Tissue and cell-specific expression of a cinnamyl alcohol dehydrogenase promoter in transgenic poplar plants (original) (raw)
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Plant Cell, 2005
During lignin biosynthesis in angiosperms, coniferyl and sinapyl aldehydes are believed to be converted into their corresponding alcohols by cinnamyl alcohol dehydrogenase (CAD) and by sinapyl alcohol dehydrogenase (SAD), respectively. This work clearly shows that CAD-C and CAD-D act as the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis thaliana by supplying both coniferyl and sinapyl alcohols. An Arabidopsis CAD double mutant (cad-c cad-d) resulted in a phenotype with a limp floral stem at maturity as well as modifications in the pattern of lignin staining. Lignin content of the mutant stem was reduced by 40%, with a 94% reduction, relative to the wild type, in conventional b-O-4-linked guaiacyl and syringyl units and incorportion of coniferyl and sinapyl aldehydes. Fourier transform infrared spectroscopy demonstrated that both xylem vessels and fibers were affected. GeneChip data and real-time PCR analysis revealed that transcription of CAD homologs and other genes mainly involved in cell wall integrity were also altered in the double mutant. In addition, molecular complementation of the double mutant by tissue-specific expression of CAD derived from various species suggests different abilities of these genes/proteins to produce syringyl-lignin moieties but does not indicate a requirement for any specific SAD gene.
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.
1998
traditional paradigm for lignification. Our view is in sharp contrast with a recent model of lignin biosynthesis requiring template dependent stereospecific control of lignin polymerization (Lewis and Davin 1998, Davin et al. 1997) [see p. 33]. Structural information has long been used to guide the search for underlying mechanisms for important biological processes, and the biosynthesis of lignin is no exception. The combination of current methods of structural chemistry, biochemistry, cell biology and genetics should continue to elucidate the nature and origin of the lignin polymer. Lignin is conventionally defined as a complex hydrophobic network of phenylpropanoid units derived from the oxidative polymerization of one or more of three types of hydroxycinnamyl alcohol precursors. These alcohols give rise to phydroxyphenyl, guaiacyl and syringyl subunits in lignin (Fig. 1). The precursors are themselves derived from phenylalanine by deamination, followed by hydroxylation of the aromatic ring, methylation, and the reduction of the terminal acidic group to an alcohol. These alcohols have long been thought to be the direct precursors for lignin (monolignols). The lignin precursors can radically couple at several sites with each other, or, more frequently, with the growing lignin oligomer, to produce a complex polymer with a variety of intermolecular linkages. Here, we focus on a mutation in the last step of the precursor pathway: the formation of the monolignol coniferyl alcohol from coniferaldehyde. This step is catalyzed by the enzyme cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) encoded by a single gene in loblolly pine. Discovery of a CAD-deficient pine mutant. The discovery of a recessive mutant allele of the cad gene in loblolly pine, cad-n1, has permitted the study of pines with severe deficiencies of CAD enzyme (MacKay et al. 1997, Ralph et al. 1997). The secondary xylem (wood) in cad-n1 homozygous seedlings acquires a brown color, distinct from the nearly white color of wild-type pine wood. CAD deficiency causes dramatic changes in the
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.
Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees
Nature Biotechnology, 1999
Because lignin limits the use of wood for fiber, chemical, and energy production, strategies for its downregulation are of considerable interest. We have produced transgenic aspen ( Populus tremuloides Michx.) trees in which expression of a lignin biosynthetic pathway gene Pt4CL1 encoding 4coumarate:coenzyme A ligase (4CL) has been downregulated by antisense inhibition. Trees with suppressed Pt4CL1 expression exhibited up to a 45% reduction of lignin, but this was compensated for by a 15% increase in cellulose. As a result, the total lignin-cellulose mass remained essentially unchanged. Leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both at the cellular and whole-plant levels in the transgenic lines. Our results indicate that lignin and cellulose deposition could be regulated in a compensatory fashion, which may contribute to metabolic flexibility and a growth advantage to sustain the long-term structural integrity of woody perennials.
Plant Cell Reports, 2009
The enzyme Cinnamyl Alcohol Dehydrogenase (CAD) catalyses the last step of lignin monomer synthesis, and is considered as a molecular marker of cell wall lignification in different plants species. Here, we report the isolation and analysis of 5 0 flanking genomic DNA regions upstream to the CAD gene, from two conifers, i.e. white spruce (Picea glauca (Moench) Voss) and loblolly pine (Pinus taeda L.). Sequence comparisons with available CAD gene promoters from angiosperms highlighted the conservation of cis-elements matching MYB, WRKY and bHLH binding sites. Functional characterization of the P. glauca CAD promoter used P. glauca seedlings stably transformed with a DNA fragment of 1,163 base pairs (PgCAD) fused to the b-glucuronidase (GUS) gene. Histochemical observations of different vegetative organs of the transgenic trees showed that this sequence was sufficient to drive GUS expression in lignifying tissues, and more specifically in differentiating xylem cells. Quantitative RT-PCR experiments also indicated that the native CAD gene was preferentially expressed in differentiating xylem both in stems and roots. In addition, GUS expression driven by the PgCAD promoter was wound-inducible which was consistent with the accumulation of CAD mRNA in response to jasmonate application and mechanical wounding. The spruce CAD promoter represents a valuable tool for research and biotechnology applications related to xylem and wood.
IAWA Journal, 2012
The transcriptional activator EgMYB2, which belongs to the large R2R3 MYB transcription factor family, plays a major role in the coordinated control of genes in the lignin biosynthetic pathway. Given that lignin genetic modification can lead to xylem alterations compromising vascular functionality, we characterised wood anatomical properties of two transgenic tobacco lines over-expressing EgMYB2, using light, fluorescence, confocal, transmission electron microscopy, immunocytochemical labelling and digital image analysis. Transgenic wood, compared with wild type, was characterised by both reduced frequency of larger vessels and lower vessel grouping; these traits are known to have physiological implications in terms of water transport efficiency and safety against embolism. Transgenic wood also appeared denser due to the occurrence of thicker cell walls and higher incidence of fibres than wild type. Increased lignin content was accompanied by a concomitant increase in cellulose and ...
Plant Journal, 2001
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 speci®c 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 con®rm that plants with a severe reduction in lignin content may undergo normal development at least in controlled conditions.