Theme 1 abstracts: Plants of the Future 

Using synthetic biology to create the next generation of energy crops
Professor Dominique Loqué
Joint BioEnergy Institute - Lawrence Berkeley National Laboratory. Berkeley, CA 94720 USA; dloque@lbl.gov

The plant cell wall represents a large source of polysaccharides that could be used to substitute for sugar derived from starchy grains, which are currently used to feed and produce biofuels. This lignocellulosic biomass, largely under-utilized, is mainly composed of sugar polymers (cellulose and hemicellulose) embedded very strong aromatic polymer called lignin. Recalcitrant to degradation, lignin inhibits efficient extraction and hydrolysis of the cell wall polysaccharide and prevents cost-effective lignocellulosic-biofuel production. Unfortunately, lignin cannot simply be genetically removed without incurring deleterious consequences on plant productivity. The cost effectiveness of the conversion of the lignocellulosic biomass into sugars is still one of the major components to produce cheap biofuels. Therefore, strategies to reduce the lignin recalcitrance and to increase polysaccharide deposition without altering plant growth should be developed to create the next generation of high sugar yielding energy crops.
In the feedstock division at the Joint Bioenergy Institute, we used synthetic biology to design new control systems to create a better spacio-temporal deposition of the lignin polymer as well as the other secondary cell polymers. We also developed new strategies to produce new monolignols in order to manipulate the lignin composition and reduce its recalcitrance. Some of these strategies result into increased cell wall polysaccharides deposition and biomass density, and other into reduced cell wall recalcitrance without affecting the plant growth. Finally, some of these approaches were combined together to further improve plant biomass characteristics. Developed approaches and preliminary data will be presented.

This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.

Exploring transgene regulation by alternative splicing
Ming Chen Hammond
Dept of Chemistry, Room 419 Latimer Hall , University of California, Berkeley, CA 94720-1460
e-mail: mingch@berkeley.edu

Compared to transcriptional activation, other mechanisms of gene regulation have not been widely exploited for the control of transgenes. One barrier to the general use and application of alternative splicing is that it has not been shown that splicing-regulated transgenes can be reliably and simply designed. We now have demonstrated that a plant-specific splicing cassette can be inserted directly and tracelessly into a variety of open reading frames (ORFs), generating transgenes whose expression is activated in response to a specific protein inducer. The surprisingly minimal sequence requirements for the maintenance of splicing fidelity and regulation indicate that this splicing cassette can be used to regulate any ORF containing one of the amino acids Glu, Gln, or Lys.
Furthermore, the level of gene activation has been improved to ~19-fold by rational design of the splicing cassette. Thus, conditional splicing has the potential to be generally useful for gene regulation. We are currently applying this new regulatory strategy toward the study of plant disease resistance and lignin biosynthesis.

Murus Ex Planta: The road to synthesizing a Plant Cell Wall Polysaccharide in vitro
Markus Pauly
Energy Biosciences Institute, 130 Calvin Hall, MC 5230 Berkeley, California 94720
e-mail: mpauly69@berkeley.edu

Markus Pauly1, Alex Schultink1, Jacob Jensen2, Sascha Gille1, Barbara Reca2,

1Energy Biosciences Institute, University of California, Berkeley 2Great Lakes Bioenergy Center, Michigan State University

Most of the biomass of plants consists of lignocellulosics, a complex aggregate of microcrystalline cellulose microfibrils, crosslinking water-soluble hemicelluloses, and a water repellent polyphenol, lignin. In order to utilize this renewable resource it is desirable to understand how the plant synthesizes these polymers. Such knowledge can also lead to tailoring the polymers for numerous applications in varying industries.

The ultimate goal of the research in my lab is to understand the complete biosynthetic pathway of a wall polymer, such as the abundant hemicellulose xyloglucan. Proof that the biosynthesis has been elucidated on a molecular level would come from reconstructing the responsible biosynthetic pathway of this polymer in e.g. a yeast.

Various strategies have been employed in our lab to identify the genes involved in the synthesis of this polymer including massive parallel pyrosequencing of relevant plants and tissues, forward genetic screens of Arabidopsis with altered xyloglucan structures, and reverse genetic approaches of analyzing insertional knock-out lines. The results demonstrate that plants could be identified that harbor advantageous properties for biorefineries. Moreover, once the function of the genes has been ascertained they are transformed into yeast in an attempt tosynthesize a heterologous xyloglucan.

Cyanogenic Glucosides
Birger Lindberg Moeller
Department of Plant Biology and Biotechnology, Thorvaldsensvej 40, Frederiksberg, 1800 Denmark
e-mail: blm@life.ku.dk 
 
For more than 420 million years, plants, insects and their predators
have co-evolved based on a chemical arms race including deployment of refined chemical defense systems by each player. Cyanogenic glucosides are produced by numerous plants (e.g. sorghum, barley, cassava, clover, flax, almonds) and by some specialized insects as part of this arms race. The biosynthetic pathway is catalyzed by multifunctional cytochrome P450s (CYP79 and CYP71E) and a UDP-glucosyltransferase with oximes as a key intermediate. The enzymes are thought to be organized within an enzyme complex (metabolon) to ensure rapid metabolism of the toxic pathway intermediates.
The genes encoding the biosynthetic enzymes are clustered on the genome. When attacked by fungi able to rapidly detoxify hydrogen cyanide, the second P450 in the pathway may be inactivated by the associated oxygen burst resulting in production of oximes with anti-fungal activity. Following plant tissue disruption the cyanogenic glucosides are hydrolyzed and release toxic hydrogen cyanide to protect the plant from generalist herbivorous insects. When present in crop or feed plants this may pose a significant problem for human and animal consumption. Forage sorghum contains the cyanogenic glucoside dhurrin and following adverse growth conditions, the amounts of HCN released may be toxic to grazing lifestock.
In a collaboration headed by Australian researchers, biochemical screens and TILLING approaches have been used to identify a single amino acid change in the CYP79A1enzyme that resulted in an inactive enzyme and acyanogenic plants. Other mutants have been identified that are cyanogenic as seedlings but where the leaves of the mature plants are acyanogenic. This would be an ideal situation for forage production.

Adam M. Takos, Camilla Knudsen, Daniela Lai et al.: “Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway”. The Plant Journal (2011) in press

C.K. Blomstedt, R.M. Gleadow, N. O’Donnell, P. Naur, K. Jensen, T. Laursen, C.E Olsen, P. Stuart, J.D. Hamill, B.L Møller and A.D. Neale “A combined biochemical screen and TILLING approach identifies mutations in Sorghum bicolor L. Moench resulting in acyanogenic forage production” Plant Biotechnology Journal pp. 1–13 (2011)

B.L. Møller: Functioning dependent metabolons. Science 330:1328-1329 (2010)

N. Bjerg-Jensen, M. Zagrobelny, K. Hjernø, C.E. Olsen, J. Houghton-Larsen, J. Borch, B.L. Møller and S. Bak: Convergent evolution in biosynthesis of cyanogenic defence compounds in plants and insects.  Nature Communications 2:273 (2011)

Improving biofuel production by modifying biosynthesis of cell wall polysaccharides
Henrik Vibe Scheller
Henrik V. Scheller1,2, Emilie A. Rennie1,2, Berit Ebert1, Jane Lau1,
Yuzuki Manabe1, Ai Oikawa1, Sara F. Hansen1, Soe M. Htwe1, Carsten Rautengarten1, Pia D. Petersen1,3, April J. M. Liwanag, Vaishali Sharma1, Dawn Chiniquy1,4, Pamela Ronald1,4, Fan Yang1, Yves Verhertbruggen1, Dominique Loque1
1. Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
2. Department of Plant & Microbial Biology, University of California, Berkeley, CA 94720, USA
3. Department of Plant Biology and Biotechnology, University of Copenhagen, Denmark.
4. Department of Plant Pathology, University of California, Davis, CA 95616, USA.
e-mail: hscheller@lbl.gov 

Plant biomass is a convenient way to harness solar energy, and biomass is already an important supplement to fossil fuels. However, there is a great need to develop new technologies that can provide fuels, especially liquid fuels for transportation, in an efficient and environmentally friendly way.
Production of biofuels from biomass requires decomposition of the polymers, which are comprised mainly of polysaccharides and lignin in the plant cell walls. These polymers are recalcitrant to degradation and some degradation products cannot be converted efficiently into fuels or may even be inhibitory. A better understanding of plant cell wall biosynthesis may enable development of crops with improved properties as biofuels feedstocks. The key enzymes are glycosyltransferases (GTs) and plants need hundreds of GTs and other transferases, hydrolases and transporters to synthesize the complex polysaccharides present in the walls. However, only a few of these enzymes have had their activity demonstrated. In addition to biosynthetic enzymes, plants need pathways to regulate the biosynthesis and to allow the plant to modulate its wall according to developmental and environmental signals.
Xylans are the major non-cellulosic polysaccharides in secondary cell walls and constitute a major component of plant biomass. Despite rather detailed information on the structure of xylans little is known about their biosynthesis. We have identified several enzymes involved in xylan synthesis, including glucuronosyltransferases, feruloyl-transferases, acetyl-transferases and putative arabinosyltransferases and confirmed their biochemical activity by heterologous expression and in vitro enzyme assays. By altering the expression of xylan biosynthetic enzymes, we have obtained plants with improved saccharification properties, a lower content of inhibitory compounds such as acetate and ferulate esters, and a higher hexose/pentose ratio. Importantly, these changes to the feedstock composition and density have been achieved without negative impact on plant growth and development.