Research Accomplishment Reports 2009

Chemical Genetic Dissection of Plant Cellulose Synthesis

S. DeBolt
Department of Horticulture

 

Non-Technical Summary

Plant cell walls are composed of highly glycosylated proteins and polysaccharides, including pectin, hemicelluloses and cellulose, which form a complex and dynamic structure that modulates cell expansion. The primary cell wall polysaccharide is cellulose, and it stands as the most abundant biopolymer in the world. Cellulose has long been utilized by the forage, pulp, paper and textile industries, and has been targeted as a vehicle for renewable energy and carbon sequestration.

In addition cellulose and cell wall structure are vital to fruit softening and ripening processes in horticulture crops worldwide. Despite the agricultural and industrial importance, cellulose biosynthesis in plants is still poorly understood.

The overarching goal of this research is to develop small molecule probes that inhibit specific aspects of the cellulose biosynthetic process, which can then be used to dissect underlying molecular processes.

2009 Project Description

INVITED PRESENTATIONS

University of Tennessee Department of Biochemistry & Cellular and Molecular Biology Spring Seminar Series 2008 International Congress for Biotechnology, Seoul Korea 2009 Gordon Research Conference Plant Cell Walls, MA, 2009 - Chemical genetic dissection of plant cell walls University of Malang Indonesia, Agricultural Biotechnology Development, Invited to give three seminars and interact with the organic and sustainable farming movement in Malang Indonesia in May, 2009 a- Developing cellulosic bioenergy agriculture in Kentucky (Campus wide faculty- 100 participants) b- Climate change and agriculture (Campus wide faculty, 100 participants) c- Molecular genetic dissection of cell wall synthesis (100 graduate students and faculty) - Scientific testimony for Kentucky legislative taskforce on bioenergy agriculture- Oct 14th, 2009- Titled Biotechnology for biofuels. - Plant and Animal Genome, San Diego, CA- Jan 5th 2010 Plant Biotechnology Series. Chair Ling Yuan - 32nd symposium on biotechnology for fuels and chemicals- April 19-22, 2010 in Clearwater Beach Fl - Bowdoin College- Biology Department, Invited Faculty seminar, Host- Prof/Chair Bruce Kohorn - Regional Climate Change Forum- Kentucky Science and Technology Center Dec 2009 - Climate Change and Agriculture for K-12 Education Webinar- for Kentucky high school teachers

GRADUATE STUDENT ADVISING

Ph.D. Darby Harris (University of Kentucky, Lexington) Molecular and chemical dissection of plant cellulose synthesis. Jan 1st-2008 to present- -Gordon Research Seminar, Plant Cell Walls, MA, 2009 - Darby Harris (Ph.D student) presented the work from (Harris et al., 2009) and won the research seminar award. Gordon research conferences represent the pinnacle of scientific meetings in our field. -He has represented the college twice at the National Agricultural Biotechnology Council annual meeting -Currently has 3 publications as first author and the biggest piece of research is currently being written up -One Patent- see patents for a specific listing. Meera Nair (University of Kentucky, Lexington) The molecular role of phyosteryl glycosides in plants Aug 2008 - present. -Currently has one peer reviewed journal publication. Arun Sampathkumar (Max Planck Institute of Molecular Plant Physiology)- Staffan Persson is the primary advisor and I am the External Advisor. "Role of actin cytoskelton during primary wall cellulose production Shawn Lucas (University of Kentucky) "Soil biology and relationships to structure.

2009 Impact

A unique aspect of my program is the use of small molecules to dissect cell wall biosynthesis. These small molecules, like herbicides, target specific areas of plant metabolism and allow us to switch on and off metabolic pathways without requiring genetic manipulation. We then use forward genetics to identify suppressors of the drug action that can be traced genetically to the target of the drug. This is a powerful tool to tease apart complex pathways where traditional genetics is limited.

There are several small molecules that we are focused on in the lab, these are quinoxyphen, morlin, dichlorobenonitrile and not yet named drugs like CBI23 and CBI24. Progress has been steady on the chemical genetics project and we have positionally cloned one resistance locus. Positional cloning is not a trivial exercise and achieving this goal and complementing the mutant with the wild type copy of the gene to restore drug sensitivity is a major achievement of the program. This mutant, which we have named AEGEUS is non-lethal, as opposed all null CESA1 alleles, since the mutation occurs in the C-terminal transmembrane region. Carrying on with the analysis, we have formed collaboration with Jocelyn Rose at Cornell and Dr Ding at NREL to provide innovative means to analyze cellulose structure changes using live cell microscopy and synchrotron analysis.

The CESA enzyme is the most important enzymes for biofuel and fiber production and discoveries in this field is a major goal of my program. The success of the cell wall projects in the lab led to an invitation to speak at the Gordon research conference for Plant Cell Walls, which was a highlight of the program, particularly to receive such an invitation so soon in the programs development. Here, both Darby Harris (Ph.D student in DeBolt lab) and myself presented talks and Darby won the top student presentation award.

Another aspect of our research into cellulose biosynthesis in the past two years has hinged on the use of X-ray diffraction (XRD). Developing XRD as a tool has led to the discovery of a low cellulose-crystallinity mutant that has greater than 50% high digestibility than wild type plants (Patent-2009). This mutation is in cellulose synthase3 (CESA3), since then we have identified additional CESA3 and CESA6 low cellulose-crystallinity mutants.

Furthermore, we characterized how cellulose structure may be an adaptive mechanism that plants modulate to adhere to abiotic stress (Harris and DeBolt, 2008).

Furthermore, developing XRD has led to laboratories elsewhere in the United States contacting us to analyze their plants using this tool (two publications arising).

2009 Publications

Harris D, DeBolt S (2009) Biosynthesis, regulation and utilization of lignocellulosic biomass. Plant Biotechnology Journal (accepted to be published in Jan 2010 issue)

DeBolt S, Campbell JE, Smith R, Stork J, (2009) Regional life cycle assessment of bioenergy agriculture on marginal land. Global Change Biology- Bioenergy 4, 360-369.

DeBolt S, Scheible WR, Schrick K, Aurer M, Carroll A, Hematy K, Bouvier P, Nair M, Schaller H, Zemla M, and Somerville C. (2009) Mutations in UDP glucose:sterol-glucosyltransferase in Arabidopsis cause transparent testa phenotype and suberization defect in seeds Plant Physiology 151: 78-87.

Stork J, Montross M, Smith R, Schwer L, Chen W, Reynolds M, Phillips T, Coolong C, DeBolt S (2009) Regional examination shows potential for native feedstock options for lignocellulosic biofuel production, Global Change Biology- Bioenergy, 2 84-96.

Harris D, Stork J, and DeBolt S (2009) Genetic modification of cellulose synthase reduces crystallinity and improves biochemical conversion to fermentable sugars Global Change Biology- Bioenergy, 1, 51-60.

Harris D and DeBolt S (2008) Relative Crystallinity in Plant Biomass: Studies on Assembly, Adaptation and Acclimation PLoS-ONE e298.

Gu Y, Deng Z, Paredez AR, DeBolt S, Wang Z, Somerville C (2008) Arabidopsis Prefoldin6 is involved in delivery of tubulin to cytosolic chaperonin Proc. Natl. Acad. Sci. USA. 105, 1109-1115.

Marak M, DeBolt S, Persson S (2008) Cellulose synthase; a complex complex. Current opinion in Plant Biology 11: 1-6

DeBolt S, Ristic R, Iland PG, Ford CM (2008) Altered light interception reduces grape berry weight and modulates organic acid biosynthesis during development HortScience 43: 957-961

Goodin MM, Chakrabarty R, Banerjee R, Yelton S, DeBolt S (2007) Update on live cell imaging: New Gateways to Discovery Plant Physiology 145: 1100-1109.

PATENTS

DeBolt S., Stork J. and Harris, D. (2008)(Provisional patent UKRF 1595) Invention title: Improved conversion of lignocellulosic biomass to fermentable sugars by altering the high-order structure of cellulose via genetic modification of cellulose synthase genes

BOOK CHAPTERS

DeBolt, S (2010) Live Cell Imaging in the Plant Cell Wall. Chapter 3: In Methods in Molecular Biology Series (Series Editor John Walker): Volume

DeBolt, S (2010) The Plant Cell Wall: Methods and Protocols Edited by Zoe Poppler. Humana Press, USA (Springer Publishing Group)

M Goodin and DeBolt S (2010)- MIMB book on BiFC/FRET techniques for transcription factor interactions