Progress 12/01/04 to 11/30/09
Outputs
OUTPUTS: Four lines of poplar (Populus tremula x P. alba) clone 717 genetically modified to over-express the Ferulate 5-Hydroxylase (F5H) enzyme were used in all studies. Over-expression of the F5H enzyme resulted in an increased syringyl:guaiacyl ratio within the cell walls. The F5H clones and wild type poplars were subjected to water stress over a 4 week period, and hydraulic conductivity and vulnerability to embolism were measured. Gas exchange was monitored throughout the experiment to assess stress levels in the plants. Initial measurements have shown an increased initial hydraulic conductivity in the F5H clones over the wild type before water stress was induced. However, as water stress increased the F5H clones showed an increase in vulnerability to embolism and a decreased hydraulic conductivity compared to the wildtype clone. This may suggest an increased brittleness and vulnerability to cavitation in the modified cell walls. No differences in gas exchange values have been observed between the F5H and wild type clones. Additionally, wild type poplars and the transgenic lines were left upright or inclined 45 from vertical for three months to induce the gravitropic responses including tension wood formation. Wild type poplar stems had 6.4% and 7.6% increase in percent syringyl in tension wood side than normal or opposite wood, respectively. Increasing syringyl formation increased percent acid soluble lignin 2.3 folds. Cell wall crystallinity was also higher in tension wood than the other types of wood tissues. Both tension and opposite woods had higher percent total sugars. Interestingly, in tension wood a 19.1% increase in percent syiringyl led to 3.6% decrease in percent total sugars and 4.1% decrease in percent glucose. Percent galactose in tension wood was also higher but dropped 0.1% in response to the lignin monomer modification in the same tissue. Xylose and rhamnose were lower in tension wood than normal wood in wild type stems. Mechanically, the stems modulus of elasticity (MOE) did not change with increased syringyl when tested with 4-point bending or under compression. A decrease in the stems modulus of rupture (MOR) in response to increased S:G ratio was detected. Trees with increased S:G ratio seemed to adjust their stems to gravity faster after inclination. Evaluating the response of these lines to inclination will improve our understanding of the role lignin monomeric composition plays in altering xylem chemical composition and mechanical properties of normal and tension wood. Data and results generated during this research project were presented at the 6th Plant Biomechanics Conference, Cayenne, French Guyana, November 16th - 21st, 2009. One undergraduate student supported by this research graduated in 2009. 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
The outcomes and impacts of this research indicate that altering lignin structure by altering the S:G ratio has a minimal impact on overall stem mechanical properties and does not impact a plant's ability to respond to wind or gravity. In fact, it appears that trees with a higher S:G will 'right' themselves faster if displaced than wildtype trees. This aspect of the current study will continue to be a focus of research beyond the completion of the current grant. However, the results of the hydraulic conductivity studies indicates that trees modified to express a higher S:G are more vulnerable to embolism and have decreased hydraulic conductivity compared to wildtype clones. This may indicate that modified poplars may be more vulnerable to water stress and could result in a reduction in productivity within a plantation.
Publications
- Al-Haddad, J., Mansfield, S., and Telewski, F.W. 2009. Wood chemical and mechanical responses to modified lignin composition in upright and inclined hybrid poplar. Page 477-481 in Proc. 6th PBM Conference, Cayenne, French Guyana.
- Pierce, J.A., Ewers, F.W., Al-Haddad, J., and Telewski, F.W. 2009. The Effects of Modified Lignin Monomer Ratios on Hydraulic Conductivity and Resistance to Embolism in Hybrid Poplar (P. tremula x P. alba). Page 164-167 in Proc. 6th PBM Conference, Cayenne, French Guyana.
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Progress 12/01/07 to 11/30/08
Outputs
OUTPUTS: Data and results generated during this research project were presented at an Annals of Botany symposium Mechanoperception and graviperseption in plants held at the at the 72nd annual meeting of the Botanical Society of Japan in Kochi, Japan (September 27, 2008) and at a seminar held at Tohoku University on October 2, 2008. This year we replicated our test of the highest expressed F5H line for drought tolerance and resistance to embolism. Results were similar to those obtained in 2007. We also tested for refilling of embolized vessels after drought stress and found there was no difference between the lines. We are in the process of analyzing stem cross sections stained using the fuchsin dye test to identify conducting vessels. We continue to make progress in developing the RNAi line of hybrid poplars with reduced syringyl content. Initial tests reporting a positive expression turned out to be false, however, recent tests conducted this fall indicate we do have inhibition. Testing to verify these results continue and samples will be sent to the University of British Columbia to verify the percent syringyl content before we incorporate the new lines into our experimental design in 2009. PARTICIPANTS: PARTICIPANTS (2008): Frank W. Telewski (PD) supervision and training of graduate students and undergraduate student working on the project. Oversee experimental design, propagation of clonal material. Conducting mechanical testing with graduate and undergraduate students. Analysis and interpretation of data. Preparation of manuscripts. Presentation of results at symposium and lectures. Frank W. Ewers (co-PD) supervision and training of graduate students and undergraduate student working on the project. Analysis and interpretation of data. Han, K.H. (co-PD) supervision of graduate student as related to RNAi transformation of hybrid poplar. Koehler, L. (co-PD) analysis of data, preparation of publications. Al-Haddad, J. (graduate student) development of RNAi line of poplars to reduce syringyl content, conducting gravitropic experiments. Pierce, J.A. (graduate student, not paid on grant) conduct hydraulic conductivity experiments, supervision of undergraduate student. Rayman, D. (undergraduate student, added Summer 2008) assist graduate students with research, greenhouse plant maintenance, anatomical analysis of woody tissues formed under mechanical stress COLLABORATORS: Mansfield, S. University of British Columbia, supply chemical analysis of wood samples. Chapple, C. Purdue University, supplied original line of F5H poplars. Ellis, D. formerly of CellFor, Canada, now USDA, Plant Genetic Resources Preservation Program National Center for Genetic Resources Preservation Fort Collins, CO. supplied original line of F5H poplars. Marks, B.P. Michigan State University, use of Instron testing machine in his lab. TRAINING OR PROFESSIONAL DEVELOPMENT: This project provided training and professional development for 3 graduate students, and 5 undergraduate students since the project was first funded. TARGET AUDIENCES: The primary target audience of this research is the scientific community working in the area of, but not limited to tree growth and development, cell wall structure and biomechanics, pulping and paper production, and biofuels conversion. As information is accumulated, this research will also be relevant to the forest products industry and any future industry based on cellulosic ethanol or other biomass to liquid fuel conversion. This research will also be of interest to those in the public sector interested in bioengineering of plants and the impact those changes may have on the ability of a plant to respond to its environment. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Under well watered conditions there was no significant difference in resistance to embolism between wildtype and the high syringyl F5H line. However, after exposure to drought stress, the high syringyl line is less resistant to further embolism, even after rewatering. No significant differences were observed in conductance between the two lines under either well watered or drought conditions. Similar results were observed for pre-dawn water potential. We also tested for refilling of embolized vessels after drought stress and found there was no difference between the lines. However, there is evidence which suggests that hybrid poplar blocks embolized vessels preventing subsequent refilling after the alleviation of drought. These results suggest that increasing the syringyl content may make these lines of poplar more susceptible to drought stress, requiring plantations to be monitored for drought and possibly irrigated. The extent to which these findings may impact tree growth will be better evaluated when we include our RNAi lines in the experimental design.
Publications
- Telewski, F.W. 2008. 205 Years of thigmomorphogenetic research: how far we've come (abstract). Annals of Botany Lecture, Botanical Society of Japan, Kochi, Japan. September, 2008
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Progress 12/01/06 to 11/30/07
Outputs
OUTPUTS: Data and results generated during this research project were presented at several national and international events: The PD presented a poster at the NRI Project Director Awardee meeting "Genes to Products" in Washington, D.C. March 2007. The PD presented an invited lecture at the "3rd International Symposium on Plant Neurobiology", Strbske Pleso, SLOVAKIA, May 2007. Two posters were presented by a co-PD and graduate student at the joint annual meeting of the American Society of Plant Biologists and the Botanical Society of America, Chicago, IL June 2007. The PD gave two oral presentations at the International Union of Forest Research Organizations' "Wind and Trees" symposium, Vancouver, B.C. held in August 2007. As an outcome of the IUFRO meeting, the PI was invited to present this research in a talk and participate in the European Union Group 4's Workshop of COST Action E50 on "Structure and function of primary and secondary cell walls" Potsdam, GERMANY, held in September
2007. In a preliminary drought study, we found a significantly higher mortality rate in the high syringyl poplar line compared to the wildtype. This finding lead to a new activity which was developed for 2007 based on a discussion with USDA NRI officers at the March 2007 NRI Project Director Awardee meeting "Genes to Products". This year we decided to test the highest expressed F5H line for drought tolerance and resistance to embolism. Under well watered there was no significant difference in resistance to embolism between wildtype and the high syringyl F5H line. However, after exposure to drought stress, the high syringyl line is less resistant to further embolism, even after rewatering. No significant differences were observed in conductance between the two lines under either well watered or drought conditions. Similar results were observed for pre-dawn water potential. However, when chlorophyll fluorescence, measured as Fv/Fm was measured, the high syringyl F5H line exhibited a
significantly greater decline in response to drought stress compared to the wildtype. We also tested for refilling of embolized vessels after drought stress and found there was no difference between the lines. However, there is evidence which suggests that hybrid poplar blocks embolized vessels preventing subsequent refilling after the alleviation of drought. We propose to further investigate this phenomenon this year by replicating this experiment and using the fuchsin dye test to identify conducting vessels. The activities originally defined for 2007 focused on replication for the previously conducted experiments and the inclusion of the recently developed F5H RNAi lines with lower syringyl to guaiacyl content.
PARTICIPANTS: INDIVIDUALS who worked on the Project: Frank W. Telewski (PD) Frank W. Ewers (Co-PD) Lothar H. Koehler (Co-PD) Jameel Al-Haddad (graduate student) Erin Nicole Getzin (undergraduate) Aimee Margaret Wilson (undergraduate) Emily Margret Flynn (undergraduate) Jeffery A. Pierce (undergraduate) COLLABORATORS: Shawn D. Mansfield (University of British Columbia) Kyu-Young Kang (University of British Columbia)
TARGET AUDIENCES: Forest Products Industry Wood Scientists Researchers in the area of Cellulosic Ethanol production The field of Plant Biomechanics Ecologists Silviculturists
PROJECT MODIFICATIONS: The activities originally defined for 2007 focused on replication for the previously conducted experiments and the inclusion of the recently developed F5H RNAi lines with lower syringyl to guaiacyl content (as originally proposed). Due to an unfortunate horticultural error imparted to our trees by the greenhouse staff and without our prior knowledge or approval, we discovered that hybrid poplar is highly sensitive to the systemic pesticide (Judo, spiromesifen insecticide/miticide) which had been applied to our research poplars to control a resistant strain of pest. This pesticide causes severe foliar, lateral bud, and meristematic burning resulting in severe the dieback of our research plants in the middle of the 2007 experiments. We were only able to complete one of several planned experiments. As a result, we requested and received a one-year no cost extension of our grant. We currently have replicated the different transgenic lines in culture and plan to
restart the experiments in 2008.
Impacts
Lignin is a major structural and chemical component of cell walls which binds to cellulose in the secondary cell wall of wood tissues. Its removal from the cellulose cell wall matrix is crucial in two major wood conversion processes: namely the pulping process to facilitate cellulosic ethanol conversion or in paper production. Research and results generated as part of this study have lead to a clearer understanding of how lignin monomer content impacts the biomechanics tree stems, wood, and cell walls. This is evidenced by the increasing interest and awareness of the scientific community studying cell wall structure and function resulting in invitations for the PD to participate in various international symposia and workshop which focus on this important and rapidly developing area of research. It has also assisted in the elucidation of the impact of wind on aspects of cell wall chemical and structural composition. The results to date indicate that trees with altered
lignin monomer content (high syringyl to guaiacyl content) will not be severely impacted by wind and other mechanical loads compared to wildtype trees. In fact, the altered trees are actually stronger than their wildtype counterparts. Altered trees have not lost their ability to respond to either wind or gravity. From a biomechanical perspective, a plantation of poplar with altered monolignol content will be able to withstand physical mechanical environmental stresses as well as their wildtype counterparts and any concern that a tree with an increased syringyl content will be mechanically inferior are unfounded. However, recent preliminary studies on drought tolerance raise a question regarding the ability of the high syringyl content trees to survive without supplemental irrigation. Addressing this question will be key to understanding the role of the different monolignols in the conduction of water through the vessels of poplar wood. If required, the need to provide supplemental
irrigation will increase the cost of fiber or energy production within a given plantation of altered trees. These results will be directly applicable to future silvicultural practices or plantation forestry for fiber or energy production using these modified tree lines. This research has also encouraged and supported post-doctorial researcher, a graduate student and four undergraduate students, providing them with advanced career development skills, experiences, and opportunities. It has also lead to the development of new collaborations with other researchers in the field for the PDs.
Publications
- Telewski, F. W. 2007. A unified hypothesis of mechanoperception in plants. 3rd International Symposium on Plant Neurobiology; Book of Abstracts, Strbske Pleso, Slovakia(p19).
- Telewski, F.W. Koehler, L., Al-Haddad, J., Mansfield, S.D., and Ewers, F.W. (2007). Flexure Wood in Hybrid Poplar. IUFRO International Conference on Wind and Trees (Abstracts), Vancouver, B.C. Canada. August 2007.
- Telewski, F.W. Koehler, L., Al-Haddad, J, Kang, K-Y., Mansfield, S.D., and Ewers, F.W. (2007). Characteristics of the Thigmorphogenetic Response in the Xylem of F5H Over-expressed Populus tremula x P. alba, Clone 717. Workshop of COST Action E50 on Structure and function of primary and secondary cell walls (Abstracts) Potsdam, Germany. September 2007.
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Progress 12/01/05 to 12/01/06
Outputs
The objective of this study is to determine 1. how altering lignin monomer content (ratio of syringyl to guaiacyl) impacts mechanical strength and allometry of trees; 2. How trees with altered lignin monomer composition will respond structurally and mechanically to wind, simulated by recurrent dynamic bending of stems; 3. How trees with altered lignin monomer content will respond structurally and mechanically to gravity (gravitropism) upon inclination. Increasing syringyl content leads to higher mechanical stiffness and a subsequent change in allometry to a relatively more slender and taller stem. This illustrates advantages due to the evolution of syringyl lignin concomitant with fiber cells in angiosperms and suggests feed-back between wood mechanical properties and growth allocation causing the tree to invest less material for stem diameter when the same flexural stiffness is reached at a smaller stem diameter due to an increased elastic modulus.
Microspectrophotometric analysis of lignin composition of controls and trees grown under influence of simulated wind reveals: (1) The typical pattern of differences in lignin monomer composition between different cell types is conserved in the mutant, showing higher relative guaiacyl content in vessel walls and higher syringyl content in fibers. Considering the bearing of syringyl content on the elastic modulus shown by our study, the conserved pattern of lignin composition according to cell type could be a hint to the importance of a gradient in the mechanical properties between vessel and fiber walls. (2) The tree actively modifies lignin monomer composition to adjust the mechanical properties of its wood to windy environment. The reaction of the transgenics to simulated wind in terms of changes in mechanical properties and syringyl content is comparable to the wild-type. (3) Increasing syringyl content does not alter cellulose microfibrilar angle (MFA), however, mechanical stress
increased MFA in all transformed clones and wild-type. These results are similar to earlier observations reported in the conifer, Abies fraseri exposed to flexing and indicate flexure wood in angiosperms share a key characteristic with compression wood and flexure wood in conifers, and not tension wood. Genetically modified trees exposed to displacement with regard to gravity express a typical gravitropic response by producing tension wood and returning to the vertical orientation. The rate of recovery to the vertical position has been documented to be faster in trees expressing an increase in S:G. Mechanical property data from displaced stems vs. vertical stems are inconclusive at the present time. These results will be clarified in replicate studies this year. Specific conductivity of inclined stems is consistently and significantly lower in displaced stems regardless of S:G content. Altering S:G does not appear to impact conductivity in tension wood. Hybrid poplar clone 717 has
been successfully transformed in 2006 using RNAi of the F5H gene to decrease S:G. Characterization of the S:G monomer content will be conducted in the early spring of 2007. These transforms will be added to our analysis in 2007.
Impacts
Lignin is a major structural and chemical component of cell walls and is removed from fibers during the pulping process of paper production. Angiosperm lignin is composed of two 'building blocks' or monomers, syringyl and guaiacyl. Recent research has focused on altering lignin monomer content by modifying the lignin biosynthetic pathway with the intent of increasing the efficiency of pulping and reducing energy requirements and pollution during the pulping process. However these studies have not addressed how altering lignin will impact tree growth, mechanical stability, or wood properties. Our study investigates how trees which were genetically modified to express differences in lignin composition will respond both biologically and structurally, to environmental stresses. This research is important in order to understand how trees respond to environmental stresses such as wind or gravity before any large scale planting of these modified trees is attempted. The
understanding of how such trees are able to withstand and respond to these stresses will be an important step providing for the adequate management of forest tree plantations and fiber farming. At a fundamental level, our research will also provide a clearer understanding of the role of the two lignin monomers (syringyl and guaiacyl) play in the biomechanics of trees, wood, and xylem cell walls.
Publications
- Telewski, F. W. 2006. A unified hypothesis of mechanoperception in plants. American Journal of Botany. 93:1306-1316
- Koehler, L. and F.W. Telewski 2006. Biomechanics of transgenic wood. American Journal of Botany. 93:1273-1278
- Al-Haddad, J., L. Koehler, F.W. Ewers, and F.W. Telewski 2006. Influence of lignin composition on gravitropic responses and mechanical properties in genetically altered poplars (Populus tremula x P. alba). in Proceedings of the 5th Plant Biomechanics Conference, vol. I, Salmen, L. (Ed.), STFI-Packforsk AB, Stockholm, 2006, 227-232.
- Koehler, L., F.W. Ewers, and F.W. Telewski 2006. Mechanical impact and function of lignin monomer composition in poplar wood. ). in Proceedings of the 5th Plant Biomechanics Conference, vol. I, Salmen, L. (Ed.), STFI-Packforsk AB, Stockholm, 2006, 255-260.
- Ewers, F.W., L. Koehler, A.L. Jacobson, S.D. Davis, R.B. Pratt, and F.W. Telewski 2006. Effect of mechanical properties on resistance to xylem cavitation. in Proceedings of the 5th Plant Biomechanics Conference, vol. II, Salmen, L. (Ed.), STFI-Packforsk AB, Stockholm, 2006, 411-417.
- Koehler, L., F.W. Ewers, and F.W. Telewski 2006. Lignin Composition, Mechanical Properties, Conductivity and Response to Wind of F5H Transgenic Poplars. ABSTRACT Botany 2006, Annual Conference of the Botanical Society of America, California State University-Chico, July 28-August 2, 2006, http://www.2006.botanyconference.org/engine/search/index.php?func=det ail&aid=800
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Progress 12/01/04 to 11/30/05
Outputs
The objective of this study is to determine 1. how altering lignin monomer content (ratio of syringyl to guaiacyl) impacts mechanical strength and allometry of trees; 2. How trees with altered lignin monomer composition will respond structurally and mechanically to wind, simulated by recurrent dynamic bending of stems; 3. How trees with altered lignin monomer content will respond structurally and mechanically to gravity (gravitropism) upon inclination. Trees expressing higher ratios of syringyl to guaiacyl (S:G) content produce wood (in the green state) with higher Modulus of Elasticity (MOE) which appears to be proportional to the S:G. Modified trees adjust their growth and wood allocation according to the altered mechanical properties (MOE) of the wood so that the stems overall flexural stiffness (EI) remains independent of S:G. For overall tree shape (allometry) trees with high S:G grow taller and have more slender stems compared to the wildtype (wt). Preliminary
results indicate that water conductivity is not impacted by alterations of the S:G. However, susceptibility to embolism (drought resistance) appears to decrease. Genetically modified trees exposed to recurrent dynamic bending as a simulation of wind sway express a typical thigomorphogenetic response of reduced height growth and increased radial growth. Additionally, trees of both wt and higher S:G express decreases in MOE and increases in EI in response to dynamic bending. Initial data derived from microspectrophotometry indicates that wt and genetically modified trees exposed to dynamic bending alter S:G as a means of adjusting overall biomechanical properties to this environmental stress. Quantitative measurements to verify these findings are underway. Genetically modified trees exposed to displacement with regard to gravity express a typical gravitropic response by producing tension wood and returning to the vertical orientation. The rate of recovery to the vertical position
appears to be faster in trees expressing an increase in S:G. Mechanical property data from displaced stems vs. vertical stems are inconclusive at the present time. These results will be clarified in replicate studies this year. Specific conductivity of inclined stems is consistently and significantly lower in displaced stems regardless of S:G content. Altering S:G does not appear to impact conductivity in tension wood.
Impacts
Lignin is a major structural and chemical component of cell walls and is removed from fibers during the pulping process of paper production. Angiosperm lignin is composed of two 'building blocks' or monomers, syringyl and guaiacyl. Recent research has focused on altering lignin monomer content by modifying the lignin biosynthetic pathway with the intent of increasing the efficiency of pulping and reducing energy requirements and pollution during the pulping process. However these studies have not addressed how altering lignin will impact tree growth, mechanical stability, or wood properties. Our study investigates how trees which were genetically modified to express differences in lignin composition will respond both biologically and structurally, to environmental stresses. This research is important in order to understand how trees respond to environmental stresses such as wind or gravity before any large scale planting of these modified trees is attempted. The
understanding of how such trees are able to withstand and respond to these stresses will be an important step providing for the adequate management of forest tree plantations and fiber farming. At a fundamental level, our research will also provide a clearer understanding of the role of the two lignin monomers (syringyl and guaiacyl) play in the biomechanics of trees, wood, and xylem cells.
Publications
- Koehler, L., Ewers, F.W. and Telewski, F.W. 2006. Optimizing for Multiple Functions: Mechanical and structural contributions of cellulose microfibrils and lignin in strengthening tissues. IN: Characterization of the Cellulosic Cell Wall. Editors D.D. Stokke and L.H. Groom. Blackwell Publishing
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