Inheritance, gene expression, and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase (original) (raw)
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Lignin Structure in a Mutant Pine Deficient in Cinnamyl Alcohol Dehydrogenase
Journal of Agricultural and Food Chemistry, 2000
Cinnamyl alcohol dehydrogenase (CAD) activity is deficient in loblolly pine (Pinus taeda L.), harboring a mutated allele of the cad gene (cad-n]). We compared lignin structure of CADdeficient and wild type pines, both types segregating within full-sib families obtained by controlled crosses. The type and frequency of lignin building units and distribution of interunit bonds were determined from the GC-MS analysis of thioacidolysis monomers and dimers. While the lignin content was only slightly reduced, the lignin structure was dramatically modified by the mutation in both mature and juvenile trees. Lignins from CAD-deficient pine displayed unusually high levels of coniferaldehyde and dihydroconiferyl alcohol (DHCA). In addition, biphenyl and biphenyl ether bonds were in large excess in these abnormal lignins. These results suggest that the CAD-deficient pines efficiently compensate for the shortage in normal lignin precursors by utilizing non traditional wall phenolics to construct unusual lignins particularly enriched in resistant interunit bonds.
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
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, 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.
Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins
Phytochemistry, 2004
A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and b-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.
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.
Journal of Biological Chemistry, 2002
Molecular marker compounds, derived from lignin by the thioacidolysis degradative method, for cinnamyl alcohol dehydrogenase (CAD) deficiency in angiosperms have been structurally identified as indene derivatives. They are shown to derive from hydroxycinnamyl aldehydes that have undergone 8 -O-4-cross-coupling during lignification. As such, they are valuable markers for ascertaining plant responses to various levels of CAD down-regulation. Their derivation illustrates that hydroxycinnamyl aldehydes incorporate into angiosperm lignins by endwise coupling reactions in much the same way as normal monolignols do, suggesting that the hydroxycinnamyl aldehydes should be considered authentic lignin precursors.
Proceedings of the National Academy of Sciences, 2013
There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure.
Lignin manipulations in the coniferous gymnosperm Pinus radiata
Plant …, 2008
Severe suppression of 4-Coumarate-CoA ligase (4CL) in the coniferous gymnosperm Pinus radiata substantially impacted plant phenotype and resulted in dwarfed plants with a 'bonsai-tree-like' appearance. Microscopic analyses of stem sections from two-year-old plants revealed substantial morphological changes in both wood and bark tissues. This included the formation of weakly lignified tracheids that displayed signs of collapse and the development of circumferential bands of axial parenchyma. Acetyl bromide-soluble lignin assays and proton NMR studies revealed lignin reductions between 36-50% in the most severely affected transgenic plants. 2D-NMR and Pyrolysis-GC/MS studies indicated that lignin reductions were mainly due to depletion of guaiacyl but not phydroxyphenyl lignin. 4CL silencing also caused modifications in the lignin interunit linkage distribution including elevated β -aryl ether (β-O-4-unit) and spirodienone (β-1) levels, accompanied by lower phenylcoumaran (β-5), resinol (β-β), and dibenzodioxocin (5-5/β-O-4) levels. A sharp depletion in the level of saturated (dihydroconiferyl alcohol) endgroups was also observed. Severe suppression of 4CL also affected carbohydrate metabolism. Most obvious was an up to ~2-fold increase in galactose content in wood from transgenic plants due to increased compression wood formation. 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