Summaries of FY 2001 Activities Energy Biosciences August 2002 ABSTRACTS OF PROJECTS SUPPORTED IN FY 2001 (NOTE: Dollar amounts are for a twelve-month period using FY 2001 funds unless otherwise stated) 1. U.S. Department of Agriculture Urbana, IL 61801 Biochemical and molecular analysis of a new control pathway in assimilate partitioning Daniel R. Bush, USDA-ARS and Department of Plant Biology, University of Illinois at Urbana- Champaign $72,666 (21 months) Plant leaves capture light energy from the sun and transform that energy into a useful form in the process called photosynthesis. The primary product of photosynthesis is sucrose. Generally, 50 to 80% of the sucrose synthesized is transported from the leaf to supply organic nutrients to many of the edible parts of the plant such as fruits, grains, and tubers. This resource allocation process is called assimilate partitioning and alterations in this system are known to significantly affect crop productivity. We recently discovered that sucrose plays a second vital role in assimilate partitioning by acting as a signal molecule that regulates the activity and gene expression of the proton-sucrose symporter that mediates long-distance sucrose transport. Research this year showed that symporter protein and transcripts turn-over with half-lives of about 2 hr and, therefore, sucrose transport activity and phloem loading are directly proportional to symporter transcription. Moreover, we showed that sucrose is a transcriptional regulator of symporter expression. We concluded from those results that sucrose-mediated transcriptional regulation of the sucrose symporter plays a key role in coordinating resource allocation in plants. 2. U. S. Department of Agriculture Raleigh, NC 27695-7631 Molecular Analysis of the Role of Sucrose Synthase in Sugar Sensing and Assimilate Partitioning Steven C. Huber, USDA/ARS and Departments of Crop Science and Botany, NCSU $111,000 The overall goals of the project are to determine the molecular bases for the localization of sucrose synthase (an important enzyme of sucrose utilization) within heterotrophic cells, the role of phosphorylation of specific sites on the enzyme, and to identify the requisite protein kinases that phosphorylate sucrose synthase. It is known that sucrose synthase is phosphorylated on a single major site close to the N- terminus (serine-15). Studies in the past year suggest that phosphorylation of serine-15 affects either the conformation or accessibility of the N-terminus of the protein. This structural change may underlie effects on activity and/or localization. In addition, we have obtained first evidence for phosphorylation of a second residue—serine-170. Both sites appear to be phosphorylated by calcium-dependent protein kinases (CDPKs). Phosphorylation-state specific antibodies that recognize phospho-serine-15 or phospho-serine- 170 have been produced to follow the phosphorylation status of these two sites. Results to date suggest that phosphorylation of serine-170 (but not serine-15 as originally thought) might be part of the mechanism that controls the membrane association of sucrose synthase. Identifying the mechanisms that control the intracellular localization of sucrose synthase is important because its localization may control how plant cells use sugars for different processes such as respiration, cell wall synthesis or starch production. 3. U.S. Department of Agriculture Urbana, IL 61801-3838 Consequences of Altering Rubisco Regulation Archie R. Portis, Jr., USDA/ARS and Departments of Crop Sciences/Plant Biology, University of Illinois $61,968 Rubisco initiates photosynthetic carbon acquisition and its activity is limiting under high light at atmospheric levels of carbon dioxide. However, under either limiting light or when adequate sinks for the products of Energy Biosciences Summaries of FY 2001 Activities / 1 photosynthesis are not available, the activity of Rubisco is reduced below its maximal capacity even though a limitation by the availability of its other substrate, RuBP, would be sufficient. The reasons for this response are unclear. The activation state of Rubisco is determined by the activity of its regulatory protein, Rubisco activase. Rubisco activase is usually present as two isoforms, differing at the carboxyl terminus and generated by alternative splicing of the pre-mRNA. Earlier work examining mutant forms of the protein in vitro indicated that reduction/oxidation of the larger isoform dramatically altered the activity of the protein and suggested that this regulation might account for the modulation of Rubisco activity by light intensity. By using transgenic Arabidopsis plants expressing only one of the two isoforms and mutant forms of the larger isoform, we found that reduction/oxidation of the larger isoform is required in order to down-regulate Rubisco activity in response to limiting light intensities. These plants and transgenic plants expressing mutant forms of the activase that have an altered response to the ATP/ADP ratio, which may account for the down- regulation when adequate sinks are not available, are currently being investigated further in order to determine the consequences of altering Rubisco regulation on photosynthesis, growth, and the response of plants to their environment. 4. U.S. Department of Agriculture Madison, WI 53706-1108 What is the Extent of Metabolic Plasticity in the Lignification Process, and Can it be Exploited? John Ralph and Ronald Hatfield, USDA Agricultural Research Service; US Dairy Forage Research Center $95,000 Lignin is a polymer that plants use to bind the fibers together and confer structural rigidity to stems as well as provide other functions for the well-being of the plant. However, utilization of plant resources is often limited by the difficulty of dealing with lignin. It is the polymer that must be removed to make fine paper, for example. This research explores the alterations to lignin’s structure and properties in a variety of natural mutant and transgenic plants; many are just becoming available as researchers seek to alter the lignin biosynthetic pathway. Such plants provide a rich source of insights into the chemistry of lignin formation that will allow more efficient uses of our plant resources in the future. Our work is revealing that deprivation of a plant’s ability to produce lignin precursors can result in increased incorporation of other plant components (phenolics) into the lignin. Some of the incorporated components were unexpected and not normally associated with the biosynthetic pathway. Others are producing new structures in the lignin that alter the pulping properties, for example. We have identified marker compounds for many of the gene deficiencies that are useful to researchers trying to assess how heavily their plants have been affected. The large compositional shifts that are possible indicates considerable plasticity in the lignification process, suggesting new approaches to plant modification for improved utilization in processes ranging from polysaccharide digestion in ruminants to industrial chemical pulping. 5. U.S. Department of Agriculture Beltsville, MD 20705 Controls on production, incorporation and decomposition of glomalin -- a novel fungal soil protein important to soil carbon storage Sara Wright, Soil Microbial Systems Laboratory (Note: see also University of Montana, M.C. Rillig) $66,491 (FY 99 funds) A group of beneficial soil fungi live on carbon supplied directly to them by plant roots. The fungi are called arbuscular mycorrhizal fungi or AM fungi. These fungi have long hair-like projections called hyphae that extend several cm from the root into soil. Glomalin is a glycoprotein that is produced on AM hyphae in large amounts, is released from hyphae, and attaches to soil particles. Glomalin is important because concentrations in soil are correlated with soil aggregate stability, and large amounts of labile soil carbon are sequestered in aggregates. Plants fix more carbon under elevated CO2 than under ambient CO2, and more carbon is transported from roots to these fungi. We continue to find larger amounts of glomalin in planted 2 / Energy Biosciences Summaries of FY 2001 Activities soils exposed long-term or short-term to increased atmospheric CO2. Preliminary evidence indicates that warming, without increased CO2, is detrimental to aggregate stability. We found that glomalin production is influenced by plant species in the field. Laboratory studies indicated that glomalin production differed among fungal species, but not between corn and crimson clover. Incubation studies indicated that glomalin levels decline more rapidly in soils from the Midwest that have been conventionally tilled compared with no- till soils. We have evidence that glomalin makes up a large part of soil organic matter in an organic soil from Hawaii. Our work shows that glomalin is in the fraction of soil organic matter called humin – a fraction that was previously thought to be composed of undefined insoluble organic matter. 6. University of Alabama Tuscaloosa, AL 35487-0336 A Combined Genetic, Biochemical, and Biophysical Analysis of the A1 Phylloquinone Binding Site of Photosystem I from Green Plants Kevin Redding, Department of Chemistry $194,001 (FY00 funds - two years) Our long-term goal is to understand how photosynthetic organisms convert electromagnetic energy in the form of light into chemical energy by studying the light-driven transport of electrons through the photosystem 1 (PS1) protein. Two phylloquinone molecules