Source: NORTH CAROLINA STATE UNIV submitted to NRP
STUDIES OF THE MOLECULAR CONTROL OF WOOD FORMATION IN LOBLOLLY PINE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
MCINTIRE-STENNIS
Reporting Frequency
Annual
Accession No.
0180147
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 1998
Project End Date
Sep 30, 2004
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
FORESTRY
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
12306111040100%
Knowledge Area
123 - Management and Sustainability of Forest Resources;

Subject Of Investigation
0611 - Conifer forests of the South;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The Objectives are to identify genes involved in controlling the process of wood formation in forest trees, and to understand the interactions between these regulatory genes and the genes required for production of various components of the cell walls. The working hypothesis is that wood formation is likely to be regulated by genes that are similar to regulatory genes previously identified in other organisms. The processes controlling wood formation may differ between hardwood and softwood trees, and a comparative approach may yield important information regarding the differences and similarities between these groups.
Project Methods
Multiple approaches will be taken to identify regulatory genes. Genetic mapping experiments will be used to screen for regions of the genome that control wood properties, including lignin content and composition. Such regions, known as quantitative trait loci, are presumed to contain genes that participate in regulating the process of wood formation. At the same time, DNA sequence analysis of cDNA clones isolated from differentiating wood will be used to identify genes similar to known regulatory factors from other species. These cloned genes can then be placed on genetic maps and the map locations compared with the locations of quantitative trait loci identified in the mapping experiments. Interactions between regulatory factors and other proteins can be assayed using a variety of biochemical as well as genetic approaches.

Progress 01/01/03 to 12/31/03

Outputs
Methods have been developed for the correlation of transcript level for specific genes with the chemical properties of wood on a population level in loblolly pine. To do this we have carried out microarray analysis for 2107 specific cDNAs and carried out chemical analysis for alpha-cellulose, hemicellulose and lignin on a set of 52 trees from the NCSU loblolly pine breeding program. We have used correlation analysis to identify specific genes whose transcript levels are positively or negatively correlated with specific wood properties. Many significant correlations are detected. The correlations may be due to metabolic relationships, genetic linkage, or due to developmental or adaptive variation in the specific population and environment. Microarrays provide a powerful tool for the investigation of molecular phenotypes in a breeding population, and these results are an important first step to demonstrate feasibility, identify genes of interest, and guide further experiments. It is the first time this strategy and technology have been applied to wood formation.

Impacts
These results provide a molecular basis for complex quantitative traits where the action of many genes contributes to the phenotype and the properties of wood.

Publications

  • Craig, D.R., Smith, C., Kirst, M., Osborne, J., Nielsen, D., Kadla, J.F., Chang, H-m., Li, L., van Zyl, L., and R.R. Sederoff. 2004 Correlation of Gene Expression in Differentiating Xylem and Specific Wood Properties in loblolly pine (Pinus taeda L.). TAPPI symposium, submitted.
  • Morris, C.R., Scott, J.T., Chang, H-M, Sederoff, R.R., OMalley, D. and Kadla, J.F. 2004. Metabolic profiling: a new tool in the study of wood formation. J. Agric. Food Chem. 52: 1427-1234.
  • Stasolla, C. Scott, J., Egertsdotter, U., Kadla, J., OMalley, D., Sederoff, R. and van Zyl, L. 2003. Analysis of lignin produced by cinnamyl alcohol dehydrogenase deficient Pinus taeda cultured cells. Plant Physiology and Biochemistry. 41, 5, 439-445.
  • Patzlaff, A., Newman, L., Dubos, C., Whetten, R.W., Smith, C., McInnis, S., Bevan, M., Sederoff, R.R and Campbell, M.M. 2003. Characterization of PtMYB1, an R2R3-MYB from pine xylem. Plant Mol. Biol. 53: 597-608.
  • Kirst, M., Myburg, A. and Sederoff, R.R. 2004 Genetic Mapping in Forest Trees: Markers, Linkage Analysis and Genomics. In Genetic Engineering, vol 24. J. Setlow ed., Plenum, NY. In press.
  • Busov, V.B., Johannes, E., Whetten, R.W., Sederoff, R.R., Spiker, S.L., Lanz-Garcia, C. and B. Goldfarb. 2004 An auxin-inducible gene from loblolly pine (Pinus taeda L.) is differentially expressed in mature and juvenile phase shoots and encodes a putative transmembrane protein. Planta (in press).
  • Brinker, M., van Zyl, L., Liu, W., Craig, D., Sederoff, R., Clapham, D., von Arnold, S., 2004. Microarray analyses of gene expression during adventitious root development in Pinus contorta, Plant Physiology (in press).


Progress 01/01/02 to 12/31/02

Outputs
Comparison of gene expression in wood forming tissues of loblolly pine with the complete genome of Arabidopsis: In this work we show that there is a high level of conservation of functional homology between loblolly pine and the distantly related dicot Arabidopsis. This result argues that there is a core genome of functional genes in all higher seed plants, and that much of the diversity in higher plants comes not from new genes but from using existing families of genes in new ways. A paper has just been accepted in PNAS. Characterization of genes and proteins associated with wood formation: Work has continued on laccases and another unusual group of cell wall associated proteins, arabinogalactan proteins (AGPs). We have identified and characterized an AGP that is highly expressed in wood forming tissues, and is particularly abundant during compression wood formation. This protein is expressed transiently in xylem differentiation, and is restricted to radially expanding cells before the complete synthesis of the wood cell wall. It is separated in time from lignification. A paper is in press in Plant Mol. Biol. Work has continued on the unusual lignin produced by a mutation in loblolly pine affecting cinnamyl alcohol dehydrogenase (CAD). We have characterized the mutant lignin in grester detail, for, example by thioacidolysis, NMR, and pulping properties. We have demonstrated that this unusual lignin, containing dihydroconiferyl alcohol, can be formed in a cell culture system following induction by phytohormone treatment. In addition, we show that the mutant cells are affected in phenolic transport, suggesting a new role for CAD. Further characterization of the lignin from mutant homozygotes has also been carried out. Pine genes have proven useful in assaying gene expression in spruce. These species are in different genera of the Pinaceae, and the level of sequence similarity between pine and spruce is very high. Therefore, we have been able to use the pine genes on microarrays to monitor expression changes during embryonic differentiation and to examine the effects of polyethylene glycol (PEG) on developing embryos. PEG is an important component of the cellular environment in vitro, and has a strong, but not well-understood effect on improvement of embryo maturation. We have also collaborated with a group studying stress responses in loblolly pine using our microarrays, and who have developed a data management system using inductive logic programming. A paper has been published on this work.

Impacts
Two types of method development have been associated with work on wood formation. Genomic mapping has been done in order to map genes of interest and DNA transformation in loblolly pine and spruce has been carried out as a basis for eventual genetic engineering of loblolly pine.

Publications

  • Marques, C.M., Carocha, V.J., Araujo, J.A., Ferriera, J.G., OMalley, D.M., Liu, B.H., and Sederoff, R.R. 1998. AFLP Genetic maps in Eucalyptus globulus and E. terecticornis. Theor. & Applied Genetics 96:727-737.
  • Marques, C.M., Vasquez-Kool, J., Carocha, V.J., Ferreira, J.G., O'Malley, D.M., Liu, B.-H., Sederoff, R.R. 1999. Genetic dissection of vegetative propagation traits in Eucalyptus tereticornis and E. globulus. Theor. Appl. Genet. 99:936-946.
  • Myburg, A.A., Remington, D.L., OMalley, D.M., Sederoff, R.R., and R. W. Whetten. 2001. High throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer. Biotechniques 30:384-357.
  • Myburg AA, Sederoff RR, 1999. Xylem structure and In: Embyronic Encyclopedia of Life Science. Nature Pub. Group: Macmillan; New York, NY.
  • Ralph, J., Hatfield, R., Fanchuang L., Marita, J.M., Ede, R.M., Junpeng, S.R., Quideau, S., Helm, R. Grabber, J., Kim, H., Jimenez-Monteon, G., Zhang, Y., Landucci, L., Sederoff, R.R. and Boudet, A. 1998. NMR applications in cell wall research. TAPPI proceedings.
  • Ralph, J., Lapierre, C., Marita, J.M., et al. 2001. Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR. Phytochemistry 57:993-1003.
  • Sato, Y., Bao, W.L., Sederoff, R., and Whetten, R. 2001. Molecular cloning and expression of eight laccase cDNAs in loblolly pine (Pinus taeda). Journal of Plant Research. 114:147-155.
  • Sederoff, R.R. 1998. The promise of Forest Biotechnology. Paper Age. August issue: pp 13-18.
  • Sederoff, R.R. 1999. Building better trees with antisense. Nature Biotechnology 17:750-751.
  • Sederoff, R.R. 1999. Surfing on AG Biotech into Genomics. Mol. Breeding. 5:485-491.
  • Sederoff. R.R. 2000. Tree genomes: what will we understand about them by the year 2020 and how might we use that knowledge? In Forest Genetics and Sustainablity C. Matyas ed., Kluwer Academic Publishers 63:23-30.
  • Sederoff, R.R., Mackay, J.J., Ralph, J., and Hatfield, R. D. 1999 Unexpected variation in lignin. Current Opinion in Plant Biology 2:145-152.
  • Stasolla, C., Scott, J., Egerstdotter, U., Kadla, J., Sederoff, R. & van Zyl, L. (2003). Analysis of lignin produced by cinnamyl alcohol dehydrogenase deficient pine (Pinus taeda L.) cultured cells. (Plant Physiol. Biochem. in press ).
  • Stasolla C., van Zyl, L., Egertsdotter, U., Craig, D., Liu, W., Sederoff, R. 2003. The effects of polyethylene glycol (PEG) on gene expression of developing white spruce somatic embryos. Plant Physiology 131:49-60.
  • Tang, W., Sederoff, R. Whetten, R., 2001. Regeneration of transgenic loblolly pine (Pinus taeda) from zygotic embryos transformed with Agrobacterium tumerfaciens. Planta 213 (6):981-989.
  • Tang, W., Whetten, R. and Sederoff R. 2001. Genotypic control of high-frequency adventitious shoot regeneration via somatic organogenesis in loblolly pine. Plant Science 161:267-272.
  • Lev-Yadun, S., and Sederoff R. 2000. Pines as model gymnosperms to study evolution, wood formation and perennial growth. J. Plant Growth Regulation. 19:290-305.
  • Lev-Yadun, S. and Sederoff, R. 2001. Grafting for transgene containment. Nature Biotechnology 19:2.
  • MacKay, J.J., Presnell, T., Jameel, H., Taneda, H., OMalley, D., and Sederoff, R. 1999. Modified lignin properties and delignification during pulping with a mutant loblolly pine. Holzforschung. 53:403-410.
  • Marques CM, Brondani RPV, Grattapaglia D, Sederoff R. 2002. Conservation and synergy of SSR loci and QTLs for vegetative propagation in four Eucalyptus species. TAG 105: 474-477.
  • van Zyl L, von Arnold S, Bozkov P, Chen Y, Egertsdotter U, MacKay J, Sederoff R, Shen J, Zelena L, Clapham D. (2002). Heterologous array analysis in Pinaceae: Hybridization of high-density arrays of Pinus taeda cDNA with cDNA from needles and embryonic cultures of P. taeda, P. sylvestris or Picea abies. Comparative and Functional Genomics 3:306-318.
  • van Zyl, L., Bozhkov, P.V., David H. Clapham, D.H., Sederoff, R.R.& von Arnold, S (2003) Up, down and up again is a signature global gene expression pattern at the beginning of gymnosperm embryogenesis. Gene Expression Patterns 3: 83-91.
  • Wenck, A.R., Quinn, M., Whetten, R., Pullman, G., and Sederoff, R.R. 1998. High efficiency Agrobacterium mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda). Plant Molecular Biology pp 1-10.
  • Whetten, R.W., Mackay, J.J., and Sederoff, R.R. 1998. Recent Advances in Understanding Lignin Biosynthesis. Annual Rev. Plant Physiol. Plant. Mol. Biol. 49:585-609.
  • Zhang, Y., Brown, G., Whetten, R., Loopstra, C.A., Neale, D.B., Kielisewski, M., and Sederoff, R.R. (2003). An Arabinogalactan-Protein Associated with Secondary Cell Wall Formation in Differentiating Xylem of Loblolly Pine. Plant Mol. Biol. In press.
  • Zhang, Y., Sederoff, R.R., and Allona, I. 2000. Differential expression of genes encoding cell wall proteins in vascular tissues from vertical and bent pine trees. Tree Physiology 20:457-466.
  • Lapierre, C, Pollet, B., MacKay, J.J., and Sederoff, R.R. 2000. Lignin structure in a mutant pine deficient in cinnamyl alcohol dehydrogenase. J. Ag. Food Chem. 48:2326-2331.
  • Allona, I., Quinn, M., Shoop, E., Swope, K., St. Cyr., S., Carlis, J., Riedl, J., Retzel, E., Campbell, M.M., Sederoff, R.R., and Whetten, R. 1998. Analysis of xylem formation in pine by cDNA sequencing. PNAS 95:9693-9698.
  • Dimmel, D.R., MacKay, J.J. Althen, E.M., Parks, C., and Sederoff, R.R. 2001. Pulping and bleaching of CAD deficient wood. J. of Wood Chemistry and Technology. 21:1-17.
  • Heath, L. S.; Ramakrishnan, N.; Sederoff, R. R.; Whetten, R. W.; Chevone, B. I.; Struble, C. A.; Jouenne, V. Y.; Chen, D. W.; Van Zyl, L.; Grene, R. (2002) Comparative and Functional Genomics. Studying the functional genomics of stress responses in loblolly pine with the Expresso microarray experiment management system. 3:226-243.
  • Kim,H., Ralph,J., Lu,F., Ralph, S.A., Boudet, A-M. John J. MacKay, J.J., Sederoff, R.R., Ito, I., Kawai, S., Ohashi, H., & Higuchi, T. (2003) NMR analysis of lignins in CAD-deficient plants. Part 1. Incorporation of hydroxycinnamaldehydes and hydroxybenzaldehydes into lignins. Org. Biomol. Chem. 1: 268-281.
  • Kirst, M., Johnson, A., Baucom, C., Bonner, E., Hubbard, K., Staggs, R., Paule, C., Retzel, E., Whetten, R., and Sederoff, R. (2003). Apparent homology of expressed genes from wood-forming tissues of loblolly pine (Pinus taeda L.) with Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA. In press.


Progress 01/01/01 to 12/31/01

Outputs
Molecular Basis for Wood Formation The purpose of this project is to understand the molecular basis of wood formation. In the past year, work has focused on the identification of genes expressed during wood formation in loblolly pine (Pinus taeda). Using DNA sequencing applied to cDNA libraries made from wood forming tissues, we identified 75,000 ESTs containing 17,000 unique sequences (comprising a unigene set). We estimate that this represents at least 10,000 unique expressed genes. A major question about genes in wood forming tissues is the extent to which they represent genes unique to woody plants, or if similar genes are expressed in all plants. In this latter case, the ability to form wood would be due, not to unique genes, but to genes common to higher vascular plants, therefore, regulated in different ways to produce specialized woody cells and tissues. Therefore, we compared our expressed genes from wood forming tissues of pine to the total genome sequence of the model herbaceous plant Arabidopsis thaliana. If we compare simple EST sequences, then only half of the pine cDNAs have apparent homology. However, as we look at assemblages of overlapping sequences (contigs), we find that the level of apparent homology increases greatly. As the length increases, we find essentially all pine expressed genes have homologs in Arabidopsis. This does not appear to be a bias due to different levels of homology for abundant sequences. The time since pine and Arabidopsis last shared a common ancestor is estimated at 300 million years ago. To maintain conservation over this time period requires that such genes are functional and maintained by selection. Our results lead to a model of higher plant evolution that proposes the existence of a core of common functional genes in all vascular plants that have diverged only at the level of expansion and divergence of members of gene families. If this is true, then the morphological, physiological and chemical diversity of higher plants is based on the differential divergence and expression of a common set of functional genes, recognizably conserved until the present day.

Impacts
The results provide the first comprehensive model for studies of the genomic evolution of all seed plants. In addition, it predicts that Arabidopsis is a valuable genetic model for forest trees. The results are the first comprehensive comparison of the expressed genes of a gymnosperm and an angiosperm. The results should have a significant impact in plant genomics, vascular plant evolution, and forest biotechnology.

Publications

  • Whetten, R. Sun, Y-H., Zhang, Y. and Sederoff, R. 2001. Functional genomics and cell wall biosynthesis in loblolly pine. Plant Mol. Biol. 47: 275-291
  • Kirst M., A. Johnson, R. Ernest, L. van Zyl, D. Craig, Z. Jun Li, R. Whetten, R., C. Baucom, E. Ulrich, K. Hubbard and R. Sederoff. 2002. Quantitative influence in fuctional genomics of loblolly pine (Pinus Taeda L.) using ESTs and microarrays. International Association for Plant Tissue Culture & Biotechnology (IAPTC&B),10th International Congress of Plant Tissue3 Culture & Biotechnology, June 23-28, 2002, Orlando, FL, USA.
  • Kirst, M., A. Johnson, E. Retzel, R. Whetten, J. Vasques-Kool, D. O'Malley, C. Baucom, E. Bonner, K. Hubbard and R. Sederoff. Homology of expressed genes from wood forming tissue of loblolly pine (Pinus taeda L.) with Arabidopsis thaliana. 2002. Manuscript


Progress 01/01/00 to 12/31/00

Outputs
Parallel studies of signal transduction pathways and transcriptional regulation have made good progress during the last year. The basic helix-loop-helix domain of a pine transcription factor was used in a yeast two-hybrid screen, and several cDNAs encoding proteins that interact with the bHLH domain in yeast were isolated. These putative interaction partners are candidates for members of the signaling and regulatory pathways upstream and downstream of the bHLH protein. Many transcription factors are regulated by interactions with other proteins, and further studies of the physiological role of the bHLH protein in loblolly pine will be greatly advanced now that candidate interaction partners are available for testing and experimental analysis. Studies of signal transduction pathways focused on a mitogen-activated protein kinase (MAPK). This kinase is present and active in differentiating pine secondary xylem, implying that the signaling cascade of which it is a part is transducing signals. The MAPK protein is detectable by antibodies in extracts of both early-stage differentiating xylem, prior to secondary cell wall deposition and lignification, and in late-stage differentiating xylem after secondary cell wall synthesis and lignification. The activity of the MAPK, as measured in immunoprecipitated proteins, decreases dramatically from early-stage tissue to late-stage tissue, suggesting that the signaling is important in the early stages of secondary xylem differentiation, but less important in later stages. This suggests that the signaling may be involved in cell division or radial expansion, as those are processes in which early-stage cells in differentiating xylem are actively engaged but late-stage cells are not. A key technology for testing hypotheses about signaling pathways and transcriptional regulation in pine is gene transfer. The ability to make transgenic pine plants is central to further studies of tree growth and development. Significant progress has been made on transformation of loblolly pine mature zygotic embryos using Agrobacterium tumefaciens. This method has two major advantages over the existing technique for gene transfer in loblolly pine. The existing method uses microprojectile bombardment as a means of introducing DNA into somatic embryogenic cultures initiated from immature embryos. Initiation and maintenance of somatic embryogenic cultures of loblolly pine requires a significant investment of time and effort that many laboratories are not equipped to make. Mature zygotic embryos can be isolated from seeds, which are available in bulk and can be stored for more than a year. This greatly simplifies the process of obtaining suitable tissue explants for transformation. Agrobacterium transformation has advantages over microprojectile bombardment as a means of introducing DNA into tissue explants; the frequency of single-copy or low-copy insertions is typically higher in Agrobacterium experiments, which means that there is a lower frequency of gene silencing and other unpredictable side effects of transformation.

Impacts
A better understanding of the limitations on forest productivity requires more knowledge of the mechanisms that allow trees to detect environmental signals such as temperature, wind, and nutrient deprivation, and respond to those changes by altering patterns of gene expression. This research project is focused on gaining an understanding of some of the mechanisms that allow loblolly pine trees to detect environmental signals and respond by changing rates of gene expression. A new method for experimental gene transfer into loblolly pine, starting with tissue obtained from mature seeds, has been developed to allow testing of new ideas about how gene regulation works in pine.

Publications

  • No publications reported this period


Progress 01/01/99 to 12/31/99

Outputs
Progress has been made on several aspects of this project. A gene transfer method has been developed for loblolly pine, a genetic map of AFLP markers has been completed for loblolly pine genotype 7-56, and molecular genetic analysis of signal transduction pathways in differentiating pine secondary xylem is proceeding. Experiments on gene transfer in loblolly pine have yielded a robust method for regeneration of transgenic pine from Agrobacterium-inoculated organogenic calli. This method will be useful in testing hypotheses about gene function during wood formation, to better understand the mechanisms that control tree growth and development under a variety of environmental conditions. Initial experiments utilizing this new gene transfer method are underway to analyze the molecular basis of regulation of a gene encoding phenylalanine ammonia-lyase (PAL), an enzyme catalyzing a key step in the biosynthesis of lignin. Genetic analysis has long played a key role in investigation of pine growth and development, and the new technology of AFLP mapping has made a much more detailed genetic map available. Pine genotype 7-56 is very important in tree breeding and genetic experiments because of the high frequency of desirable characteristics in its progeny, and the availability of a detailed genetic map for this individual will enable a thorough analysis of the genetic basis for this trait. Other areas of progress include investigations of the role that protein kinase pathways may play during xylem differentiation, and of interactions between transcription factors and other proteins in differentiating xylem cells. These projects are focused on understanding the ways in which cells in differentiating pine xylem perceive and respond to environmental and developmental signals.

Impacts
A better understanding of the process of wood formation in loblolly pine can contribute to more efficient utilization of forest products, both for existing uses such as paper and lumber and for new applications such as renewable energy and chemical feedstocks. Sustainable use of forest resources requires a balance between intensively managed plantations and natural stands, and plantation-grown trees can be better-adapted to commercial use only in the context of knowledge about how wood formation is controlled at the molecular level.

Publications

  • Wenck, A. R., Quinn, M., Whetten, R. W., Pullman, G., and Sederoff, R. 1999. High efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda). Plant Mol. Biol. 39:407-416
  • Remington, D.L., Whetten, R.W., Liu, B.-H., and O'Malley, D. M. 1999. Construction of an AFLP genetic map with nearly complete genome coverage in Pinus taeda. Theor. Appl. Genet 98:1279-1292