cells poor in S-type lignin (primary xylem vessels and immature secondary xylem cells) and also in cells that later become rich in S-type lignin (mature secondary xylem cells). #
Proceedings of the National Academy of Sciences, 1997
We have discovered a mutant loblolly pine (Pinus taeda L.) in which expression of the gene encoding cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) is severely reduced. The products of CAD, cinnamyl alcohols, are the precursors of lignin, a major cell wall polymer of plant vascular tissues. Lignin composition in this mutant shows dramatic modifications, including increased incorporation of the substrate of CAD (coniferaldehyde), indicating that CAD may modulate lignin composition in pine. The recessive cad-n1 allele, which causes this phenotype, was discovered in a tree heterozygous for this mutant allele. It is inherited as a simple Mendelian locus that maps to the same genomic region as the cad locus. In mutant plants, CAD activity and abundance of cad RNA transcript are low, and free CAD substrate accumulates to a high level. The wood of the mutant is brown, whereas the wood in wild types is nearly white. The wood phenotype resembles that of brown midrib (bm) mutants and some transgenic plants in which xylem is red-brown due to a reduction in CAD activity. However, unlike transgenics with reduced CAD, the pine mutant has decreased lignin content. Wood in which the composition of lignin varies beyond previous expectations still provides vascular function and mechanical support.
Genome-wide analysis of the lignin toolbox of Eucalyptus grandis
New Phytologist, 2015
Lignin, a major component of secondary cell walls, hinders the optimal processing of wood for industrial uses. The recent availability of the Eucalyptus grandis genome sequence allows comprehensive analysis of the genes encoding the 11 protein families specific to the lignin branch of the phenylpropanoid pathway and identification of those mainly involved in xylem developmental lignification.
Tree Genetics & Genomes, 2011
One hundred genotypes of Eucalyptus globulus were ranked according to specific consumption of wood (cubic meters of wood needed to produce 1 ton of pulp). Ten of the most contrasting genotypes were separated in two groups of five clones each; group 1 (G1) with high wood density, high pulp yield, and low specific consumption, and group 2 (G2) with low density, low pulp yield, and high specific consumption. The contrasting genotypes also had significant differences in lignin content, percent syringyl unit composition, and frequency of β-O-4 linkages. Gene expression for phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), 4coumarate:CoA ligase (4CL) and ferulate 5-hydrolase (F5H) was analyzed in the contrasting genotypes. In both groups, transcript abundance for CAD, PAL, and 4CL were similar and only F5H presented significant differences between groups, with high values in the best ranked genotypes G1 in comparison to G2. Correlations between traits were estimated for lignin content vs. pulp yield (R 2 =0.97), pulp yield vs. syringyl units (R 2 =0.82), β-O-4 linkages vs. pulp yield (R 2 =0.84), and β-O-4 linkages vs. syringyl units (R 2 =0.97). Correlations between chemical composition and transcript abundance for F5H were calculated, finding correlation values with lignin content (R 2 =0.81), syringyl units (R 2 =0.83), and pulp yield (R 2 =0.81). The measurement of transcript abundance of F5H represents a potential genomic tool for tree improvement programs to select trees with high pulp yield.
A candidate gene for lignin composition in Eucalyptus: cinnamoyl-CoA reductase (CCR)
Tree Genetics & Genomes, 2012
Lignin content and composition are considered as mandatory traits of eucalyptus breeding programs, especially for pulp, paper, and bioenergy production. In this article, we used 33 Eucalyptus urophylla full-sib families of an 8× 8 factorial design to provide estimates of genetic parameters for lignin-and growth-related traits. Secondly, from the sequencing of the 16 unrelated founders, we described the nucleotide and haplotype variability of cinnamoyl-CoA reductase (CCR), a candidate gene for lignin-related traits encoding the cinnamoyl-CoA reductase. Finally, we tested the association between CCR polymorphisms and trait variation using a mixed linear model. A high value of narrow sense heritability was obtained for lignin content (h²=0.85) and S/G ratio (h²=0.62) indicating that these traits are under strong genetic control. High levels of nucleotide (θ π =0.0131) and haplotype (Hd=0.958) diversity were detected for CCR. From an initial set of 152 biallelic single nucleotide polymorphisms (SNPs), a subset of 65 nonredundant loci was selected. Three intronic SNPs were found to be associated to the variation of S/G ratio after multiple testing correction. In the line of what has been obtained in forest trees, these SNPs explained between 2.45% and 2.87% of the genetic variance of the trait. This study demonstrates the interest of the candidate gene approach for quantitative trait nucleotide detection in Eucalyptus and paves the way to gene assisted selection of lignin composition in E. urophylla.
Biotechnology for biofuels, 2015
Lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance. Eucalyptus trees with down-regulated C4H or C3'H expression displayed lowered overall lignin content. The control samples had an average of 29.6 %, the C3'H reduced lines had an average of 21.7 %, and the C4H reduced lines had an average of 18.9 % lignin from wet chemical analysis. The…
Plant Molecular Biology, 1995
Cinnamyl alcohol dehydrogenase (CAD) which catalyses the synthesis of the cinnamyl alcohols, the immediate precursors of lignins, from the corresponding cinnamaldehydes is considered to be a highly specific marker for lignification We have isolated and characterized a CAD genomic clone from eucalyptus, a woody species of economic importance. The full-length promoter (EuCAD, 2.5 kb) and a series of 5′ deletions were fused to the β-glucuronidase (GUS) reporter gene. These constructs were tested in a homologous transient expression system of eucalyptus protoplasts which enabled the identification of several regions involved in transcriptional control. In order to study the spatial and developmental regulation of the CAD gene, the chimeric gene fusion (EuCAD-GUS) was then transferred via Agrobacterium tumefaciens-mediated transformation into poplar, an easily transformable woody angiosperm. Quantitative fluorometric assays conducted on eight independent in vitro transformants showed that GUS activity was highest in roots followed thereafter by stems and leaves. Histochemical staining for GUS activity on both in vitro primary transformants and more mature greenhouse-grown plants indicated a specific expression in the vascular tissues of stems, roots, petioles and leaves. At the onset of xylem differentiation, GUS activity was detected in parenchyma cells differentiating between the xylem-conducting elements. After secondary growth has occurred, GUS activity was localized in xylem ray cells and parenchyma cells surrounding the lignified phloem and sclerenchyma fibers. This first characterization of a woody angiosperm CAD promoter provides functional evidence for the role of CAD in lignification and suggests that parenchyma cells expressing CAD may provide lignin precursors to the adjacent lignified elements (vessels and fibres).
Plant Journal, 2005
EgMYB2, a member of a new subgroup of the R2R3 MYB family of transcription factors, was cloned from a library consisting of RNA from differentiating Eucalyptus xylem. EgMYB2 maps to a unique locus on the Eucalyptus grandis linkage map and co-localizes with a quantitative trait locus (QTL) for lignin content. Recombinant EgMYB2 protein was able to bind specifically the cis-regulatory regions of the promoters of two lignin biosynthetic genes, cinnamoyl-coenzyme A reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), which contain MYB consensus binding sites. EgMYB2 was also able to regulate their transcription in both transient and stable expression assays. Transgenic tobacco plants over-expressing EgMYB2 displayed phenotypic changes relative to wild-type plants, among which were a dramatic increase in secondary cell wall thickness, and an alteration of the lignin profiles. Transcript abundance of genes encoding enzymes specific to lignin biosynthesis was increased to varying extents according to the position of individual genes in the pathway, whereas core phenylpropanoid genes were not significantly affected. Together these results suggest a role for EgMYB2 in the co-ordinated control of genes belonging to the monolignol-specific pathway, and therefore in the biosynthesis of lignin and the regulation of secondary cell wall formation.
A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression
Plant Molecular Biology, 1998
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 35790 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.