Source: CLEMSON UNIVERSITY submitted to
STUDY OF CINNAMYL ALCOHOL DEHYDROGENASE GENES IN TULIP-POPLAR AND MODIFICATION OF CELL WALL TO ENHANCE FIBER EXTRACTABILITY IN POPLAR
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0210317
Grant No.
(N/A)
Project No.
SC-1700324
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Feb 1, 2007
Project End Date
Jun 30, 2012
Grant Year
(N/A)
Project Director
Liang, HA.
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
Genetics and Biochemistry
Non Technical Summary
Lignin degradation is of central importance in biomass utilization. It is essential to explore approaches that can help break down lignin more efficiently. The purposes of this proposal are to characterize cinnamyl alcohol dehydrogenase genes from tulip-poplar and their promoters, and to test a novel approach to facilitate lignin digestibility.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2010699104050%
2040699100050%
Goals / Objectives
1) Cloning and characterization of cinnamyl alcohol dehydrogenase (CAD) genes from tulip-poplar (Liriodendron tulipifera L.): A. Cloning of full length cDNA encoding cinnamyl alcohol dehydrogenese (CAD) from tulip-poplar and its promoter; B. Transformation of Arabidopsis CAD double mutant with Ltu CAD cDNA sequence; C. Characterization of CAD promoter in Arabidopsis using a uidA gene. D. Genomic sequence comparison of Ltu CAD with homologs from Arabidopsis, rice, Medicaco, and poplar. 2) Modification of lignin by protein cross-linking to facilitate production of biofuels from poplar: A. Design of tyrosine-rich peptide genes and vectors; B. Poplar transformation with tyrosine-rich peptide genes; C. Characterize the transgenic plants for plant fitness, tissue strength, lignin morphology and viscoelastic properties, lignin composition and structure; D. Small scale processing tests for the effect of the protease treatment on both pulping and ethanol production.
Project Methods
1) Cloning and characterization of cinnamyl alcohol dehydrogenase (CAD) genes from tulip-poplar (Liriodendron tulipifera L.) Full length cDNA sequence of putative CAD gene(s) will be obtained with a SMARTTM RACE cDNA Applification kit (Clontech) and cloned into pCAMBIA1301 vector gene by replacing the GUS gene. The CAD gene will be driven by a constitutive CaMV 35S promoter. The cinnamyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD) activities of the Liriodendron CAD gene will be tested by molecular complementation in an Arabidopsis CAD-C, D double mutant. Expression patterns of the Ltu CAD promoter will be characterized in Arabidopsis. The LtuCAD promoter will be cloned into pCAMBIA 1391 xb binary vector to drive the expression of uidA gene. The functions of the LtuCAD will be further tested by suppression of CAD expression in Liriodendron RNAi lines. Four CAD positive BAC clones have been identified with LtuCAD EST sequence as probe. These four clones will be fingerprinted. If they represent different CAD genes, these BAC clones will be sequenced by using advanced sequencing technology GS20 system. Sequence comparative studies will be conducted within tulip-poplar CAD genes and among tulip-poplar, rice, Arabidopsis, Medicao, and Populus (which represent major lineages in phylogenetic tree). 2) Modification of lignin by tyrosine-rich protein cross-linking to facilitate production of biofuels from poplar To determine the effect of primary and secondary structure of the Tyr-rich peptides, several tyrosine-rich peptide genes will be created, varying in length and sequence. Hybrid poplar Ogy will be transformed with the Tyr-rich protein genes via Agrobacterium tumefaciens. Each Tyr-rich gene will be fused to a leader sequence to direct secretion into the cell wall. A promoter from a hybrid poplar PAL2 will be used to drive the expression of transgenes. Analysis of lignin in transformed plants will include lignin histological staining, Klason lignin analysis, and thioacidolysis. Localization of tyrosine-rich peptides will be determined by immunolocalizations. Evaluating lignin cross linking and lignin tyrosine interactions will be investigated. Small scale pulping and ethanol production tests will be conducted. To determine whether the introduction of a tyrosine-rich peptide gene would compromise tree health, disease susceptibility of the transgenic plants to Septoria musiva will be evaluated.

Progress 02/01/07 to 06/30/12

Outputs
OUTPUTS: We have built a comprehensive Expressed Sequence Tag (EST) dataset for yellow-poplar (Liriodendron tulipifera) from 10 different tissue types, mined this dataset for cinnamyl alcohol dehydrogenase (CAD) family genes, and conducted phylogenetic analysis. Spatial expression of the LtuCADs has been determined by real-time RT-PCR in leaves, roots, buds, and xylem. We have also characterized LtuCAD1 and LtuCAD2 in Arabidopsis CAD4 and CAD5 double mutant, examined the promoter of LtuCAD1 and LtuCAD2 through silico motif mining and expressing GUS fusion construct in Arabidopsis, and compared gene order in a 122kb contig harboring the LtuCAD1 gene with its corresponding region from other angiosperm species. We have mined the yellow-poplar EST dataset for SSR makers. Currently we are testing the SSR markers for polymorphism. We will use 200 markers to build the first framework genetic linkage map of yellow-poplar with a full sib population of 400 progeny. Meanwhile, we have generated the first generation of transgenic poplar transformed with the gene encoding the tyrosine-rich protein. We have characterized these transgenic poplars in respect to fitness (growth and Septoria musiva susceptibility) and their use for biofuels. We also conducted microarray experiments on these transgenic poplars to identify genes that have been affected by the introduction of the tyrosine-rich peptide gene. A total of 10 genes from the affected gene list were chosen to validate the microarray results by real-time RT-PCR. Lastly, we have generated at least 20 transgenic lines for 8 newly constructed binary vectors. We will characterize these new lines for fitness and bioethanol production. This project helped train one postdoctoral fellow, one technician, one visiting scholar, two Ph.D. students, five undergraduate students, and five high school students. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This project focused on understanding the lignin biosynthesis and its regulatory mechanism, which are essential for developing cultivars with desired lignin traits for biofuel and timber production. Both tulip-poplar and poplar are trees with significant ecological and economic values and have the potential to be domesticated for renewable energy production. With support from PSA and other resources, the research project has enriched people's lives and provided education. The yellow-poplar EST dataset has facilitated gene discovery, evolutionary study, and molecular breeding in Liriodendron. The identification of the LtuCADs, especially the unraveling of the functions of LtuCAD1 and LtuCAD2, have filled in the gap of our knowledge with CAD gene family in basal angiosperm species. The LtuCAD1 and LtuCAD2 promoters can be utilized in genetic engineering to drive the expression of genes of interest. The SSR markers identified in the yellow-poplar EST dataset are being applied in assisting analyses of parental exclusion, genetic diversity and structure, and genetic mapping. Our approach of lignin modification via introducing crosslinkings in cell wall provides a unique way to facilitate production of biofuels from poplar without dramatic change in lignin quantity. The transgenic poplars we obtained are more accessible to lignin extraction by pretreatment with proteases. This will increase biomass conversion efficiency and decrease processing cost. Currently pretreatment is one of the most expensive steps in the conversion of lignocellulosic material to fermentable sugars. Additionally, the overall conversion is poor, due largely to the efficacious nature of lignin in limiting enzymatic access to cellulose and hemicelluloses.

Publications

  • No publications reported this period


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We successfully germinated 40 open-pollinated yellow-poplar seedlings. Using these materials, we have tested 66 molecular markers (EST-SSR) and identified 35 pairs that are polymorphic. Parentage exclusion analysis to generate a full-sib population and a framework genetic linkage map will be the next goals. We have generated transgenic Arabidopsis and poplar plants expressing the yellow-poplar cinnamyl alcohol dehydrogenase 1 (LtuCAD1) or the tyrosine-rich peptide gene (TYR). Wood chemistry analyses (lignin content, S/G lignin ratio, 5-carbon and 6-carbon sugars) on these analyses were complete. Previously a gene expression study using whole genome DNA microarrays revealed a group differentially expressed transcripts in the TYR transgenic poplar plants, with all their transcript abundance decreased. Our recent analyses showed that these reduced transcripts were highlighted with biochemical pathways in biosynthesis of secondary metabolites, amino acid metabolism and energy metabolism. Transcript abundance was found decreased primarily for five types of genes presumptively encoding proteins involved in cell wall organization and in lignin biosynthesis. Validation close to 90% of 20 selected genes from the microarray results were replicated by qPCR experiments, suggesting the robustness of the gene expression pattern. One technician, one visiting scholar, two Ph.D. students, three undergraduate students, and one high school student were trained in 2011. The PD team taught two graduate courses (GEN 814 Advanced Genetics, GEN 890 Special Topics on Genetics) and one large undergraduate course (GEN 302 Fundamental Genetics, 110 students) and its accompanying lab course (GEN303). Lastly, our results provide the first gene expression evidence for the basis of the flexible and degradable cell wall phenotype obtained in the transgenic poplar plants. Because of the great progress on yellow-poplar, the PD is now a part of a multi-institutional NSF-funded project on genomics resource development for several hardwood species, in which yellow-poplar is included. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project focuses on understanding the lignin biosynthesis and its regulatory mechanism, which are essential for developing cultivars with desired lignin traits for biofuel and timber production. Both tulip-poplar and poplar are trees with significant ecological and economic values and have the potential to be domesticated for renewable energy production. With support from PSA and other resources, this project has enriched people's lives and provided education. I have provided research experience for 17 high school and undergraduate students either through a summer internship, or an independent research course, or volunteering. I have taken nine (9) Clemson undergraduate students and two (2) faculty members to experience woody plant genomics research in China during the summers of 2010 and 2011 for 10-14 days per trip. Seven of the participating students were female, five were African American, and five were South Carolina residents. Such experience has not only helped students appreciate differences in people and cultures, but also provided an opportunity for the students to relate what they learned in class to real research projects and applications in the field. One of the participating students is now working as a lab technician in a plant molecular lab at North Carolina A&T State University, and two are currently conducting independent research projects with plants at Clemson University. The trip has also encouraged one African American female student, who had not previously traveled outside South Carolina, to participate in an internship that was in another state and to consider attending graduate school. The participating faculty members were rewarded with potential collaboration with Chinese scientists working with fruit and forest trees. The involved projects have also helped create jobs by supporting one technical specialist and one postdoctoral fellow, in addition to the financial support and training of two Ph.D. students.

Publications

  • (2011) H. Liang, S. Ayyampalayam, N. Wickett, A. Barakat, Y. Xu, L. Landherr, P. Ralph, T. Xu, S.E. Schlarbaum, J. H. Leebens-Mack, C.W. dePamphilis. Generation of a large-scale genomic resource for functional and comparative genomics in Liriodendron. Tree Genetics and Genome. 7: 941-954.
  • (2011) H. Liang, A. Barakat, S.E. Schlarbaum, J.E. Carlson.: Organization of the chromosome region harboring a FLORICAULA/LEAFY gene in Liriodendron. Tree Genetics and Genomes. 7: 373-384.
  • (2011) Y Jiao, N Wickett, S Ayyampalayam, A Chanderbali, L Landherr, PE Ralph, LP Tomsho, H Liang, PS Soltis, DE Soltis, SE Clifton, SE Schlarblum, SC Shuster, H Ma, J Leebens-Mack, CW dePamphilis. Phylogenomic detection of ancient polyploidy in seed plants and angiosperms. Nature 473:97-100 (2011) H Liang, J Romero-Severson, SE Schlarbaum, MVCoggeshall, O Gailing, KL Shumaker, M Staton, NC Wheeler, JE Carlson. Comparative Genomics of Environmental Stress Responses in North American Hardwoods. Southern Forest Tree Improvement Conference, Biloxi, Mississippi, June (Oral presentation)
  • (2011) Y Jiao, NWickett , S Ayyampalayam, A Chanderbali , L Landherr, P Ralph , L Tomsho , Y Hu , H Liang, D Soltis, S Clifton, S Schlarbaum, S Schuster, H Ma, J Leebens-Mack, CdePamphilis: Ancestral Polyploidy In Seed Plants And Angiosperms. Plant and Animal Genome conference, San Diego, Jan, 16-19, 2011. (Oral presentation)
  • (2011) H Liang, T Xu, S Thammannagowda. RNA isolation from several hardwood species for transcriptome sequencing and SSR marker development for yellow-poplar. Hardwood Genomics project annual meeting, Penn State Mont Alto, July.(Oral presentation)
  • (2011) Yi Xu, Abdelali Barakat, Scott E. Schlarbaum, H Liang: Molecular Characterization of A Cinnamyl Alcohol Dehydrogenase Homolog In Liriodendron tulipifera L., A Basal Angiosperm Species. Plant and Animal Genome Conference, Jan. 16-19, 2011, San Diego; Southern Forest Tree Improvement Conference, Biloxi, Mississippi, June (poster presentation)
  • (2011) Xi Xu, S.E. Schlarbaum, H Liang. Characterization of a cinnamyl alcohol dehydrogenase homolog in Liriodendron tulipifera. Journal of Systematics and Evolution. 49 (5): 396-405.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: We have built the most comprehensive Expressed Sequence Tag (EST) dataset for yellow-poplar (Liriodendron tulipifera) from 10 different tissue types, mined this dataset for cinnamyl alcohol dehydrogenase (CAD) family genes, and conducted phylogenetic analysis. Spatial expression of the LtuCADs has been determined by real-time RT-PCR in leaves, roots, buds, and xylem. We have also characterized the LtuCAD1 in Arabidopsis CAD4 and CAD5 double mutant and compared gene order in a 122kb contig harboring the LtuCAD1 gene with its corresponding region from other angiosperm species. Real-time RT-PCR with 25 genes obtained from microarray experiments on the transgenic poplar plants with a flexible wall has been completed. One technician, one visiting scholar, one Ph.D. student, two undergraduate students, and one high school student were trained in 2010. The Ph.D. co-taught one graduate course (GEN 814 Advanced Genetics) and guest-lectured one undergraduate course (GEN/BIOCH 101 Careers in Genetics and Biochemistry). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Built from ten different types of tissues (premeiotic flower buds, post meiotic flower buds, open flowers, developing fruit, terminal buds, leaves, cambium, xylem, roots and seedlings), the new Liriodendron tulipifera EST dataset contained 137,923 unigenes (132,905 contigs and 4,599 singletons). About 50% of the unigenes had significant matches to publicly available plant protein sequences, representing a wide variety of putative functions. Seven full-length cinnamyl alcohol dehydrogenase (CAD) family genes were identified, representing the three classes of CAD genes found in other plant species. Real-time RT-PCR indicated that LtuCAD1 and LtuCAD4 shared a similar expression pattern, with higher expression in xylem. LtuCAD3 showed a similar expression level in xylem and root, with much lower expression in bud (1/100 of the expression level in root). In comparison, LtuCAD2, LtuCAD5, LtuCAD6, and LtuCAD7 had a high expression level in roots. LtuCAD1 and LtuCAD6 had the highest expression in xylem and root, respectively. The phylogenetic tree suggests that LtuCAD1 is the primary CAD genes involved in lignifications since it is the only Liriodendron CAD grouped with the bona fide CADs class, which include the well characterized Arabidopsis CAD-C and CAD-D. When over-expressed in the Arabidopsis Cad4 and 5 double mutant, the LtuCAD1 was able to partially complement the phenotypes of the double mutant. Besides the LtuCAD1, the 122-kb BAC contig contained fragmented sequences for one integrase, eight hypothetical proteins, two gag-pol polyproteins, one RNase H family protein, and one chromatin binding protein. The transposable elements covered 9.0% of the contig. Comparative analysis with other angiosperm species suggests that the genomic segment in this BAC has undergone frequent arrangement. Microarray analysis identified 102 down-regulated genes. No up-regulated genes were found. Detailed functional annotation of the affected genes was obtained by Gene Ontology slim terms: 64.7% have predicted cellular components, 69.6% can be assigned putative molecular functions, and 53.2% are given a biological process prediction. Four biological processes are significantly enriched: lignin metabolic process, phenylpropanoid metabolic process, cellular amino acid derivative metabolic process, and defense response. The four genes involved in lignin metabolic process were identified as cinnamoyl-CoA reductase 1 (CCR1), hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyltransferase gene, laccase 4 (LAC4), and laccase 17 (LAC17). An association analysis of Gene Ontology terms with FatiGO also singled out lignin metabolic process as significantly affected. When validated by real-time RT-PCR with 25 genes, 80% of the genes showed down-regulated in the transgenic poplar plants when compared to the wild-type control.

Publications

  • H Liang, A Barakat, SE Schlarbaum, JE Carlson (2011) Organization of the chromosome region harboring a FLORICAULA/LEAFY gene in Liriodendron. Tree Gen. Genet. DOI 10.1007/s11295-010-0338-2.
  • H Liang. Genetic engineering for Septoria disease resistance in hybrid poplar, THE CONSORTIUM FOR PLANT BIOTECHNOLOGY RESEARCH, INC. Washington DC, February. 2010 (Poster)
  • H Liang, A Barakat, JE Carlson: Organization Of The Chromosome Region Containing A Floricaula/Leafy Gene In Liriodendron. Presentation, Plant and Animal Genomes XVIII Conference, January, 2010. (Poster)
  • (2010) NJ Wickett, Y Zhang, Y Jiao, S Ayyampalayam, AS Chanderbali, PK Wall, H Liang, LLScheaffer, P Ralph, S Schuster, H Ma, PSSoltis, DE Soltis, SClifton, JE Carlson, J Leebens-Mack, CW dePamphilis. Evolution Of Floral Development Genes And Gene Families In Basal Angiosperms. Plant and Animal Genomes XVIII Conference, January, 2010 oral presentation
  • H Liang, Zhebentyayeva T, Olukolu B, Wilde D, Reighard GL, Abbott A (2010) Comparison of gene order in the chromosome region containing a TERMINAL FLOWER 1 homolog in apricot and peach reveals microsynteny across angiosperms. Plant Science 179:390-398.
  • H Liang, Barakat A, Schlarbaum SE, Mandoli DF, Carlson JE (2010) Comparison of gene order of GIGANTEA loci in yellow-poplar, monocots, and eudicots. Genome 53: 533544.
  • Authored or co-authored oral or poster abstracts at international and national scientific meetings: H Liang, Y. Xu, C-F Chen, M. Marshall, N.R. Brown, J.E. Carlson, M. Tien. Molecular dissection of a poplar mutant with a more flexible and digestible cell wall. 2nd Pan American Congress on Plants and BioEnergy, August 8-13, Sao Pedro, SP, Brazil. 2010 (Poster)
  • J. Carlson, F Cong, C Frosta, Y Xu, H Liang, B Diehla, N Browna, M Tien. Transgenic Hybrid Poplar with Enhanced Accessibility of Cellulose and Hemicellulose. 2010 International Workshop on Wood Biorefinery and Tree Biotechnology, 21-23 June 2010, Sweden. 2010 (Oral presentation)


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: We have finished the characterization of the yellow-poplar sinapyl alcohol dehydrogenase (SAD) promoter. A 122kb contig harboring a cinnamyl alcohol dehydrogenase (CAD) gene has been sequenced and assembled. Collection of 10 different tissue types, premeitotic flower buds, post meiotic buds, open flowers, developing fruit, terminal buds, leaves, roots, seedling, cambium, xylem, has been complete for yellow-poplar. In collaboration with Dr. C. DePamphilis at the Penn State, 454 sequencing has been carried out for the xylem and cambium tissues, along with other tissue types. For the poplar lignin modification project, we have generated at least 10 transgenic lines for 8 newly constructed binary vectors. Microarray experiments have completed for the first generation of the transgenic poplar plant. Pathogen tests and growth measurements are also complete. One Ph.D. student, five undergraduate students and one high school student were trained in the year of 2009. A CAREER proposal has been submitted to NSF for yellow-poplar based on the results achieved to date and two manuscripts are expected in 2011. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Characterization of the yellow-poplar SAD promoter indicated that SAD is mainly expressed in vascular tissues, in consistence with its functions in lignin biosynthesis. This promoter can be used to drive the expression of beneficial genes in yellow-poplar and other plant species. Sequencing and assembly of the yellow-poplar CAD BAC clone generated a 122 kb contig. The structure of a CAD gene from a basal angiosperm species is revealed for the first time. This big contig will provide the necessary information to study the genome organization in a basal angiosperm species. The sample collections for 10 different yellow-poplar tissue types have provided the materials for the NSF-funded project "Ancestral Angiosperm Genome Project" led by Dr. C. DePamphilis at the Penn State. A new deep EST database is now available for yellow-poplar, which include 2,012,281 reads and 113,473 unigenes. Microarray analysis indicated that no genes were unregulated in the transgenic plants while there were 176 genes were down-regulated. qRT-PCR is being conducted to confirm the expression of these genes. The information obtained from this will shed light in to the mechanisms behind the more flexible cell wall achieved in the transgenic plants. The pathogen tests with Septoria musiva and growth measurements showed that there was no significant difference between wildtype and transgenic plants, implying that we can achieve a more flexible cell wall which is beneficial to lignin extraction, while the fitness of the plants unaffected. In addition, new transgenic poplar plants have been generated for 8 newly constructed vectors. These are the materials we will focus on in 2010. A renewal proposal has been submitted to DOE.

Publications

  • Wall PK, Leebens-Mack J, Chanderbali A, Barakat A, Liang H, Landherr L., Tomsho LP, Hu Y, Carlson JE, Ma H, Schuster S, Soltis DE, Soltis PS, Altman N, and dePamphilis CW. (2009) Comparison of Next Generation sequencing technologies for de novo transcriptome characterization. BMC Genomics 10:347.
  • Four poster abstracts at international and national scientific meetings: The IUFRO Tree Biotechnology Conference (Whistler, Canada, June 2009) The Plant Biology meeting (Hawaii, July, 2009) The 9th International Plant Molecular Biology (IPMB) Congress (St. Louis, MO, October, 2009)
  • Manuscript in Review: Comparison of gene order of GIGANTEA loci in yellow-poplar, monocots and eudicots. Haiying Liang, Abdelali Barakat, Scott E. Schlarbaum, Dina F. Mandoli, John E. Carlson. Submitted to Genome in October, 2009
  • H. Liang, P.A. Kumar, V. Nain, W.A. Powell, J.E. Carlson (2009 accepted) Selection and Screening Strategies. In: C. Kole, C.H. Michler, A.G. Abbott and T.C. Hall (eds) Development and Deployment of Transgenic Plants. Springer-Verlag, Berlin, Heidelberg, New York, Tokyo, pages 85-144.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: We have finished the pBIN-based binary cloning of the full-length cDNA sequences of yellow poplar cinnamyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD), as well as the pCAMBIA1369 binary vector cloning of the CAD and SAD promoters. Currently the pBIN/CAD_cDNA and pCAMBIA1369/CAD_promoter construct s have been transferred in to Arabidopsis and/or poplar. Characterizations are being conducted. For the poplar lignin modification project, we compared the digestibility of the transgenic and wild-type stem tissue using a sequential treatment of protease K followed by cellulase/hemicellulase. The concentration of reducing sugar in each sample was detected by tetrazolium blue method and represented as milligrams of reducing sugar per milliliter per gram of stem tissue. Seven transgenic lines were included, and there were at least two experimental repeats for each sampling plant. Of the lines we surveyed, two (lines T1 and T7) showed significant differences in the amount of sugar released from stem digestions pre-treated with protease K, relative to those without protease treatment (p=0.0017, 0.0380, respectively for T1 and T7). In one of these cases (line T1), the digestibility of the non-protease treated stem tissue was similar to the wildtypes. However, in most of the transgenic lines, significantly more polysaccharides were released than with wildtype, independent of protease K treatment. We also analyzed the wood tensile strength with a Dynamic Mechanical Analyzer DMA 2980 on wildtype and five transgenic poplar plants. We included two different stem samples for each transgenic line, each from the same sapling. Our data indicated reduced tensile strength in transgenic lines, except in transgenic line T22 (Alpha=0.05, as tested by ANOVA with the Tukey pairwise comparisons test). We are continuing to maintain and propagate transgenic and wild-type plants in cultures and greenhouse. We are currently conducting pathogen/herbivore tests and generating more transgenic plants carrying different versions of tyrosine-rich peptide genes.In the study of plant/herbivore interactions, we have cloned the full-length cDNA of an allene oxide synthase (AOS) gene and a Gal83 encoding the beta-subunit of SnRk1 (sucrose nonfermenting 1-related kinase) from poplar. Allene oxide synthase is a member of the cytochrome P450 family. This enzyme converts lipoxygenase-derived fatty acid hydroperoxide to allene epoxide, which is the precursor for jasmonic acid (JA) formation. JA is a plant growth regulator and involved in development and in response to biotic and abiotic stress. Gal83 is involved in plants and herbivory tolerant response through sucrose repression. We have insert the full-length cDNA sequences of these two genes in an overexpression vector, respectively. Two Ph.D. students, five undergraduate students and one high school student were trained in the year of 2008. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Yellow-poplar (Liriodendron tulipifera L.) and Populus species are of economic and ecological importance, and have the potential to be a feedstock resource for renewable energy production. Our studies focus on the molecular mechanisms of lignin biosynthesis and interactions with herbivores. Our findings will provide necessary information to improve biomass production and bioconversion efficiency in these two species.

Publications

  • H Liang, CJ Frost, X Wei, NR Brown, JE Carlson, M Tien (2008) Improved sugar release from lignocellulosic material by introducing a tyrosine-rich cell wall peptide gene in poplar. Clean-Soil, Air, Water 36 (8): 662-668
  • One oral and two poster abstracts at international and national scientific meetings: The Pan American Congress on Plants and BioEnergy (Mexico, June, 2008), The Plant Biology meeting (Mexico, June/July, 2008).


Progress 01/01/07 to 12/31/07

Outputs
We have 1) cloned full-length cDNA sequences of cinnamyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD) from yellow poplar, and prepared two over-expression binary vectors for these two genes, driven by a constitutive 35S promoter; 2) finished a RNAi construct for the yellow poplar CAD gene; 3) cloned approximately 2kb promoter regions of the CAD and SAD genes by Genome Walking, and inserted them into a pCAMBIA1369 vector, respectively, to drive the expression of a scoreable beta-glucuronidase (GUS) maker gene. Characterization of the CAD/SAD genes and promoters in poplar and Arabidopsis are ongoing. Meanwhile, we have generated the first generation of transgenic poplar transformed with the gene encoding the tyrosine-rich protein (called glycine rich protein). We have initiated the work to characterize this first generation of poplar in respect to fitness and also to their use for biofuels. We are preparing materials for pathogen tests, in vitro digestibility, immuno-locaization, small scale pulping and ethanol production studies. We also have initiated antibody production using the first tyrosine-rich peptide as the antigen through Promab Biotechnology, Inc. The antibody will be used to localize the crosslinking peptide in the cell wall and confirm its expression in the cell wall. In preparation for the fitness tests, we have requested USDA-APHIS ePermit to move poplar pathogen Septoria musiva from Wisconsin. Other progress involves acclimating micropropated (tissue culture) transgenic and wild-type poplar plants in real soil and grew them in greenhouse. We are also active in maintaining the transgenic and wild-type poplar plants in tissue culture and propagating explants for transformation. Three undergraduate students (2 semesters), one high school student (one summer), and a Ph.D. student (one summer) were trained in this project. The high school intern and PI were interviewed by The Greenville News and appeared in the newspaper. A Ph.D. student has been recruited.

Impacts
Yellow-poplar (Liriodendron tulipifera L.), a basal angiosperm species, is one of the feedstock materials being studied for the conversion of woody biomass to biofuels, such as ethanol. Because of its rapid growth and biomass accumulation, unusual resistance to insects and disease, and its niche as an aggressive pioneer tree species on disturbed lands, yellow-poplar has great future potential to be domesticated as a source of fiber for biologically-based products, for biofuels, and chemicals. To date, little is known about cell wall biosynthesis in basal angiosperm species, our best living record of the origin of all hardwood tree species. A functional study of genes and proteins involved in cell wall biosynthesis in yellow-poplar will help fill that gap and get us much closer to understanding the key, universal features of cell wall biosynthesis in woody plants. The functions of CAD/SAD genes and their promoters are currently being characterized in planta. Meanwhile, sequence comparisons and expression profiles of the CAD and SAD genes are being investigated. Our findings will provide necessary information as to how to improve yellow poplar as a feedstock resource for renewable energy production. Our proposed study on modification of lignin structure by tyrosine-rich protein cross-linking to facilitate production of biofuels from poplar will: 1) Increase biomass conversion efficiency and decrease processing cost. Currently pretreatment is one of the most expensive steps in the conversion of lignocellulosic material to fermentable sugars. Additionally, the overall conversion is poor, due largely to the efficacious nature of lignin in limiting enzymatic access to cellulose and hemicelluloses; 2) Better use of lignoceelulosic material, which is one of the most abundant biopolymers on earth, but largely underutilized; 3) Decrease use of harsh chemicals like strong acid and alkaline in pulping and paper industry; 3) Fitness of modified plants won't be compromised; 4) The technology generated can be transferred to other biomass-important species

Publications

  • Wall PK, Leebens-Mack J, Chanderbali A, Barakat A, Liang H, Landherr L., Tomsho LP, Hu Y, Carlson JE, Ma H, Schuster S, Soltis DE, Soltis PS, Altman N, and dePamphilis CW. 2008.(Submitted to Genome Research) Comparison of Next Generation sequencing technologies for de novo transcriptome characterization.
  • Liang, H., JE Carlson, JH Leebens-Mack, PK Wall, LA Mueller, M Buzgo, LL Landherr, Y Hu, DS DiLoreto, DC Ilut, D Field, SD Tanksley, H Ma, CW dePamphilis. 2008 (in press) An EST Database for Liriodendron tulipifera L. floral buds: the first EST resource for functional and comparative genomics in Liriodendron. Tree Genetics and Genomics.
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