Brief Summary of Minutes of Annual Meeting

This was the first meeting of Multistate Research Project NC1168 (Regulation of Photosynthetic Processes), which is the approved renewal of NC1142. The meeting was hosted by the Nebraska AES and held at the Beadle Center of the University of NebraskaLincoln on November 17, 2007. Attending members included Christoph Benning, Raymond Chollet, Gerald Edwards, Glenda Gillaspy, Irwin Goldman, Karen Koch, Wayne Loescher, John Markwell, Gail McLean, Archie Portis, Jack Preiss, Mary Rumpho, Michael E. Salvucci, Robert J. Spreitzer, Donald P. Weeks, and Marna Yandeau-Nelson (for Guiltinan). Attending guests included Gary Cunningham (Dean and Director of the Nebraska AES) and Julie Stone (University of Nebraska). The meeting convened at 8:50 AM in room E228 in the Beadle Center. Bob Spreitzer (Nebraska, AES) provided opening remarks and introduced Dr. Gary Cunningham, Dean and Director, Nebraska AES. Bob Spreitzer then gave the first presentation on Rubisco engineering studies. A new member, Mary Rumpho, Maine AES, then presented her work on a sea slug that engulfs and maintains chloroplasts. Bob Spreitzer then introduced Irwin Goldman, NC1168 project administrator, who drove in from Madison, WI. Archie Portis, Illinois ARS, presented his work on genetic engineering of Rubisco in plants and member Hans Bohnerts work on a new algorithm to correlate gene expression patterns with gene function. At 10:35 AM a break occurred, and, at 10:55, Mike Salvucci, Arizona ARS, spoke about next years meeting in Arizona, an upcoming Gordon Conference on photosynthesis, and his work on Rubisco activase and heat stress. Don Weeks, Nebraska AES, spoke about carbon-concentrating mechanisms, a collaboration with member Martin Spalding. Lunch occurred in the atrium of the Beadle Center at 11:35 (hosted by the director of the Nebraska AES). The meeting reconvened at 1:00. Gerry Edwards, Washington AES, described his work and also member Tom Okitas work on the AGPase enzyme. Karen Koch, Florida AES, spoke about sorbitol and carbon partitioning, followed by Marna Yandeau-Nelson, who spoke for member Mark Guiltinan, Pennsylvania AES, on the starch branching enzymes. After a break, Jack Preiss, Michigan AES, described his structural work, and added that this is perhaps his last meeting. Wayne Loescher, Michigan AES, spoke about sugar alcohols and plant stress. After a break, Glenda Gillaspy, Virginia AES, followed with a talk on sugar-alcohol synthesis genes. Christoph Benning, Michigan AES, spoke about plans to produce oils in canola that can be used for biodiesel production. After a break, Irwin Goldman, Administrative Advisor, Wisconsin AES, provided an update on the last project (NC-1142), relaying that Bob Spreitzer was key in getting the new project (NC-1168) approved. Irwin then outlined the process of NCRA and encouraged members to let their Experiment Station directors know about NC1168 activities and the importance of this project. Irwin described how he could help organize an NC1168 meeting in the future and could provide assistance to the members of NC-1168. Irwin also announced that a progress report for NC1142 would be due soon, and that he would send inquires out by email at the beginning of the year requesting information for this report. Gail McLean, USDA/CSREES Representative then presented an update on USDA/NRI programs relevant to members. She outlined the current funding level and outlook for several programs. She added that it is important for this group to file annual reports. At 5:05 PM the business meeting convened. Bob Spreitzer brought up the next and future meeting dates as related to whether the group should stay with the second weekend in November or switch to the third. Mike Salvucci remarked that hotel rooms in Arizona would be cheaper earlier in the season. After a discussion, the group decided to retain the second weekend in November as the meeting time for future meetings. Bob Spreitzer then remarked on the status of attending and non-attending members, adding that all members are active in attending the meetings. Jack Preiss inquired about Doug Randall, and Bob Spreitzer replied that Randall had retired. Bob Spreitzer announced a need for a new secretary, who would host the meeting in 2011, and who would be in charge of the project renewal. Christoph Benning was nominated and then elected to serve in this position. The renewal will be due in the Spring of 2012. The schedule for future meetings was determined: 2008 Arizona AES Mike Salvucci 2009 Ohio AES J. C. Jang 2010 Virginia AES Glenda Gillaspy 2011 Michigan AES Christoph Benning The meeting was adjourned at 5:20 PM.

A. Plastid Function and Intracellular Communication. IA-AES has continued to investigate the role of the var2 gene of Arabidopsis (which encodes a chloroplast AtFtsH metalloprotease) in the repair of photodamaged photosystem II. Selection of suppressors has now identified genes encoding a polyribonucleotide phosphorylase and a chaperonin 60a that may also contribute to the assembly and regulation of the photosynthetic apparatus. ME-AES has continued to study the endosymbiotic relationship between the marine mollusk Elysia chlorotica (sea slug) and chloroplasts of the heterokont alga Vaucheria litorea. Sequences for phosphoribulokinase and the photosystem II Mn-stabilizing protein have been found in sea slug DNA, indicating that horizontal gene transfer may have occurred as a means for maintaining photosynthesis in the endosymbiotic algal chloroplasts. B. Photosynthetic Capture and Photorespiratory Release of CO2. IA-AES has identified a gene (lciB) responsible for the CO2-concentrating mechanism in the green algae Chlamydomonas reinhardtii. Further analysis, in collaboration with NE-AES, will evaluate the potential for transferring all or part of the CO2-concentrating mechanism into crop plants. NE-AES is using RNAi to study a possible bicarbonate transporter that may be involved in the CO2-concentrating mechanism. WA-AES is studying unique species in the family Chenopodiaceae that perform C4 photosynthesis without Kranz anatomy. Microtubules are critical for the maintenance of two cytoplasmic compartments. One type of chloroplast has Rubisco, starch biosynthesis, and well-developed grana, while the other type of chloroplast is specialized for supporting CO2 fixation in the C4 cycle. AZ-ARS and IL-ARS have continued to collaborate on Rubisco activase. An S-tagged b-isoform of activase has been engineered in Arabidopsis and crossed with an Arabidopsis plant expressing only the a-isoform. The new line will be used to examine the effects of temperature on interactions among activase subunits, and the effect of these interactions on Rubisco regulation. IL-ARS has transformed more-thermostable Rubisco activase into Arabidopsis to demonstrate improved photosynthesis and growth at elevated temperature. IL-ARS is attempting to eliminate rbcS gene expression in tobacco by employing RNAi technology. The Amaranthus rbcS gene has been introduced as a first step to see whether C4 Rubisco may provide a benefit for CO2 fixation. NE-AES has produced hybrid Rubisco enzymes in Chlamydomonas comprised of crop-plant small subunits and Chlamydomonas large subunits. Small subunits influence large-subunit catalysis. Phylogenetic analysis and directed mutagenesis have shown that large-subunit regions far from the active site can also influence catalysis. KY-AES has determined the polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase using a fusion protein construct between the first 23 amino acids of the large subunit of Rubisco and human carbonic anhydrase II. RNAi-mediated knockdown of the methyltransferase in transgenic tobacco resulted in a two-fold decrease of a-tocopherol, the product of g-tocopherol methyltransferase, indicating that its consensus sequence may also be methylated. The crystal structure of Arabidopsis peptide deformylase 2 has been determined, and its substrate specificity is being defined. C. Mechanisms Regulating Photosynthate Partitioning. FL-AES has investigated a role for sorbitol metabolism and/or shuttling within developing kernels, possibly involving dual contributions to carbon transport and modulation of reducing power. Analysis of maize mutants has shown that sorbitol metabolism can contribute prominently to kernel capacity for sucrose import and use. FL-AES has also developed a method for 3'-UTR profiling that resolves expression to the level of individual gene family members and facilitates gene discovery. By whole-genome analysis, IL-AES has discovered that genes involved in nitrogen sensing and signaling may limit productivity in crop plants grown under elevated CO2 conditions. NV-AES has been identifying targets in nitrogen and carbon metabolism that interact with affinity-tagged 14-3-3 regulatory proteins in Arabidopsis. OH-AES is studying bZIP transcription factors involved in sugar signaling. SC-AES has shown that F-actin mediates some aspects of glucose signaling in Arabidopsis by interacting with mitochondrial hexokinase 1. Directed mutagenesis indicates that the hexokinase-like proteins, which lack glucose phosphorylation activity, may have specialized roles as regulatory components for glucose signaling. VA-AES is studying the synthesis and signaling functions of the sugar alcohol myo-inositol, which is linked to osmoprotective functions, signal transduction, and ascorbic acid synthesis, among other functions. Arabidopsis mutants lacking various members of the myo-inositol phosphate synthase and inositol monophosphatase enzyme families indicate that there are different subcellular locations for the first and second steps in myo-inositol synthesis. MI-AES is investigating the function and molecular biology of WRINKLED 1 and the genes controlled by this transcription factor in Arabidopsis. Analysis of mutants and engineered genes are clarifying the role of WRINKLED 1, pyruvate kinase, and glucose-6-phosphate dehydrogenase in the partitioning of photosynthetic carbon to oils. Understanding the conversion of photosynthetic sugars into triacylglycerols will be essential for the engineering of novel biofuel crops. MI-AES and WA-AES continue to define the structure-function relationships of ADP-glucose pyrophosphorylase, which catalyzes the first step in starch synthesis. Families of genes encoding the subunits have been analyzed in Arabidopsis. Hybrid enzymes and directed mutagenesis are being used to define regions involved in catalysis and regulation. This information will be used for the further engineering of improvements in the production of starch. WA-AES has used up-regulated forms of ADP-glucose pyrophosphorylase to increase leaf starch and biomass of Arabidopsis and rice. PA-AES is studying the starch branching enzymes of maize, which are critical in determining the molecular fine-structure of starch. Recent data indicate that starch branching enzyme activity in leaf extracts can be modulated by redox reactions. D. Developmental and Environmental Limitations to Photosynthesis. AZ-ARS and IL-ARS have shown that inhibition of photosynthesis and loss of Rubisco activation under moderate heat stress is more acute when Rubisco activase levels are suboptimal for photosynthesis, regardless of the isoform of activase. This finding is consistent with a proposed mechanism for inhibition of photosynthesis by heat stress based on the acute sensitivity of activase to thermal denaturation. New data indicate that chaperonin-60b plays a role in acclimating photosynthesis to heat stress, possibly by protecting Rubisco activase from thermal denaturation. IL-AES has found that chemical treatments promoting leaf greening in maize can disrupt ear development by altering ethylene sensing. IL-AES continues to exploit soyFACE (http://www.soyface.uiuc.edu) for investigating the effects of elevated CO2 on plant productivity. Metabolic profiling and gene-mining methods are being developed to analyze environmental stress effects. Gene networks are being defined. MI-AES and WA-AES have shown that expression of the mannitol biosynthetic enzyme gene, M6PR, in Arabidopsis improves salt tolerance. MI-AES is also studying the constitutive expression of the abiotic stress responsive transcription-factor gene, CBF3, and the plasma-membrane sodium antiporter gene, SOS1, in transgenic Arabidopsis to see whether further improvements in salt tolerance can be achieved. VA-AES is studying the role of myo-inositol signaling in abiotic stress. Protein-protein interactions in sugar signaling pathways are being identified. NV-AES has used microarray transcript profiling, quantitative RT-PCR, and metabolite profiling to show that metabolite differences found under long-term salinity or water deficit stress are linked to differences in transcript abundance of many genes involved in energy metabolism and nitrogen assimilation, particularly photosynthesis, gluconeogenesis, and photorespiration. Water-deficit-treated plants also appeared to have a higher demand than salinized plants to adjust osmotically, detoxify free radicals, and cope with photoinhibition. NV-AES is also screening through Arabidopsis mutants to identify genes that affect a plant's ability to grow under extreme environments. These genes provide important leads towards a goal of engineering higher-yield crops.