Association of the cad-n1 allele with increased stem growth and wood density in full-sib families of loblolly pine

Stem growth and wood density associated with a mutant null (cad-n1) allele were examined in three 15-year-old loblolly pine half-diallel tests established on two sites in the southern United States. In each half-diallel test, one or two cad-n1 heterozygous parents were crossed with five unrelated wild-type parents to produce five or ten full-sib families. In all, 839 trees from 20 full-sib families in four genetic backgrounds (a cad-n1 heterozygote × five unrelated trees) were sampled, genotyped at the cad locus, and assessed for growth and wood density traits. In a combined analysis of all four genetic backgrounds, we found evidence for effects of increased wood density associated with the cad-n1 allele at age 15 years (p=0.03) and height growth at ages 6 (p=0.03) and 15 (p=0.005). There were differences in the cad-n1 effects for the various growth and wood traits among the half-diallel tests. This variation may be due to either different genetic backgrounds among the parents of the different half-diallel tests or for different growing environments at the field sites. Even though the cad-n1 effect on growth and wood density was significant across genetic backgrounds, the effect was variable among full-sib families within backgrounds. We speculate that certain wild-type alleles from second parents specifically interact with cad-n1 producing large positive effects. In addition, pleiotropic effects on growth and wood density appear to be associated with the cad-n1 allele. While substantial gains are possible through deployment of trees carrying cad-n1, these gains may be family-specific and should be verified for each cross through field-testing.     

Performance of the loblolly pine fusiform rust disease resistance gene (Fr1) in a slashXloblolly pine hybrid family

Unexpectedly high levels of field susceptibility to the fusiform rust fungus observed for slashXloblolly hybrid families in the Cooperative Forest Genetics Research Program Pine Hybrid Trials led to several hypotheses concerning causation of the observed susceptibility. One of these hypotheses involved the failure of major resistance genes to appropriately function in this particular hybrid combination. This current work, involving the loblolly pine resistance gene Fr1 and a fusiform rust inoculum avirulent against Fr1 resistance in a greenhouse trial, delineates the investigation of major gene resistance for a particular slashXloblolly hybrid family. In this trial, the Fr1 resistance allele, derived from the heterozygous (Fr1/fr1) loblolly male parent and transferred to hybrid offspring that should have been segregating 1:1 for resistance, was fully penetrant. Likewise, in the pure-species loblolly control, the Fr1 resistance allele was again fully penetrant, and the performances of our hybrid family and the loblolly control family (both of which segregated for Fr1 resistance) were comparable. These results by inductive reasoning refute the hypothesis that major resistance genes are not appropriately functional in a slashXloblolly hybrid background.     

Effect of forest thinning and wood quality on the short-term wood decomposition rate in a <Emphasis Type="Italic">Pinus tabuliformis</Emphasis> plantation

The effects of forest thinning and wood quality on wood decomposition in the mineral soil were investigated in a Chinese pine (Pinus tabuliformis Carriére) plantation in northern China by measuring mass loss and changes in wood properties (carbohydrates, lignin and nitrogen (N) concentrations) in wood stakes of two tree species—loblolly pine (Pinus taeda L.) and trembling aspen (Populus tremuloides Michx.). Stakes were inserted to a 20 cm soil depth in stands with three thinning levels (low, moderate, and heavy) and an unharvested control and removed after 1 year. There were significant differences in stake mass loss among the treatments. The species effect on the stake mass loss was marginally significant. Wood N content of both species increased during decomposition in all thinning treatments, and was only correlated with aspen mass loss. Wood properties of stakes placed in each stand before insertion (t?=?0) were similar, except for pine lignin concentration and aspen lignin: N ratio, but neither had any effect on thinning treatment results. Lignin concentration increased and carbohydrate concentration decreased in both aspen and pine wood stakes during decomposition across all thinning treatments, which suggests that brown-rot fungi are dominant wood-decomposers on our study site. We conclude that thinning has a significant influence on the wood decomposition in the mineral soil of this Chinese pine plantation.     

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 (h_e=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.     

LtuCAD1 Is a Cinnamyl Alcohol Dehydrogenase Ortholog Involved in Lignin Biosynthesis in Liriodendron tulipifera L., a Basal Angiosperm Timber Species

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis and catalyzes the final step in the synthesis of monolignols. Seven CAD homologs (LtuCAD1 to LtuCAD7) have been previously identified from a basal angiosperm species Liriodendron tulipifera L., which is an important timber tree species with significant ecological and economic values. The phylogenetic analysis indicates that LtuCAD1 is the only Liriodendron CAD grouped with the bona fide CADs, the primary CAD genes involved in lignification. In this study, the predicted protein sequence of LtuCAD1 was found to have conserved domains and the same key determinant site with the bona fide CADs in other plant species. Additionally, LtuCAD1 had the highest expression level in xylem as revealed by quantitative RT-PCR analysis. The expression of beta-glucuronidase (GUS) driven by the LtuCAD1 promoter was largely localized in vascular tissues in Arabidopsis. In stem cross sections, GUS staining was found exclusively in xylem and phloem. When expressed in the Arabidopsis cad4 cad5 double mutant, LtuCAD1 was able to restore the total lignin content and decrease the S/G lignin ratio. Our data indicate that LtuCAD1 is a CAD ortholog involved in lignin biosynthesis in Liriodendron.     

Wood properties and ring width responses to long-term atmospheric CO2 enrichment in field-grown loblolly pine (Pinus taeda L.)

Loblolly pine (Pinus taeda L.) were grown in the field, under non-limiting nutrient conditions, in open-top chambers for 4 years at ambient CO₂ partial pressures (pCO₂) and with a CO₂-enriched atmosphere (+ 30 Pa pCO₂ compared to ambient concentration). A third replicate of trees were grown without chambers at ambient pCO₂. Wood anatomy, wood density and tree ring width were analysed using stem wood samples. No significant differences were observed in the cell wall to cell lumen ratio within the latewood of the third growth ring formed in 1994. No significant differences were observed in the density of resin canals or in the ratio of resin canal cross-sectional area to xylem area within the same growth ring. Ring widths were significantly wider in the CO₂-enrichment treatment for 3 of 4 years compared to the ambient chamber control treatment. Latewood in the 1995 growth ring was significantly wider than that in the ambient control and represented a larger percentage of the total growth-ring width. Carbon dioxide enrichment also significantly increased the total wood specific gravity (determined by displacement). However, when determined as total sample wood density by X-ray densitometry, the density of enriched samples was not significantly higher than that of the ambient chamber controls. Only the 1993 growth ring of enriched trees had a significantly higher maximum latewood density than that of trees grown on non-chambered plots or ambient chambered controls. No significant differences were observed in the minimum earlywood density of individual growth rings between chambered treatments. These results show that the most significant effect of CO₂ enrichment on wood production in loblolly pine is its influence on radial growth, measured as annual tree ring widths. This influence is most pronounced in the first year of growth and decreases with age.     

Functional characterization of CCR in birch (Betula platyphylla × Betula pendula) through overexpression and suppression analysis

We cloned a Cinnamoyl‐CoA Reductase gene (BpCCR1) from an apical meristem and first internode of Betula platyphylla and characterized its functions in lignin biosynthesis, wood formation and tree growth through transgenic approaches. We generated overexpression and suppression transgenic lines and analyzed them in comparison with the wild‐type in terms of lignin content, anatomical characteristics, height and biomass. We found that BpCCR1 overexpression could increase lignin content up to 14.6%, and its underexpression decreased lignin content by 6.3%. Surprisingly, modification of BpCCR1 expression led to conspicuous changes in wood characteristics, including xylem vessel number and arrangement, and secondary wall thickness. The growth of transgenic trees in terms of height was also significantly influenced by the modification of BpCCR1 genes. We discuss the functions of BpCCR1 in the context of a phylogenetic tree built with CCR genes from multiple species.     

Effects of propagule type on genetic parameters of wood density and growth in a loblolly pine progeny test at ages 10 and 11?years

Nine full-sib families of loblolly pine (Pinus taeda L.) were produced by a 3 × 3 factorial mating design. Rooted cuttings and seedlings of full-sib families were tested together in two field locations. Twelve-millimeter wood increment cores were collected from 10- and 11-year-old test trees. On each of the two sites, there were six blocks and a split-plot design, with propagule type as the whole plot and family as the sub-plot. In addition to the collection of wood samples, height and diameter of 1,600 trees were measured. No significant differences were found between cuttings and seedlings for wood density and growth traits. Significant family variation was found for growth and wood density. Genetic parameters estimated for wood density and growth traits using seedlings and rooted cuttings showed that individual-tree and family heritability estimates from rooted cuttings were similar to or higher than those from seedlings for all traits. Half-sib breeding values for parents were highly correlated based on seedling and rooted cutting estimates for height (0.95) and wood density (0.99) but not for diameter (0.56), which suggests that wood density and height breeding value estimates from rooted cuttings in clonal progeny tests can be estimated by traditional seedling tests, but not for tree diameter.     

Phenotypic analysis of first-year traits in a pseudo-backcross {(slash x loblolly) x slash} and the open-pollinated families of the pure-species progenitors

A single test, including one pseudo-backcross (Pinus elliottii x Pinus taeda) x P. elliottii and open-pollinated families of the pure species progenitors, was established in North Central Florida in December 2007 to study the transfer of the fast-growing characteristics from a P. taeda L. (loblolly pine) parent into the P. elliottii Engelm. (slash pine) background. Several traits were measured in the first growing season: height growth, phenology, tip moth incidence, stem traits, crown architectural and needle traits. Heterosis was evaluated for each trait using analyses of variance by fitting a linear mixed model. All traits were significantly (p value < 0.05) different among families while the significance for heterosis varied by trait. Positive heterosis was found for average rate of shoot elongation (ASRE), total growth (TG), total height and number of needles per fascicle while the opposite was true for base diameter, top diameter, fascicle length, fascicle diameter, crown projected area and phenological traits (cessation, duration and day to reach 50% of the height). Average performance (i.e., no heterosis) was found for initiation of growth, number of branches, number of nodes, tip moth incidence, sheath length and specific leaf area. The analyses indicated that introgression of loblolly pine alleles into slash pine was effective and novel trait combinations were achieved. The pseudo-backcross had larger variation in early height growth than the slash pine families and was taller than all open-pollinated families at the end of the first season. Tip moth incidence was much lower than the loblolly pine family.     

A sequence mutation in the cinnamyl alcohol dehydrogenase gene associated with altered lignification in loblolly pine

Evidence for the molecular basis of a null allele of cinnamyl alcohol dehydrogenase (CAD) has been discovered in the loblolly pine (Pinus taeda L.) clone 7-56. The mutation is a two-base pair adenosine insertion located in exon 5 that causes a frame-shift which is predicted to result in premature termination of the protein. For routine detection of the mutation, a diagnostic assay was developed utilizing Template-directed Dye-terminator Incorporation and Fluorescence Polarization detection (FP-TDI). Loblolly pine is the most important commercial tree species in the USA, being harvested for pulp and solid wood products. Chemical pulping could be increased in efficiency by selecting for trees having a two-base pair adenosine insertion, by use of the rapid diagnostic assay developed in this study.     

The cinnamyl alcohol dehydrogenase gene family in Populus: phylogeny, organization, and expression

Background  

Lignin is a phenolic heteropolymer in secondary cell walls that plays a major role in the development of plants and their defense against pathogens. The biosynthesis of monolignols, which represent the main component of lignin involves many enzymes. The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. The CAD gene family has been studied in Arabidopsis thaliana, Oryza sativa and partially in Populus. This is the first comprehensive study on the CAD gene family in woody plants including genome organization, gene structure, phylogeny across land plant lineages, and expression profiling in Populus.     

Enhanced expression of glutamine synthetase (GS1a) confers altered fibre and wood chemistry in field grown hybrid poplar (Populus tremula X alba) (717-1B4)

Hybrid poplar (Populus tremula X P. alba) genetically engineered to express the pine cytosolic glutamine synthetase gene (GS1a) has been previously shown to display desirable field performance characteristics, including enhancements in growth and nitrogen use efficiency. Analysis of wood samples from a 3‐year‐old field trial of three independently transformed GS1a transgenic hybrid poplar lines revealed that, when compared with wild‐type controls, ectopic expression of GS1a resulted in alterations in wood properties and wood chemistry. Included were significant enhancements in wood fibre length, wood density, microfibre angle, per cent syringyl lignin and elevated concentrations of wood sugars, specifically glucose, galactose, mannose and xylose. Total extractive content and acid‐insoluble lignin were significantly reduced in wood of GS1a transgenics when compared with wild‐type trees. Together, these cell wall characteristics resulted in improved wood pulping attributes, including improved lignin solubilization with no concurrent decrease in yield. Trees with increased GS1a expression have improved characteristics for pulp and paper production and hold potential as a feedstock for biofuels production.     

Involvement of cinnamyl-alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench

The lignin structure and enzyme activities of normal and brown-midrib (BMR-6) mutant lines of Sorghum bicolor have been compared to identify the enzyme(s) involved in the reduction of the lignin content of the mutant. The results indicate that cinnamyl-alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase are depressed in the BMR-6 line, whereas the structural modifications correspond only to a reduction of CAD activity. Apparently, the change in the Sorghum lignin content, caused by depression of CAD activity, is accompanied by the incorporation of cinnamaldehydes into the core lignin.Abbreviations CAD cinnamyl-alcohol dehydrogenase - HPLC high-performance liquid chromatography - m/z mass number - OMT caffeic acid O-methyltransferase     

Genetic improvement of the chemical composition of Scots pine (Pinus sylvestris L.) juvenile wood for bioenergy production

Chemical composition is one of the key characteristics that determines wood quality and in turn its suitability for different end products and applications. The inclusion of chemical compositional traits in forest tree improvement requires high‐throughput techniques capable of rapid, non‐destructive and cost‐efficient assessment of large‐scale breeding experiments. We tested whether Fourier‐transform infrared (FTIR) spectroscopy, coupled with partial least squares regression, could serve as an alternative to traditional wet chemistry protocols for the determination of the chemical composition of juvenile wood in Scots pine for tree improvement purposes. FTIR spectra were acquired for 1,245 trees selected in two Scots pine (Pinus sylvestris L.) full‐sib progeny tests located in northern Sweden. Predictive models were developed using 70 reference samples with known chemical composition (the proportion of lignin, carbohydrates [cellulose, hemicelluloses and their structural monosaccharides glucose, mannose, xylose, galactose, and arabinose] and extractives). Individual‐tree narrow‐sense heritabilities and additive genetic correlations were estimated for all chemical traits as well as for growth (height and stem diameter) and wood quality traits (density and stiffness). Genetic control of the chemical traits was mostly moderate. Of the major chemical components, highest heritabilities were observed for hemicelluloses (0.43–0.47), intermediate for lignin and extractives (0.30–0.39), and lowest for cellulose (0.20–0.25). Additive genetic correlations among chemical traits were, except for extractives, positive while those between chemical and wood quality traits were negative. In both groups (chemical and wood quality traits), correlations with extractives exhibited opposite signs. Correlations of chemical traits with growth traits were near zero. The best strategy for genetic improvement of Scots pine juvenile wood for bioenergy production is to decrease and stabilize the content of extractives among trees and then focus on increasing the cellulose:lignin ratio.     

Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy

A quantitative approach to characterize lignin–carbohydrate complex (LCC) linkages using a combination of quantitative ¹³C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.     

Secondary xylem-specific expression of caffeoyl-coenzyme A 3-O-methyltransferase plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine

Two types of structurally distinct O-methyltransferases mediate the methylation of hydroxylated monomeric lignin precursors in angiosperms. Caffeate 3-O-methyltransferase (COMT; EC 2.1.1.68) methylates the free acids and caffeoyl CoA 3-O-methyltransferase (CCoAOMT; EC 2.1.1.104) methylates coenzyme A esters. Recently, we reported a novel hydroxycinnamic acid/hydroxycinnamoyl CoA ester O-methyltransferase (AEOMT) from loblolly pine differentiating xylem that was capable of methylating both acid and ester precursors with similar efficiency. In order to determine the possible existence and role of CCoAOMT in lignin biosynthesis in gymnosperms, a 1.3 kb CCoAOMT cDNA was isolated from loblolly pine that showed 79–82% amino acid sequence identity with many angiosperm CCoAOMTs. The recombinant CCoAOMT expressed in Escherichia coli exhibited a significant methylating activity with hydroxycinnamoyl CoA esters whereas activity with hydroxycinnamic acids was insignificant. Moreover, 3.2 times higher catalytic efficiency for methylating caffeoyl CoA over 5-hydroxyferuloyl CoA was observed which could serve as a driving force towards synthesis of guaiacyl lignin. The secondary xylem-specific expression of CCoAOMT was demonstrated using RNA blot analysis, western blot analysis, and O-methyltransferase enzyme assays. In addition, Southern blot analysis indicated that CCoAOMT may exist as a single-copy gene in loblolly pine genome. The transgenic tobacco plants carrying loblolly pine CCoAOMT promoter-GUS fusion localized the site of GUS activity at the secondary xylem tissues. These data suggest that CCoAOMT, in addition to AEOMT, plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine.     

Purification, Characterization, and Cloning of Cinnamyl Alcohol Dehydrogenase in Loblolly Pine (Pinus taeda L.)

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1. 195) has been purified to homogeneity from differentiating xylem tissue and developing seeds of loblolly pine (Pinus taeda L.). The enzyme is a dimer with a native molecular weight of 82,000 and a subunit molecular weight of 44,000, and is the only form of CAD involved in lignification in differentiating xylem. High levels of loblolly pine CAD enzyme were found in nonlignifying seed tissue. Characterization of the enzyme from both seeds and xylem demonstrated that the enzyme is the same in both tissues. The enzyme has a high affinity for coniferaldehyde (K_m = 1.7 micromolar) compared with sinapaldehyde (K_m in excess of 100 micromolar). Kinetic data strongly suggest that coniferin is a noncompetitive inhibitor of CAD enzyme activity. Protein sequences were obtained for the N-terminus (28 amino acids) and for two other peptides. Degenerate oligonucleotide primers based on the protein sequences were used to amplify by polymerase chain reaction a 1050 base pair DNA fragment from xylem cDNA. Nucleotide sequence from the cloned DNA fragment coded for the N-terminal protein sequence and an internal peptide of CAD. The N-terminal protein sequence has little similarity with the λCAD4 clone isolated from bean (MH Walter, J Grima-Pettenati, C Grand, AM Boudet, CJ Lamb [1988] Proc Natl Acad Sci USA 86:5546-5550), which has homology with malic enzyme.     

Gene silencing of cinnamyl alcohol dehydrogenase in Pinus radiata callus cultures

Xylem-derived Pinus radiata cell cultures, which can be induced to differentiate tracheary elements (TEs), were transformed with an RNAi construct designed to silence cinnamyl alcohol dehydrogenase (CAD), an enzyme involved in the biosynthesis of monolignols. Quantitative enzymatic CAD measurements revealed reduced CAD activity levels in most transclones generated. TEs from transclones with approximately 20% residual CAD activity did not release elevated levels of vanillin, which was derived from coniferyl-aldehyde through a mild alkali treatment. However, the activation of the phenylpropanoid pathway in transclones with approximately 20% residual CAD activity through the application of non-physiological concentrations of sucrose and l-phenylalanine produced phenotypic changes. The accumulation of metabolites such as dihydroconiferyl-alcohol (DHCA), which also accumulates in the P. taeda CAD mutant cad-n1, was observed. These results indicate that a substantial reduction in CAD activity is necessary for this enzyme to become a rate-limiting step in lignin biosynthesis in conifers such as P. radiata and confirm that transformable P. radiata callus cultures can be useful to investigate the function of xylogenesis-related genes in conifers.