MODELING AND MANIPULATING THE REGULATION OF TENSION WOOD, AN ECONOMICALLY IMPORTANT TRAIT FOR FOREST PRODUCTS, BIOFUELS AND NANOTECHNOLOGY.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1004866
• Grant No.: 2015-67013-22891
• Cumulative Award Amt.: $490,000.00
• Proposal No.: 2014-04041
• Project Start Date: Mar 1, 2015
• Project End Date: Dec 6, 2019
• Grant Year: 2015
• Program Code: [A1101]- Plant Health and Production and Plant Products: Biology of Agricultural Plants

Recipient Organization
FOREST SERVICE
201 14TH ST SW YATES BLDG
WASHINGTON,DC 20002-6405

Performing Department
ConBio

Non Technical Summary
Angiosperm trees respond to gravitational and mechanical forces by producing tension wood, which pulls the stem or branches upright. Tension wood is a primary factor influencing lumber quality and pulping efficiency for forest industry. More recently, the inherently high cellulose/low lignin content of tension wood has been recognized as ideal for bioenergy feedstocks. In preliminary experiments, we provide evidence describing new, fundamental insights into the regulation of tension wood including the identification of the gravity sensing tissues of woody stems, changes in PIN-mediated auxin transport during tension wood induction, and the interacting roles of the transcription factor ARK2, auxin and gibberellin in regulating tension wood. We have also established a large collection of Populus gene dosage variants that can be used to identify mutants and associated genes conditioning novel tension wood phenotypes.We propose here to: 1) Characterize the roles of ARK2, auxin and gibberellin during tension wood formation 2) Screen an existing Populus gene dosage functional genomics resource to identify useful genotypes and chromosomal regions conditioning tension wood phenotypes, and 3) Produce rigorous models of transcriptional networks regulating tension wood. Integration of information across these three goals will provide an unprecedented view of tension wood regulation, including identification of genotypes with novel tension wood properties within a commercially important hybrid.USDA program priorities addressed include: Improving plant composition (in this case, through tailoring wood properties). Understanding plant response to abiotic stress (in this case, though understanding the development of water conducting tissues in trees).

Animal Health Component

10%

Research Effort Categories

Basic

70%

Applied

10%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
123	0120	1050	100%

Knowledge Area
123 - Management and Sustainability of Forest Resources;

Subject Of Investigation
0120 - Land;

Field Of Science
1050 - Developmental biology;

Keywords

forest trees

populus

bioenergy.

forest industry

wood formation

Goals / Objectives
Tension wood is formed by trees in responose to gravitational stimulus. Tension wood has dramtically different biochemical properties in comparison to "normal" wood, including altered ratios of cellulose and lignin that are conducive to conversion of wood to liquid biofuels and other value added products. We propose here experiments that will provide fundamental new insights into the hormonal and transcriptional regulation of tension wood formation. For the proposed experiments, we will use uniform conditions and calibrated tools for monitoring the development of Populus tension wood in controlled conditions, allowing integration of data across all experiments. We propose three key goals: 1) Characterize the roles of auxin and gibberellin during tension wood formation; 2) Screen an existing resource of poplar dosage variants to identify genotypes with altered tension wood formation; and 3) Model the transcriptional networks underlying tension wood formation. Together these results will provide information fundamental to altering wood properties for both economic and ecological benefits.

Project Methods
For the proposed experiments, we will use uniform conditions and calibrated tools for monitoring the development of Populus tension wood in controlled conditions, allowing integration of data across all experiments. We propose three key goals: 1) Characterize the roles of auxin and gibberellin during tension wood formation; 2) Screen an existing resource of poplar dosage variants to identify genotypes with altered tension wood formation; and 3) Model the transcriptional networks underlying tension wood formation. We have established all of the basic informatics tools, laboratory techniques, reagents, and germplasm required for the proposed experiments. We have an excellent team of researchers and collaborators contributing to the research. Lead PI Andrew Groover has extensive experience in the genetics and cell biology of plant development and woody growth. Vladimir Filkov of UC Davis Computer Science is an expert in network reconstruction and computational biology, and will lead the informatics and computational efforts. Shawn Mansfield of the University of British Columbia will lead the wood biochemistry efforts, and is an expert in the properties, biochemistry, and biotechnology of wood and fibre quality.

Progress 03/01/15 to 02/29/20

Outputs
Target Audience:Research during the reporting period was directly primarily to researchers, but also to bioenergy industry and forest industries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Multiple undergraduate students have received field and lab-based research experience in the course of this project. Two postdocs have received extensive training in genomics and bioinformatics, and both were able to secure positions for their next career steps. How have the results been disseminated to communities of interest?This work has resulted in multiple major data and review papers as reported. The research has also resulted in the establishment of a new model system for the study of wood formation that is being used now by multiple labs to advance forest biology research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? All of the major goals of the project were accomplished. We modeled the genetic regulatory networks controlling tension wood formation, including identification of functional gene modules that gives tension wood specific biochemical and chemical properties that are of direct relevance to forest industries and bioenergy application for trees. We also determinied how trees perceive and respond to gravity through homrome related processes that result in reprogramming of wood formation to result in changes to cellulose and lignin content. Together our results combine genetic, cell biology and hormonal measures of tree growth to understand wood formation at a fundmanetal level necessary for targetting manipulation of ecologically and economically important traits.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Du J, Gerttula G, Li Z, Zhao S, Liu Y, Liu Y, Lu M, Groover A. (2019). Brassinosteroid regulation of wood formation in poplar. New Phytologist. doi:10.1111/nph.15936

Progress 03/01/15 to 12/06/19

Outputs
Target Audience:Research during the reporting period was directly primarily to researchers, but also to bioenergy industry and forest industries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Multiple undergraduate students have received field and lab-based research experience in the course of this project. Two postdocs have received extensive training in genomics and bioinformatics, and both were able to secure positions for their next career steps. How have the results been disseminated to communities of interest?This work has resulted in multiple major data and review papers as reported. The research has also resulted in the establishment of a new model system for the study of wood formation that is being used now by multiple labs to advance forest biology research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? All of the major goals of the project were accomplished. We modeled the genetic regulatory networks controlling tension wood formation, including identification of functional gene modules that gives tension wood specific biochemical and chemical properties that are of direct relevance to forest industries and bioenergy application for trees. We also determinied how trees perceive and respond to gravity through homrome related processes that result in reprogramming of wood formation to result in changes to cellulose and lignin content. Together our results combine genetic, cell biology and hormonal measures of tree growth to understand wood formation at a fundmanetal level necessary for targetting manipulation of ecologically and economically important traits.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Du J, Gerttula G, Li Z, Zhao S, Liu Y, Liu Y, Lu M, Groover A. (2019). Brassinosteroid regulation of wood formation in poplar. New Phytologist. doi:10.1111/nph.15936

Progress 03/01/18 to 02/28/19

Outputs
Target Audience:Our results have been shared to a broad audience of researchers in the areas of plant science, forest biology and genomic science through both publications and seminars. Changes/Problems:Our work has been impacted by the government shutdown but we still antocipate completing the project by the end of this calendar year. What opportunities for training and professional development has the project provided?One postdoc completed their studies and went on to a faculty position. A current postoc received extensive training in statistics and bioinformatics. Two recent graduates received extensive training in molecular biology, histology, moicroscopy and computer programming. Three undergrads received training in molecular biology, plant histology and microscopy. How have the results been disseminated to communities of interest?We have published papers in primary plant science journals and also presented findings at international conferences. What do you plan to do during the next reporting period to accomplish the goals?We are currently completing making sequencing libraries and preparing wood samples for biochemical analysis, prior to final integration of datasets and computational analyses to model economically important wood anatomical and biochemical traits as a function of gene regulation.

Impacts
What was accomplished under these goals? We have accomplished all of the initial goals of the project, and are now working on the final goal of producing extensive biochemical and gene expression datasets describing tension wood formation and properties in poplar. To date we have identified the hormonal mechanisms responsible for the change in wood properties associated with tension wood, which is both the primary defect in lumber but also associated with cellulose-enriched cell walls with ideal properties for bioenery applications. We have also extensively described the anatomical changes in wood formation between tension wood and "normal" wood, and developed extensive models of changes in gene expression that underlie tension wood regulation and development. Currently we are working to complete extensive new gene expression and wood biochemical datasets that will allow us to link gene regulation with biochemical properties of wood central to properties important for forest industry and bioenergy applications of wood.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zinkgraf M, Groover A, and Filkov V (2018). Reconstructing gene networks of forest trees from gene expression data: Toward higher resolution approaches. In: Kalajdziski S., Ackovska N. (eds) ICT Innovations 2018. Engineering and Life Sciences. ICT 2018. Communications in Computer and Information Science, vol 940. Springer.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Tuskan G, Groover A., et al (2018). Hardwood tree genomics: Unlocking woody plant biology. Frontiers in Plant Science. doi.org/10.3389/fpls.2018.01799
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Tomescu A, and Groover A. (2018). Mosaic modularity: an updated perspective and research agenda for the evolution of vascular cambial growth. New Phytologist. doi.org/10.1111/nph.15640
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Rodriguez-Zaccaro D, and Groover A. Wood and water: How trees modify wood development to cope with drought. In review Plants People Planet.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: A. Groover, H. Bastiaanse, M. Zinkgraf, I. Henry, L. Comai. Functional genomics for quantitative trait dissection in forest trees. Agriculture Bioscience International Conference. Weifang, China. September 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: A. Groover, S. Gerttula, M. Zinkgraf, H. Bastiaanse, V. Filkov, S. Zhou, MZ Lu. Genes and mechanisms underlying the evolution and development of wood in angiosperm trees. 42nd New Phytologist Symposium Biology of wood, from cells to trees. Tahoe, CA. July 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: A. Groover. Molecular and genomic insights into the evolution and development of wood in forest trees. University of G�ttingen, Germany. April 2018.

Progress 03/01/17 to 02/28/18

Outputs
Target Audience:Forest industry. Lumber and vaneer manufacturers. Other researchers in the field of forest biology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One postdoc had extensive training in bioinforatics and was hired as a faculty member at Western Washington University. A second postdoc is currently being trained in advanced statistics and bioinformatics. How have the results been disseminated to communities of interest?Yes. Results have been shared in both highly visable publications and in lectures at conferences and at universities. What do you plan to do during the next reporting period to accomplish the goals?Complete all final proposed goals of the project.

Impacts
What was accomplished under these goals? During this period we extended previous genomic and bioinformatics approaches to complete the proposed time course of gene expression during development. This involved significant effort to develop the bioinformatics and provided significant new insights into the biology of wood formation. We published multiple papers on the methods developed throughout the project including both lab and bioinformatics-based techniques, as well as papers providing details of the regulation of traits central to wood properties for forest industry and biofuels applications.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zinkgraf M, Gerttula S, Zhao S, Filkov V, Groover A (2018). Transcriptional and temporal response of Populus stems to gravi-stimulation. Journal of Integrative Plant Biology. doi: 10.1111/jipb.12645
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Groover A (2017). Age-related changes in tree growth and physiology. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0023924
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Zinkgraf M, Gerttula S, Groover A (2017). Transcript profiling of a novel plant meristem, the monocot cambium. Journal of Integrative Plant Biology 59(6):436-449. doi: 10.1111/jipb.12538.
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Gerttula S, and Groover A (2017). Immunolocalization in secondary xylem of Populus. In: Xylem: Methods and Protocols, Methods in Molecular Biology vol 1544. De Lucas and Etchells (ed.), Springer.
• Type: Book Chapters Status: Accepted Year Published: 2016 Citation: He X and Groover A (2016). The genomics of wood formation in angiosperm trees. In: Evolutionary and comparative genomics of angiosperm trees. Groover and Cronk (Eds.) Springer.

Progress 03/01/16 to 02/28/17

Outputs
Target Audience:Our publications and presentations reached a wide array of researchers, as well as land managers, industry partners, and students. Changes/Problems:No major changes or problems. What opportunities for training and professional development has the project provided?Three postdocs have received extensive training and experience in bioinformatics and genomics. One Masters student has received training in plant molecular biology and general plant biology. Three undergraduates have received training in laboratory techniques. How have the results been disseminated to communities of interest?Results are being disseminated primarily through publications in scientific jorunals and through presentations at universities and research conferences. What do you plan to do during the next reporting period to accomplish the goals?We are next planning experiments to visualize changes in gene expression and cell wall synthesis at high resolution during development, determining the role of plant hormones during wood development, and extending these findings to additional tree species of economic and ecological importance.

Impacts
What was accomplished under these goals? We have published major papers describing primary molecular and genetic mechanisms controlling how trees alter wood development in response to gravity and mechanical stress. Because these changes in development result in wood with properties directly relevant to forest industry and biofuels production from trees, they are directly relevant to making trees that are tailored towards induststry needs. In brief, we found that woody stems alter the transport of the plant hormone, auxin, in response to gravity. This change in auxin transport in turn results in changes in transcriptional regulation of a large number of genes involved in remodeling the cell walls of wood cells, resulting in changes in the lignin and collulose composition of the walls. We are next planning experiments to visualize changes in gene expression and cell wall synthesis at high resolution during development, and extending these findings to additional tree species of economic and ecological importance.

Publications

• Type: Book Chapters Status: Accepted Year Published: 2017 Citation: He X and Groover A (2016). The genomics of wood formation in angiosperm trees. In: Evolutionary and comparative genomics of angiosperm trees. Groover and Cronk (Eds.) Springer.
• Type: Books Status: Awaiting Publication Year Published: 2017 Citation: Evolutionary and comparative genomics of angiosperm trees. Groover and Cronk (Eds.) Springer.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: A. Groover, S. Gerttula, M. Zinkgraf, H. Brumer, S. Mansfield, V. Filkov. Transcriptional and hormonal control of gravitropisms in forest trees. Society of Developmental Biology 74th Annual Meeting, Snowbird Utah, July 2015.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: A. Groover. Genomic-enabled insights into growth and wood formation in Populus. Ume� Plant Science Centre, Ume� University, Sweden, March 2016.

Progress 03/01/15 to 02/29/16

Outputs
Target Audience:Target audiences include researchers studying forest tree biology, forest tree genomics and computational biology, and wood formation. Forest industry partners have been briefed on our findings and have collaborated to help us obtain biological samples and measure phenotypes relevant to industry. Undergraduate and graduate students have been introduced to new concepts in forest genomics through lectures and a bioinformatics training class. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three postdocs have benefitted from training in molecular biology and bioinformatics. One new graduate student in basing part of her thesis project on resources being developed as part of the funded project. How have the results been disseminated to communities of interest?We are interacting with forest industry to make them aware of the potential of our genomic approaches to inform directed changes in wood properties for specific applications. Results have also been presented in lectures at major scientific conferences, as well as in talks to graduate students at UC Davis. What do you plan to do during the next reporting period to accomplish the goals?We plan to perform the remaining proposed hormone treatment - gene expression studies, and complete a larger screen of the Populus dosage variation population for gravitropism and wood formation phenotypes. We will undertake ambituous imaging studies to visualize genes and proteins directly in wood foring tissues. We will beging the challenging task of integrating all of our phenotypic, gene expression, and imaging data to produce a comprehensive description of the regulation of wood development at the molecular genetic level.

Impacts
What was accomplished under these goals? We have made excellent progress on all of our original goals. Extensive RNAseq data was produced for Populus trees from experiments testing the effects of hormones (gibberellic acid) and gravity stress on wood formation. These data were then used to model co-expression gene modules, which are groups of genes that are expressed in a similar way across the different treatments and samples. We then identified individual gene modules that were correlated with different wood development traits, including traits directly associated with wood properties of ecological and economic importance. These modules were then further dissected to identify candidate genes encoding transcriptional regulators that may be responsible for regulating expression of genes within each module. In so doing, we were able to start with the ~45,000 genes in Populus and narrow to a modest-sized list of regulators that can now be experimentally studied. Also during this period, we charaterized the mechanisms by which woody stems perceive and respond to gravity. These mechanisms involve gravity sensing cells in the endodermis and seconodary phloem of the stem, which express PIN-like auxin transport proteins. These proteins reorient in response to gravity, changing the flow of auxin radially across the stem, which results in changes in wood development. Specifically on the upward facing side of the stem, specialized "tension wood" forms that serves to pull the stem upright. This process is of fundamental importance for normal tree development, but also changes wood properties that are central to forest indistries and use of trees as biofuels feedstocks. We also performed a pilot screen for Populus dosage variants and have identified genotypes with potential alterations in gravitropisms and wood formation. A larger screen will be performed in the coming growing season.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Gerttula S, Zinkgraf M, Muday G, Lewis D, Ibatullin F, Brumer H, Hart F, Mansfield, S, Filkov V, and Groover A (2015). Transcriptional and hormonal regulation of gravitropism of woody stems in Populus. Plant Cell 27: 2800-2813
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Groover, A (2016). Gravitropisms and reaction woods of forest trees  evolution, functions and mechanisms. submitted to New Phytologist