Brief Summary of Minutes of Annual Meeting

Annual Meeting Date and Location: November 22, 2003; Nevada AES host; Best Western Airport Plaza Hotel, Reno, Nevada

Attending members: Dr. Robert Aiken (Kansas AES), Dr. Ray Chollet (Nebraska AES), Dr. John Cushman (Nevada AES), Dr. Larry Daley (Oregon AES), Dr. Gerry Edwards (Washington State AES), Dr. Steve Huber (Illinois AES), Dr. Karen Koch (Florida AES), Dr. Wayne Loescher (Michigan State AES), Dr. John Markwell (Nebraska AES), Dr. Brandon Moore (South Carolina AES), Dr. Archie Portis (Illinois AES), Dr. Jack Preiss (Michigan State AES)

New Members: Dr. Robert Aiken (Kansas AES)

Retiring members of the NC-1-142 Group: Dr. Jack Shannon (Pennsylvania AES)

Attending guests interested in NC-1-142 membership: Dr. Jeff Harper (Nevada AES), Dr. Ron Mittler (Nevada AES)

The annual meeting was called to order by Dr. John Cushman, Chair. It was attended by all members listed above, although our Administrative Advisor, Forrest Chumley and CSREES Representative, Gail McLean were both unable to attend. Each member presented a 25 min overview of their previous years accomplishments, followed by questions and comments following earch presentation. In his place, Dr. Ron Pardini, Associate Director, Nevada AES welcomed the group to Reno and gave a short overview of the activities of the Nevada AES. Each member the presented a 25 min overview of their previous years accomplishments, followed by questions and comments following earch presentation. Scientific discussions began during the informal breakfast period, and continued during breaks, lunch, and dinner. The on-site arrangements for these events facilitated the extent of discussions and ease of interactions. Presentations occurred from 8:30 am to 6:00 pm, although collectively the talks continued for about an hour longer than anticipated. This was due largely to the extent of interactive discussion generated in association with the presentations.

The business meeting was convened after the talks and lasted approximately an hour. The primary issues were to 1) election of new officers, 2) appraise sites and formats for future meetings, 3) increase attendance, 4) discuss candidates for incoming and outgoing membership, and 5) discuss new business.

The group concurred that the previously-outlined plan for identifying meeting sites and candidates for the Executive Committee needed to be revisited. After some discussion, the Florida AES was volunteered as a meeting site for 2005 by Dr. Karen E. Koch, who also agreed to serve as the new secretary. The period of service for this position was advanced to that of the current year by the absence of Dr. Robert Houtz of Kentucky AES. Dr. Robert Spreitzer of Nebraska AES was nominated to serve as secretary in 2006.

For future meetings, the recurring issue was raised of whether it would be better to visit actual AES campus sites or to use hotels at readily accessible locales. All concurred that there were considerable advantages to meeting at campus sites, because this facilitated interactions with scientists at the host institution (especially collaborations with the hosting NC-1-142 member). However, the group also felt that these advantages were generally outweighed by the greater time and travel costs involved in reaching the universities. It was also noted that ease of accessibility and proximity to a major airport have been important to members past decisions of whether to attend or not, since significant differences in time can be involved. A final consideration was that of the broad geographic distribution of our now nation-wide project, and the travel challenges this poses for some members. The discussion was especially relevant to arrangements for the 2004 meeting to be hosted by the South Carolina AES. Brandon Moore agreed to investigate sites with these considerations in mind, yet also noted that the difficulties involved in hosting an off-site meeting could increase markedly with distance.

Several potential means of increasing attendance were discussed. -- Ease of travel and efficient time use were considered important. The possibility of shifting the meeting date was also raised. -- The date of the meeting was also tentatively shifted to one week earlier. Several members reported travel difficulties apparently due to the weekend of the meeting being so close to Thanksgiving. Costs were higher and availability limited. A date one week earlier was suggested, and there was a general consensus that this was worth trying. -- Membership issues were also viewed as relevant to attendance and received additional attention.

Significant discussion was dedicated to means of identifying vigorous, new members (especially in the area of Objective 1: Photochemistry and the Biogenesis of the Photosynthetic Apparatus) and also of linking attendance to viable membership. The group was enthused about research presented by the two prospective new members from the Nevada AES, and their work was discussed. In addition, members were asked to think about potential new collaborators in the area of photobiology.

The issue of linking membership to attendance was discussed. It was suggest that our Administrative Advisor, Dr. Forrest Chumley, could help by officially contacting non-attending members (see list above) and their Experiment Station Directors to request an explanation of their lack of attendance. Initially this would be to encourage attendance, and if non-involvement persisted, to suggest or discuss resignation. This has been a consistently important issue

Several items of new business were discussed. For the new five year NC-1-142 project initiated in October 2002, the development of a project website to be located at Kansas State University was discussed. Possible information to be presented on the website might include a lay summary of the NC-1-142 project, citations of publications, a summary of key impacts (usefulness of findings) that could be presented as bullet points. It was suggested that these items be included in the annual reports to facilitate the posting of this information to the website. It was also suggested that the project website have links to the Long laboratory, in particular, as well the websites of all project participants.

Most discussion addressed Objectives 2, 3, and 4, which respectively were Photosynthetic capture and photorespiratory release of CO2, Mechanisms regulating photosynthate partitioning, and Developmental and environmental limitations to photosynthesis. The greatest number of attendees at this years meeting worked in some way on Objective 3 (partitioning), but overlapping interests and vigorous discussion was evident in all three areas. As noted for the previous meeting (2002) concern was expressed at our need to either acquire greater expertise in the area of Objective 1 (Photochemistry and Biogenesis of the Photosynthetic Apparatus) or omit this area from the project.

Key discussions centered on the science outlined in reports from the attending AES representatives and the visiting candidates for new membership. The primary issues of the business meeting are enumerated above.

As decided by NC-1-142 participants, the leadership succession will progress from Dr. John Cushman (Nevada AES) departing Chair of the 2003 meeting, to Dr. Brandon Moore (South Carolina AES) incoming Chair and host for the 2004 meeting. Dr. Karen Koch (Florida AES) will become vice-chair, and will also continue to serve as secretary for the 2004 meeting unless a suitable candidate can be identified at its opening. As noted above, the weekend selected for the 2004 meeting will be ONE WEEK EARLIER than in past years, now two weekends prior to the Thanksgiving holiday. The date will be Saturday, November 13, 2004.

Photosynthesis is a unique and fundamental process on which most life forms on earth depend. However, this process can be dramatically limited by environmental constraints resulting in rates of agricultural productivity that are far lower than optimal rates. The research objectives of this regional project are to improve our understanding of the biochemical regulation of important photosynthetic enzymes and how environmental and developmental signals affect photosynthetic performance at the molecular genetic level across four different areas of photosynthetic research. Objective 1: Photochemistry and the biogenesis of the photosynthetic apparatus. Collaborating units include AES and/or ARS-USDA: IA-AES (Rodermel). A major focus of research for IA-AES (Rodermel) continues to be improving our understanding of the control of thylakoid membrane biogenesis and the mechanisms that coordinate gene expression between the nucleus-cytoplasm and the plastid. The model system being used the immutans (im) variegation mutant of Arabidopsis, which contains green and white sectored leaves. White cells contain plastids with abnormal plastids that lack internal membrane structures and accumulate phytoene, a colorless C40 carotenoid intermediate, indicating that im is impaired in the activity of phytoene desaturase (PDS), the plastid enzyme that converts phytoene to zeta-carotene. The IMMUTANS locus encodes for a chloroplast homolog of the mitochondrial alternative oxidase (AOX) and this gene product may act as a terminal oxidase in plastid membranes. This same terminal oxidase is expected to function in the redox pathway that desaturates phytoene. The tomato ghost and Arabidopsis immutans variegation mutants define orthologous genes, opening the possibility of using tomato fruit ripening as a model to explore GHOST (IMMUTANS) protein function. The GHOST gene product plays a critical role in the biogenesis of chloroplasts and chromoplasts and likely is involved in carotenogenesis. To identify other gene products that may interact with IM, suppressor screens have been initiated and have resulted in the isolation of three suppressor mutations. Map-based cloning of one of the suppressors is near completion. Objective 2: Photosynthetic Capture and Photorespiratory Release of CO2. Collaborating units include AES and/or ARS-USDA: IL (Portis), MO (Randall), NE (Chollet, Spreitzer), NV (Cushman), OR-AES (Daley). Investigations at IL-ARS (Portis) into the response of Rubisco activase to high temperature, its interactions with Rubisco, and improved strategies for genetically engineering Rubisco have progressed significantly this past year. At elevated temperatures, Rubisco activity becomes inactivated by alterations or reductions in Rubisco activase enzyme. These results suggest that activase enzyme plays an important role in limiting the inhibition of photosynthesis at high temperatures. Manipulation of the degree of polyunsaturation of thylakoid lipids also was found to inhibit net photosynthesis. As part of a long-term collaboration with NE-AES (Spreitzer) the effects of replacing specific amino acid residues of the Chlamydomonas Rubisco large subunit residues with tobacco residues unique to the Solanaceae were investigated. To date, such changes have had only minimal effect in altering the specificity with spinach and tobacco activase. Efforts to continue the identification of residues at the site of Rubisco interaction with activase remain under investigation. In collaboration with Dr. Long's (IL-AES) group, a steady-state biochemical model for leaf photosynthesis was coupled to a canopy biophysical microclimate model and used to explore how theoretical changes in the kinetic parameters of Rubisco or replacing the typical crop Rubisco with naturally occurring Rubiscos will increase total crop carbon gain. As the genetic engineering of Rubisco is proving to be difficult and time consuming, such modeling studies will facilitate the selection of modified Rubiscos. Studies at MO-AES (Randall) investigating the mitochondrial pyruvate dehydrogenase complex (mtPDC), which links glycolysis to the Krebs cycle by catalyzing the oxidative decarboxylation of pyruvate to acetyl-CoA. The reversible phosphorylation of mtPDC, by pyruvate dehydrogenase kinase (PDK), provides a cardinal regulatory mechanism for the interaction of respiratory and photorespiratory metabolism. PDK lacks the 12 signature domains/motifs of the Ser/Thr protein kinase family, and instead, has the 5 signature motifs of the histidine protein kinases (HPKs) or two-component protein kinases. However, PDKs are a unique type of protein kinase having a His-kinase like sequence, but Ser-kinase activity. PDK is a member of the ATPase/kinase superfamily, which includes ATPases, Gyrase, HSP90, and the PHKs. In silco analysis has identified Lys241 as a potential catalytic residue and mutagenesis to Ala yielded a PDK with highly reduced affinity for ATP and low catalytic efficiency. Phosphorylation sites on the Arabidopsis E1alpha subunit of mtPDC have been mapped and the influence of this phosphorylation by E2 subunits have been investigated. To investigate the functional role of PDK in vivo, homozygous knockout lines of PDK have been generated and phenotypic characterization is underway. Investigations by NE-AES (Spreitzer) into the structure-function relationships of Rubisco have continued to add new insights into the catalytic efficiency and CO2/O2 specificity of Rubisco. These investigations have been performed in the green algal model, Chlamydomonas reinhardtii, because non-functional mutants of Rubisco can be maintained and subunits can be replaced at will in order to conduct genetic screens for the isolation of second-site suppressor mutations as a means for identifying complementing structural interactions. Comparative phylogenetic analysis of Rubisco large-subunit sequences has revealed 13 residues that differ in regions previously shown by mutant screening to influence CO2/O2 specificity. Mutant screens to identify compensatory changes in the small subunit of Chlamydomonas are underway. In collaboration with Dr. Inger Andersson (Swedish Agricultural University, Uppsala), X-ray crystal data sets have been obtained for more than 10 large-subunit mutant enzymes, and complete structures have been solved for six of them. Collaborative research with IL-ARS (Portis) has continued to focus on a Rubisco large-subunit loop that interacts with Rubisco activase. Additional substitutions in this loop have failed to alter specificity for activase, indicating that overall structure may be more important than individual residues. Work is in progress to replace this entire loop with the loops characteristic of divergent land-plant Rubisco enzymes. Research by NE-AES (Chollet) had resulted in significant new insights into the structure-function relationships and regulatory phosphorylation of phosphoenolpyruvate carboxylase (PEPC) by PEPC kinase (PpcK). Two different PEPC isogenes were characterized from Chlamydomonas reinhardtii, representing the first PEPC genes described from any algal species. In related work on green-plant PEPC, detailed investigation of the sorghum recombinant C4 enzyme using site-directed mutagenesis revealed that the conserved, C-terminal QNTG-tetrapeptide is important for its influence on catalysis. Chollet's group also reported on the molecular cloning and expression analysis of phosphoenolpyruvate carboxylase kinase (PPCK) in Soybean (Glycine max). Four members of this small multigene family with varying expression patterns have now been described. Investigations into the biochemical, kinetic, and immunological analyses of a soluble, recombinant form of ice plant CAM PpcK with NV-AES (Cushman) were completed. Collaborative studies with Prof. Chris Chastain and his many undergraduate researchers at Minnesota State University-Moorhead into the striking up-/down-regulation by reversible phosphorylation of a strictly conserved Thr residue of plastidic pyruvate orthophosphate dikinase (PPDK), one of a few flux-controlling enzymes in C4 photosynthesis, have shown that the posttranslational modification of plastidic PPDK by its bifunctional regulatory protein (RP) is not restricted to green leaves of C4 and C3 plants, but also is present in plastids of developing cereal seeds. NV-AES (Cushman) has continued to conduct gene discovery through large-scale expressed sequence tag (EST) sequencing in the common ice plant, Mesembryanthemum crystallinum as a Crassulacean acid metabolism (CAM) model. This work has facilitated the molecular cloning of genes encoding circadian clock components in collaboration with the Hartwell (University of York, UK) and Bohnert (IL-NAES) laboratories. Collaboration with the NE-AES (Chollet) resulted in the molecular cloning, expression and purification of a partially soluble, recombinant form of phosphoenolpyruvate carboxylase kinase (PPCK1) from the common ice plant. NV-AES has also continued analysis of CAM-defective mutants of M. crystallinum obtained by fast neutron (Nf) irradiated plant populations. Finally, in collaboration with the Winter group (Smithsonian Tropical Research Institute), we have conducted molecular phylogenetic analysis of 31 Panamanian Clusia species in parallel with photosynthetic pathway mapping along the C3 to CAM continuum based on 13C/12C ratios of plant carbon and found that CAM species distribute on distinct branches of the phylogenetic trees consistent with the polyphyletic origins of CAM allowing exploitation of an extended range of ecological opportunities. OR-AES (Daley) has continued investigations into the possible mechanisms capable of explaining the polyphyletic evolutionary origins of CAM. Finally, several NC-1-142 researchers, including those at NE-AES, KY-AES, and USDA/ARS-Urbana, have edited and/or co-authored a minireview series on the CO2/HCO3_-fixing enzymes in plants that is related directly to the overall focus of Objective 2. Objective 3: Mechanisms regulating photosynthate partitioning. Collaborating units include AES and/or ARS-USDA: FL-AES (Koch), IA-AES (Knapp and Rodermel), IL-AES/ARS (Huber), MI-AES (Loescher and Preiss), SC-AES (Moore), WA-AES (Edwards and Okita). Sucrose cleavage by invertase generates hexoses essential for growth and sugar signaling during development of maize kernels. FL-AES (Koch) has tested ABA responsiveness on the mRNA abundance changes of invertases during early kernel development in maize wildtype ovaries and those of vp1 (viviparous1) ABA-insensitive mutants. The difference in invertase expression between WT and vp1 kernels provides evidence for a role of ABA-signaling in crosstalk with sugar sensing during early seed development. The interface between sugar and ABA signaling is also being studied in Arabidopsis in collaboration with IA-AES (Rodermel) in which the mRNA abundance patterns of 8 invertase family members is under investigation. The role of sucrose synthase and its regulation is also being investigated through the study of the growth, development, and cell wall composition of double and single mutants deficient in functional Sus1, Sh1, or both sucrose synthase genes. QTLs for plant height have been identified near both the SUS1 and SH1 loci. Work is in progress to determine the degree to which sucrose synthase may be contributing to these QTLs. The Koch lab, in collaboration with IL-AES (Huber), is also studying the effects of specifically-altered sucrose synthase transgenes in a double mutant sucrose synthase background to examine the effects of alterations in sucrose synthase phosphorylation in vivo. The IA-AES (Knapp) laboratory continues to investigate the responses of maize to various low temperature stress. Studies completed this year evaluated stress duration on the growth, electrical conductivity, and potassium leakage from the fourth leaf of maize inbreds that putatively differ in low temperature stress tolerance. The percent electrical conductivity (EC) values of inbreds from the control treatments were not significantly different under normal temperature conditions, significant differences were observed under low temperature conditions. While EC values provide a useful indirect measure of relative cold tolerance, potassium leakage does not appear to be as good a screening tool as EC. However, EC evaluation appeared to be influenced by leaf structural characters and there was not a good correlation between EC values and dry weight measurements. Thus, more work is needed to refine stress scenarios and screening techniques to reliably rank inbreds. IL-AES/ARS (Huber) have continued their investigations into the regulatory control mechanisms of sucrose synthase, an important enzyme responsible for sucrose metabolism and partitioning of carbon in various metabolic and biosynthetic pathways in growing plant organs, such as developing seeds. Sucrose synthase is phosphorylated at Ser15, close to the N-terminus. Sequence-specific antibodies were used to show that phosphorylation affects the conformation of the amino terminus of the protein, most likely by favoring an open-coil conformation rather than an alpha-helix. This conformational change may be responsible for an increase in enzyme activity and changes in binding to membranes that are observed. Calcium-dependent protein kinases (CDPKs) are thought to phosphorylate metabolic enzymes, such as nitrate reductase (NR), sucrose synthase (SUS) and sucrose-phosphate synthase (SPS). The Huber lab has characterized several new recognition motifs for phosphorylation sites in cellular proteins by CDPKs. The eukaryotic regulatory protein 14-3-3 is involved in many important plant cellular processes including regulation of nitrate assimilation, through inhibition of phosphorylated nitrate reductase (pNR) in darkened leaves. Structure-function studies of the 14-3-3 protein showed that the C-terminal tail functions as an autoinhibitor, that blocks binding to nitrate reductase when magnesium is not present. These results increase our understanding of the molecular mechanisms that control 14-3-3 binding, and also provide unique experimental tools to use in future experiments to study the function of 14-3-3 proteins in vivo using transgenic plants. MI-AES (Loescher) has continued their investigations on alcohol metabolism in the family Rosaceae, which includes the majority of the world's temperate tree fruits. Progress to date includes identification and characterization of a number of sorbitol transporters in fruit and other sink tissues in apple and cherry. Genes for these sorbitol transporters are quite similar, but they are often expressed at different stages of leaf and fruit development and in some cases their expression is correlated with changes in the sink's capacity to attract and utilize photosynthetic products. Expression of the gene encoding mannitol 6-phosphate reductase mannitol metabolism from celery in Arabidopsis showed that the transgenic plants produced mannitol and had higher tolerance to salinity than wildtype plants that did not produce mannitol. Analysis of Arabidopsis transformants was conducted in collaboration with WA-AES (Edwards) laboratory. WA-AES (Edwards and Okita) have collaborated with MI-AES (Loescher) to improve the salt tolerance of Arabidopsis by expression of the mannitol 6-P reductase gene from celery. The transformed plants produced mannitol and had higher tolerance to salinity than wildtype plants that did not produce mannitol. Other research by Edwards and Okita was directed at understanding the significance of starch biosynthesis in source (leaves) and sink (seed) tissues to photosynthesis, plant growth and crop production. Mutants with down-regulated forms ADP-glucose pyrophosphorylase (AGP), a key enzyme in regulation of starch biosynthesis in plants, showed reduced starch synthesis, with corresponding reductions in CO2 assimilation rates, and cumulative leaf area when compared to wildtype plants. Mutants containing single amino acid changes in the large subunit L1 gene of AGP were expressed in a mutant background that lacks the large subunit of AGP and only produces about half as much starch as the wildtype. Transformants were selected showing high AGP activities compared to wildtype plants and are now the subject of current investigations. MI-AES (Preiss) has investigated structure-function relationships of glycogen synthase (GS) from Escherichia coli and ADP-glucose pyrophosphorylase (AGP) from Arabidopsis. These enzymes catalyze the rate-limiting and committed steps in glycogen biosynthesis in bacteria and starch biosynthesis in plants, respectively. Comprehensive mutagenesis studies have identified specific residues critical for the affinity of glycogen synthase for ADPGlc, glucose-1-P binding site, and catalysis. Recent studies have shown that the Arabidopsis AGP catalytic subunit in its interaction with each of the four regulatory large subunits differentially present in a tissue-specific manner, confer different kinetic properties with respect to apparent affinities for the allosteric activator, 3PGA and for the inhibitor, Pi. The large subunit mainly present in leaf confers on the catalytic subunit the highest affinity for the allosteric effectors as well as for the substrates ATP and glucose-1-P when compared to the effects of the large subunits mainly present in the sink tissues. In an exciting development, the crystal structure of the potato tuber AGP catalytic subunit was recently been determined at 2.3 Å resolution and will now permit the location of enzyme substrates within the context of the three-dimensional structure and its relation to the catalytic residue, Asp145. SC-AES (Moore) is a new addition to the project this year. Research efforts focus on the characterization of the regulatory mechanisms that control sucrose and starch synthesis and degradation. Studies in Arabidopsis have identified hexokinase (HXK1) as a key protein involved both in glucose metabolism and glucose signaling. The cellular context for glucose signaling by HXK1 remains unclear. Transiently expressed HXK1-GFP is associated with mesophyll mitochondria, suggesting that glucose signaling somehow involves a mitochondrial function. To identify other possible interacting proteins, an affinity tagged version of HXK1 was used to isolate about 30 proteins, including a number of chloroplast and mitochondrial proteins, but no obvious transduction proteins. A related possibility for mitochondria functioning as an organizing center is that they may move directly to or from the nucleus, thereby facilitating signal transduction. Both Arabidopsis and pea leaf mitochondria have protein that cross-reacts with anti-F-actin antibody. Vegetative actin also was identified from the microsequencing experiment, as being associated with an HXK1-protein complex. Thus, a working hypothesis is that glucose signal transduction may involve mitochondria movement by actin filaments to/from the nucleus. Objective 4: Developmental and environmental limitations to photosynthesis. Collaborating units include AES and/or ARS-USDA: Guam AES (Marler), IL-AES (Below), KS-AES (Aiken and Knapp), MN-AES (Jones), NE-AES (Markwell), NV-AES (Cushman), PA-AES (Guiltinan). Research efforts at NE-AES (Markwell) are directed toward environmental limitations included collaboration with entomologists at the University of Nebraska-Lincoln and the University of Montana. Following up on the initial observation that methanol treatment may enhance plant growth under water-limiting conditions, through increased leaf formate dehydrogenase (FDH) enzyme activity, more detailed molecular genetic and biochemical investigations into the regulation of this enzyme were conducted. FDH is an NAD-dependent enzyme that catalyzes the oxidation of formate to CO2 in the mitochondria of higher plants. Although the enzyme is specific for its NAD cofactor and did not utilize NADP+ for the oxidation of formate, NADPH appeared to be an inhibitor of the NAD+-dependent formate oxidation. Upon heating, however, it is able to use NADP+ as a substrate for the oxidation of formate. This ability of the FDH to assume a conformation able to use NADP+ in a chloroplastic compartment is of interest and will be the focus of future in vitro mutagenesis studies. To be able to initiate in vitro mutagenic studies on cofactor specificity and stability of the Arabidopsis FDH, optimal conditions for the production of an active enzyme in Escherichia coli are being investigated. MN-AES (Jones) investigated the effects of high temperature during maize endosperm cell division on protein accumulation. This study confirmed that a 2 or 4-day heat stress caused a 20 to 48% reduction in total protein content. Specifically, zein content was reduced by an average of 53%, but zein composition was only mildly affected whereas, the concentration of glutelins and albumins plus globulins was negatively affected. Cytokinin oxidase (CKO) is the principal enzyme involved in cytokinin catabolism. In collaboration with Pioneer Hi-Bred International, a study characterizing cytokinin oxidase gene expression, localization and induction confirmed that cytokinin oxidase was expressed in the vascular bundle of kernels, coleoptiles and leaves and was developmentally regulated in a manner that is correlated with cytokinin levels and cytokinin oxidase activity. Research also continued in the use of transgenic plants over-expressing the bacterial form of the cytokinin synthesis gene (isopentyl transferase-ipt) to manipulate maize kernel endogenous cytokinin levels in planta. Gene dose experiments also revealed that only one dose of the ipt gene was sufficient to result in the highest level of ipt expression. These data suggest that expression or over-expression of cytokinin synthesis genes in specific kernel component tissues may be a viable molecular approach to increasing endogenous cytokinin levels during kernel development and thus stabilizing grain yield of maize against the periodic occurrence of heat stress or more long-term global climate change. KS-AES (Knapp) has continued to analyze the limitations and environmental factors that influence photosynthetic productivity at the whole plant and canopy levels with particular emphasis on the study of ecosystem responses to altered precipitation and temperature patterns (climate change factors). Precipitation has been manipulated experimentally to identify, quantify, and understand water limitations to photosynthesis and productivity in the native tallgrass prairie ecosystem of Kansas. Increasing the variability of rainfall patterns altered photosynthetic rates and aboveground productivity in this grassland. The primary mechanism identified was increased water stress as soil nitrogen availability was actually increased under a more variable rainfall regime. A warming treatment was also nested within current rainfall treatments to study the interactive effects of these two predicted climate change factors on carbon uptake at the plant and ecosystem level. Impacts of climate change on the establishment of trees in this grassland ecosystem were also investigated. Woody plant encroachment into these productive ecosystems represents a major threat to their long-term sustainability. Results indicated that even though photosynthetic rates were negatively impacted in drought years by temperature and water stress, survivorship was high. This suggests that projected climate changes will not slow the expansion of forest into grassland. KS-AES (Aiken) has focused on temperature stress effects on the germination and heterotrophic growth as well as photosynthesis and fluorescence characteristics in a population of recombinant inbred lines of sorghum. Grain sorghum is known for drought tolerance, though not cold tolerance. Studies of temperature and germplasm effects on sorghum germination and heterotrophic growth demonstrated that both male and female parents contributed to cold tolerance. Mapping of the relative positions of thermotolerance traits in the sorghum genome is underway using molecular markers developed for 112 recombinant inbred lines. Candidate traits include leaf development rate, photosynthetic and fluorescence characteristics and responses to chilling, electrolyte leakage following chilling, and potential release of hydrocyanic acid. Greenhouse and field results are under analysis, in collaboration with M. Tuinstra's lab. Photosynthesis and fluorescence measurements on leaves of selected RILs indicate a negative correlation between internal leaf pCO2 and transpiration efficiency, normalized for vapor pressure deficits. Principle component analysis of six photosynthesis and fluorescence parameters indicated diverging characteristics. NV-AES (Cushman) showed that transgenic wheat plants producing mannitol exhibit improved tolerance to artificial water-deficit and salinity stress presumably through reducing the accumulation of hydroxyl radicals. Additional studies were focused on understanding the functional roles of abiotic stress-protective proteins known as hydrophilins or late embryogenesis abundant (LEA) proteins. Detailed structural characterization of group 1 and group 2 LEA proteins showed that both proteins exist in equilibrium between two conformational states: unordered and left-handed extended helical or poly-L-proline-type II (PII) structures. PII structures are solvent exposed, and like unordered structures, can interact efficiently with water. Such proteins may carry out a variety of functional roles including the stabilization of enzymes and membranes during tissue desiccation or the binding of divalent metals to reduce the production of free radical formation and resultant oxidative damage to macromolecules. Guam AES (Marler) studied the fluctuations in non-structural carbohydrates in papaya plants following manipulation of source-sink balance by leaf removal in collaboration with Cecil Stushnoff, Colorado State University. Source-sink relationships play a key role in papaya plant recovery from any environmental stress, especially one that severely impairs source size or function. Starch pools were minimal and unchanged following defoliation. Lateral roots had higher concentration than taproots or stems, but maximum levels were only 5 mg/g. Glucose and fructose concentration was similar for taproot and stem tissue, but was lower in lateral root tissue. Alternatively, sucrose concentration was similar for lateral root, taproot, and stem tissue. Other saccharides were minimal or undetectable in papaya tissues. The increased need for carbohydrates for reconstructing source leaves following foliage injury was not apparently met by mobilization of soluble carbohydrates from stem or root tissue. IL-AES (Below) employed the Illinois High Protein (IHP) and Illinois Low Protein (ILP) strains in a variety of studies investigating the relative contributions of genotype and vegetative source versus kernel sink control over maize grain composition. The interactive effects of genotype and N fertilizer on grain yield, yield components, and grain composition were investigated by evaluating hybrids. All hybrids except ILP showing increased grain protein in response to N. Protein concentration was inversely proportional to starch concentration and grain yield. The yield decreases observed for Protein Strain hybrids relative to a current elite hybrid were due primarily to reductions in kernel weight rather than kernel number. The three hybrids with low grain protein concentrations (ILP, IRHP, and elite) exhibited a consistent strong yield response to N. These results demonstrate the potential genetic variability for grain protein and starch concentrations in maize hybrids and the interactive effects of both genotype and N supply on grain yield and composition. A separate study examined the role of the cob in the movement of N assimilates into developing maize ovules to improve our understanding of how N assimilates move through the young earshoot to assist in kernel set and subsequent kernel growth. The young cob was found to have active enzymes that transform amino acids during early reproductive development, which may allow for a continual flow of these principal amino acids from the phloem for sustained reproductive growth. PA-AES (Guiltinan) continued the molecular genetic analysis of the maize starch branching enzymes (SBEs) by determining the effect of SBE gene dosage on starch branching architecture, both for single genes and for multiple dosage combinations of three different starch branching enzyme genes in order to test the hypothesis that manipulation of dosage of these three SBE isoforms will produce starches with novel branching architectures. In addition, the effects of a starch debranching enzymes (SDEs) and starch synthase on starch branching enzyme isoforms and starch branching architecture was also studied in order to test the hypothesis that varied dosage of this gene in combination with varied dosage for individual SBE genes will produce novel starch with different branch lengths and also different branch patterns. Various combinations of SBE, SDE, and starch synthase mutants were constructed. Methods for the isolation and analysis of starch from single kernels were developed to follow starch structural phenotypes in segregating populations. Using modified zymogram analysis no pleiotropic effects within single SBE mutants on the activity level of any of the SBE isoforms were observed. However, mutation in the gene encoding SBEIIb does result in a decrease in three starch synthase isoforms and a corresponding increase in a separate starch synthase activity. Analysis of the multiple mutant combinations will allow new insights into the interactions of the various starch biosynthetic enzymes.

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