Abnormal lignin in a loblolly pine mutant.

Novel lignin is formed in a mutant loblolly pine (Pinus taeda L.) severely depleted in cinnamyl alcohol dehydrogenase (E.C. 1.1.1.195), which converts coniferaldehyde to coniferyl alcohol, the primary lignin precursor in pines. Dihydroconiferyl alcohol, a monomer not normally associated with the lignin biosynthetic pathway, is the major component of the mutant's lignin, accounting for approximately 30 percent (versus approximately 3 percent in normal pine) of the units. The level of aldehydes, including new 2-methoxybenzaldehydes, is also increased. The mutant pines grew normally indicating that, even within a species, extensive variations in lignin composition need not disrupt the essential functions of lignin.

Unexpected variation in lignin.

Recent studies on mutant and transgenic plants indicate that lignification may be far more flexible than previously realized. Pines with a mutation affecting the biosynthesis of the major lignin precursor, coniferyl alcohol, show a high level of an unusual subunit, dihydroconiferyl alcohol. These results argue in favor of an increased potential for genetic modification of lignin and indicate that our knowledge of the biosynthesis of lignin is far from complete.

Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR.

Studying lignin-biosynthetic-pathway mutants and transgenics provides insights into plant responses to perturbations of the lignification system, and enhances our understanding of normal lignification. When enzymes late in the pathway are downregulated, significant changes in the composition and structure of lignin may result. NMR spectroscopy provides powerful diagnostic tools for elucidating structures in the difficult lignin polymer, hinting at the chemical and biochemical changes that have occurred. COMT (caffeic acid O-methyl transferase) downregulation in poplar results in the incorporation of 5-hydroxyconiferyl alcohol into lignins via typical radical coupling reactions, but post-coupling quinone methide internal trapping reactions produce novel benzodioxane units in the lignin. CAD (cinnamyl alcohol dehydrogenase) downregulation results in the incorporation of the hydroxycinnamyl aldehyde monolignol precursors intimately into the polymer. Sinapyl aldehyde cross-couples 8-O-4 with both guaiacyl and syringyl units in the growing polymer, whereas coniferyl aldehyde cross-couples 8-O-4 only with syringyl units, reflecting simple chemical cross-coupling propensities. The incorporation of hydroxycinnamyl aldehyde and 5-hydroxyconiferyl alcohol monomers indicates that these monolignol intermediates are secreted to the cell wall for lignification. The recognition that novel units can incorporate into lignins portends significantly expanded opportunities for engineering the composition and consequent properties of lignin for improved utilization of valuable plant resources.

Lignin structure in a mutant pine deficient in cinnamyl alcohol dehydrogenase.

Cinnamyl alcohol dehydrogenase (CAD) activity is deficient in loblolly pine (Pinus taeda L.) harboring a mutated allele of the cad gene (cad-n1). We compared lignin structure of CAD-deficient 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. 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 nontraditional wall phenolics to construct unusual lignins particularly enriched in resistant interunit bonds.

Genetic analysis of cinnamyl alcohol dehydrogenase in loblolly pine: single gene inheritance, molecular characterization and evolution.

The gene encoding the monolignol biosynthetic enzyme cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) can be expressed in response to different developmental and environmental cues. Control of Cad gene expression could involve either differential regulation of more than one Cad gene or, alternatively combinatorial regulation of a single Cad gene. In loblolly pine (Pinus taeda L.), we found several electrophoretic variants (allozymes) of CAD and a high level of heterozygosity (he = 0.46). Analysis of inheritance patterns of pine CAD allozymes gave segregation ratios that were consistent with Mendelian expectations for a single functional gene. The identity of the full-length Cad cDNA sequence was confirmed by alignment with peptide sequences obtained from purified active enzyme and by extensive similarity to Cad sequences from other species. Southern blot analysis of genomic DNA using the Cad cDNA as a hybridization probe gave simple patterns, consistent with our interpretation that pine Cad is a single-copy gene. Phylogenetic analysis and evolution rate estimates showed that Cad sequences are diverging less rapidly in the gymnosperms than in the angiosperms. The Cad mRNA was present in both lignifying tissues and a non lignifying tissue (the megagametophyte) of pine. The presence of a single gene suggests that different regulatory mechanisms for a single Cad gene, rather than differential regulation of several genes, can account for its expression in response to different cues.

Average effect of a mutation in lignin biosynthesis in loblolly pine.

Cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) is a monolignol biosynthetic enzyme that catalyzes the final step of lignin subunit biosynthesis in higher plants. Recently, a mutant allele of the cad gene, cad-n1, encoding for the CAD enzyme, was discovered in loblolly pine. By reducing the expression of the cad gene, this mutant has a decreased lignin content and major changes in the lignin composition in wood. In this study, we found that the substitution of a wild-type allele by cad-n1 was associated with a significant effect on 2nd-year shoot elongation in a half-sib family of loblolly pine (designated family 7-1037). The average effect of cad-n1 appeared to increase with tree growth and was greater for stem radial growth than height growth. An increase of 14.1% in de-barked volume in year 4 was associated with cad-n1. Co-segregation analysis indicated that the cad locus itself might represent a gene that governs stem growth in pine. The significance of the mutation cad-n1 for tree growth and wood processing is discussed.

Inheritance, gene expression, and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase.

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.