Progress 04/09/06 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Define the key regulatory elements controlling photosynthate partitioning and nitrate assimilation and their interactions; develop and begin to test strategies to modify those processes for agricultural purposes. Determine the mechanistic basis for limitations on photosynthetic performance including those imposed by agriculturally significant stresses. Establish the major features controlling the response of photosynthetic productivity in soybean to elevated atmospheric CO2, tropospheric ozone, and their interactions with drought, explore the bases for genetic variability in responses, and test potential transgenic amelioration strategies. Sub-objective 3.5 to the 416/417 as �Use genomic and metabolomic data obtained at the plant community level to interpret the interaction of elevated CO2 and drought on the structure and function of soybean agrosystem.� Approach (from AD-416) Investigate isoform specificity for nitrate reductase (NR) posttranslational modification in vivo, and elucidate the impact of 14-3- 3 binding on NR protein degradation. Localize the membrane binding site(s) on sucrose synthase and identify factors that may control the interaction. Investigate the biochemical basis of the light regulation of Rubisco/activase in species that lack the redox-regulated large isoform of activase. Further elucidate the mechanism of the interaction between Rubisco and activase. Use high-resolution spatial and temporal analysis of leaf growth to identify specific areas where leaf growth is occurring. Determine the biochemical factors responsible for the lower activation state of Rubisco, at high temperatures and test potential transgenic amelioration strategies. Determine the biochemical basis for the "Green Seed Problem" of canola. Perform metabolite analysis of growing leaves under elevated CO2 and O3 to identify key components that may be involved in controlling growth. Determine the factors that lower the activation state of Rubisco under sink- and/or N-limited conditions, which are often encountered when plants are grown under high CO2. Explore the interaction of elevated O3 with drought on soybean performance. Determine if growth at elevated CO2 enhances or ameliorates oxidative stress. Over the lifetime of the project Identifying and Manipulating Determinants of Photosynthate Production and Partitioning, progress was made toward accomplishing the three objectives: 1) Define elements controlling photosynthate partitioning; 2) Determine the mechanistic basis for limitations on photosynthetic performance including those imposed by agriculturally significant stresses; 3) Establish the major features controlling the response of photosynthetic productivity in soybean to elevated atmospheric CO2, tropospheric ozone, and their interactions with drought, explore the basis for genetic variability in responses, and test potential transgenic amelioration strategies. Experiments were initiated to investigate the effect of the predicted increase in the frequency and severity of heat waves on soybean production. Three meter diameter plots of soybean were warmed by 6�C for three consecutive days during three different developmental stages. A full suite of physiological and agronomic measurements were conducted just prior to, during and after the heat wave treatments. End of season yield measurements will be taken. It is hypothesized that warming during reproductive stages of soybean development will have the largest negative effects. Experiments were initiated to investigate the interaction of season long warming and elevated CO2 (585 ppm) on the physiology and agronomy of corn. In a fully factorial and replicated experimental design, three meter diameter plots of corn were warmed by 3.5�C for the entire growing season at both ambient and elevated CO2. It is hypothesized that the warming will impact growth and productivity less at elevated CO2 than at ambient due to improved water use efficiency at elevated CO2. Accomplishments 01 The response of crops to their dynamic environment depends on intricate signaling processes that transmit information and initiate adaptive or defense responses. A form of protein modification known as tyrosine phosphorylation was shown to play an important and previously unknown ro in a form of signaling that involves receptor kinase enzymes. These plan receptor kinases transmit information about the environment to the interior of plant cells via signal transduction cascades that control processes ranging from plant development to responses to the environment Hence understanding how receptor kinases function is of fundamental importance and may provide new approaches to improve crop productivity. While it had been known for some time that plant receptor kinases are regulated by protein phosphorylation of serine and threonine residues an the role of tyrosine residue phosphorylation in this signaling is entire new. Specifically, we demonstrated that tyrosine phosphorylation of the receptor kinase, BAK1, is required for proper response to steroid hormon signaling, and unexpectedly, to the response to stress and pathogens. These results show that tyrosine phosphorylation plays an important role in plant signal transduction, and may yield new strategies to develop crops with improved stress tolerance and pathogen defense mechanisms. 02 Discovery of natural variation in ozone tolerance in soybean. Effects of the atmospheric pollutant ozone on soybean antioxidant capacity, photosynthesis and seed yield was investigated in 10 different soybean cultivars. In order to breed for ozone tolerance, the identification of variation in the response of current soybean cultivars to ozone pollutio is a critical element. Ten cultivars of soybean were grown at elevated [O3] from germination through to maturity at the Soybean Free Air Concentration Enrichment facility in 2007 and six were grown in 2008. Doubling background [O3] decreased soybean yields on avearage by 17%, bu the variation in response among cultivars and years ranged from 8 to 37% Chlorophyll content and photosynthetic parameters were positively correlated with seed yield, while antioxidant capacity was negatively correlated with photosynthesis and seed yield, suggesting a trade-off between antioxidant metabolism and carbon gain. Ozone exposure response curves indicated that there has not been a significant improvement in th tolerance of commercial soybean cultivars to [O3] in the past 30 years. Our discovery of genetic variation in ozone tolerance in non- commercial soybean cultivars is an important step toward improving ozone tolerance commercial soybeans. 03 Spring leaf flush in aspen (Populus tremuloides) is changed by long-term growth at elevated carbon dioxide and elevated ozone concentrations. How long-term growth of aspen trees at elevated [CO2] and elevated ozone concentrations [O3], which are predicted for the middle of this century, affect leafing out of aspen trees in the spring and density of leaves th formed over the growing season was investigated. This work was done at t Aspen Free Air Concentration Enrichment experiment in Rhinelander, WI where aspen tress have been grown since 1997 in conditions simulating th [CO2] and [O3] predicted for 2050. Trees grown in elevated [CO2] showed increased leaf density, while trees grown in elevated [O3] had lower lea density. Two different aspen types were investigated and found to respon differently to long-term growth at elevated [O3]. This is important as i indicates that increasing ozone will affect competition between these tw types of aspen and change the composition in forests of the future. 04 Growth at elevated ozone [O3] alters antioxidant metabolism of soybean. Antioxidants are important defense molecules for crop plants and their synthesis within crops is highly responsive to environmental conditions and is proposed to be a critical element of ozone pollution tolerance. A controlled environment study and work at the Soybean Free Air Concentration Enrichment facility investigated the response of soybean antioxidant synthesis to elevated [O3]. Both studies demonstrated that soybean antioxidant synthesis is dramatically increased by ozone polluti Genomic evidence showed that increases in antioxidant synthesis require increased energy production within the plant to fuel the energy demands increased synthesis. This work demonstrates that in antioxidant synthesi crops have a natural defense against ozone, a man-made pollutant. These results predict that increasing antioxidant synthesis in crops will be a important strategy for adapting plants to this element of global change.
Impacts
(N/A)
Publications
- Rogers, A., Ainsworth, E.A., Leakey, A.D.B. 2009. Will Elevated Carbon Dioxide Concentration Amplify the Benefits of Nitrogen Fixation in Legumes? Plant Physiology. 151:1009-1016.
- Sun, J., Yang, L., Wang, Y., Ort, D.R. 2009. FACE-ing The Global Change: Opportunities for Improvement in Photosynthetic Radiation Use Efficiency and Crop Yield. Plant Science. 177(6):511-522.
- Long, S., Ort, D.R. 2010. More Than Taking the Heat: Crops and Global Change. Current Opinion in Plant Biology. 13(3):241-248.
- Oh, M., Clouse, S.D., Huber, S.C. 2009. Tyrosine Phosphorylation in Brassinosteroid Signaling. Plant Signaling and Behavior. 4(12):77-81.
- Ainsworth, E.A., McGrath, J.M. 2009. Direct effects of rising atmospheric carbon dioxide on crop yields. In: Loebell, D. and Burke, M., editors. Climate Change and Food Security: Adapting Agriculture to a Warmer World. New York, NY: Springer. p. 109-132.
- Calfapietra, C., Ainsworth, E.A., Beier, C., De Angelis, P., Ellsworth, D. S., Godbold, D.L., Hendrey, G.R., Hickler, T., Hoosbeek, M.R., Karnosky, D. F., King, J., Korner, C., Leakey, A., Lewin, K.F., Liberloo, M., Long, S.P. , Lukac, M., Matyssek, R., Miglietta, F., Nagy, J., Norby, R.J., Oren, R., Percy, K.E., Rogers, A., Mugnozza, G.S., Stitt, M., Ceulemans, R. 2010. Challenges in Elevated CO2 Experiments on Forests. Trends in Plant Science. 15(1):5-10.
- McGrath, J.M., Karnosky, D.F., Ainsworth, E.A. 2010. Spring Leaf Flush In Aspen (Populus tremuloides) Clones Is Altered By Growth At Elevated Carbon Dioxide and Elevated Ozone. Environmental Pollution. 158(4):1023-1028.
- Rascher, U., Biskup, B., Leakey, A.D.B., McGrath, J.M., Ainsworth, E.A. 2010. Altered Physiological Function, Not Structure, Drives Increased Radiation-Use Efficiency of Soybean Grown at Elevated CO2. Photosynthesis Research. 105(1):15-25.
- Zhu, X., Long, S.P., Ort, D.R. 2010. Improving Photosynthetic Efficiency for Greater Yield. Annual Reviews of Plant Biology. 61:235-261.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Define the key regulatory elements controlling photosynthate partitioning and nitrate assimilation and their interactions; develop and begin to test strategies to modify those processes for agricultural purposes. Determine the mechanistic basis for limitations on photosynthetic performance including those imposed by agriculturally significant stresses. Establish the major features controlling the response of photosynthetic productivity in soybean to elevated atmospheric CO2, tropospheric ozone, and their interactions with drought, explore the bases for genetic variability in responses, and test potential transgenic amelioration strategies. Sub-objective 3.5 to the 416/417 as �Use genomic and metabolomic data obtained at the plant community level to interpret the interaction of elevated CO2 and drought on the structure and function of soybean agrosystem.� Approach (from AD-416) Investigate isoform specificity for nitrate reductase (NR) posttranslational modification in vivo, and elucidate the impact of 14-3- 3 binding on NR protein degradation. Localize the membrane binding site(s) on sucrose synthase and identify factors that may control the interaction. Investigate the biochemical basis of the light regulation of Rubisco/activase in species that lack the redox-regulated large isoform of activase. Further elucidate the mechanism of the interaction between Rubisco and activase. Use high-resolution spatial and temporal analysis of leaf growth to identify specific areas where leaf growth is occurring. Determine the biochemical factors responsible for the lower activation state of Rubisco, at high temperatures and test potential transgenic amelioration strategies. Determine the biochemical basis for the "Green Seed Problem" of canola. Perform metabolite analysis of growing leaves under elevated CO2 and O3 to identify key components that may be involved in controlling growth. Determine the factors that lower the activation state of Rubisco under sink- and/or N-limited conditions, which are often encountered when plants are grown under high CO2. Explore the interaction of elevated O3 with drought on soybean performance. Determine if growth at elevated CO2 enhances or ameliorates oxidative stress. Significant Activities that Support Special Target Populations Progress was made in meeting or substantially meeting milestones that contribute to accomplishing the three objectives of the project Indentifying and Manipulating Determinants of Photosynthetic Production and Partitioning. The three objectives are: 1) Define the elements controlling photosynthate partitioning for which research this year revealed important information about a newly discovered control mechanism for novel tyrosine kinases that function in signal transduction; 2) Determine the mechanistic basis for limitations on photosynthetic performance including those imposed by agriculturally significant stresses for which research this year in conjunction with a CRADA produced gene expression arrays for 7 cultivars of soybean exposed to elevated concentrations of ozone under field conditions; 3) Establish the major features controlling the response of productivity in soybean to atmospheric change for which this year the response of antioxidant metabolism to elevated CO2 was investigated showing that this treatment surprisingly reduces total antioxidant capacity. Technology Transfer Number of Active CRADAS: 1 Number of Other Technology Transfer: 2
Impacts
(N/A)
Publications
- Li, P., Ainsworth, E.A., Leakey, A.D.B., Ulanov, A., Lozovaya, V., Ort, D. R., Bohnert, H.J. 2008. Arabidopsis Transcript and Metabolite Profiles: Ecotype-specific Acclimation to Open-air Elevated [CO2]. Plant Cell and Environment. 31(11):1673-1687.
- Feng, Z., Kobayashi, K., Ainsworth, E.A. 2008. Impact of elevated ozone on growth, physiology and yield of wheat (Triticum aestivum L.): A meta- analysis. Global Change Biology. 14(11)2696-2708.
- Leakey, A.D.B., Ainsworth, E.A., Bernacchi, C.J., Rogers, A., Long, S.P., Ort, D.R. 2009. Elevated CO2 Effects on Plant Carbon, Nitrogen and Water Relations: Six Important Lessons From FACE. Journal of Experimental Botany. 60(10):2859-2876.
- Leakey, Andrew D.B., Xu, Fangxiu., Gillespie, Kelly M., McGrath, Justin M., Ainsworth, Elizabeth A., Ort, Donald R. 2009. Genomic Basis for Stimulated Respiration by Plants Growing Under Elevated Carbon Dioxide. Proceedings of the National Academy of Sciences USA. 106:3597-3602.
- Leakey, A.D.B., Ainsworth, E.A., Bernard, S.M., Markelz, R., Ort, D.R., Placella, S.A., Rogers, A., Smith, M.D., Sudderth, E.A., Weston, D.J., Wullschleger, S.D., Yuan, S. 2009. Gene expression profiling � opening the black box of plant ecosystem responses to global change. Global Change Biology. 15(5):1201-1213.
- Wittig, V.E., Ainsworth, E.A., Naidu, S.L., Karnosky, D.F., Long, S.L. 2009. Quantifying the impact of current and future tropospheric ozone on tree biomass, growth, physiology and biochemistry: A quantitative meta- analysis. Global Change Biology. 15(2):396-424.
- Clouse, S.D., Goshe, M.B., Huber, S.C., Li, J. 2008. Functional Analysis and Phosphorylation Site Mapping of Leucine-Rich Repeat Receptor-Like Kinases. In: Agrawal, G.K., Rakwal, R., editors. Plant Proteomics: Technologies, Strategies, and Applicatioins. Hoboken, NJ: John Wiley & Sons, Inc. p. 469-484.
- Ainsworth, E.A., Beier, C., Calfapietra, C., Cuelemans, R., Durand-Tardif, M., Farquhar, G.D., Godbold, D.L., Hendrey, G.R., Hickler, T., Kaduk, J., Karnosky, D.F., Kimball, B.A., Koerner, C., Koornneef, M., Lafarge, T., Leakey, A. D. B., Lewin, K.F., Long, S.P., Manderscheid, R., McNeil, D.L., Meis, T.A., Miglietta, F., Morgan, J.A., Nagy, J., Norby, R.J., Norton, R. M., Percy, K.E., Rogers, A., Soussana, J., Stitt, M., Weigel, H., White, J. W. 2008. Next generation of elevated [CO2] experiments with crops: A critical investment for feeding the future world. Plant Cell and Environment. 31:1317-1324.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Define the key regulatory elements controlling photosynthate partitioning and nitrate assimilation and their interactions; develop and begin to test strategies to modify those processes for agricultural purposes. Determine the mechanistic basis for limitations on photosynthetic performance including those imposed by agriculturally significant stresses. Establish the major features controlling the response of photosynthetic productivity in soybean to elevated atmospheric CO2, tropospheric ozone, and their interactions with drought, explore the bases for genetic variability in responses, and test potential transgenic amelioration strategies. Sub-objective 3.5 to the 416/417 as �Use genomic and metabolomic data obtained at the plant community level to interpret the interaction of elevated CO2 and drought on the structure and function of soybean agrosystem.� Approach (from AD-416) Investigate isoform specificity for nitrate reductase (NR) posttranslational modification in vivo, and elucidate the impact of 14-3- 3 binding on NR protein degradation. Localize the membrane binding site(s) on sucrose synthase and identify factors that may control the interaction. Investigate the biochemical basis of the light regulation of Rubisco/activase in species that lack the redox-regulated large isoform of activase. Further elucidate the mechanism of the interaction between Rubisco and activase. Use high-resolution spatial and temporal analysis of leaf growth to identify specific areas where leaf growth is occurring. Determine the biochemical factors responsible for the lower activation state of Rubisco, at high temperatures and test potential transgenic amelioration strategies. Determine the biochemical basis for the "Green Seed Problem" of canola. Perform metabolite analysis of growing leaves under elevated CO2 and O3 to identify key components that may be involved in controlling growth. Determine the factors that lower the activation state of Rubisco under sink- and/or N-limited conditions, which are often encountered when plants are grown under high CO2. Explore the interaction of elevated O3 with drought on soybean performance. Determine if growth at elevated CO2 enhances or ameliorates oxidative stress. Accomplishments Improving the heat tolerance of plants. The rate of photosynthesis declines at moderately high temperatures in temperate plants like Arabidopsis and the decline is due to deactivation of Rubisco which in turn is due to a reduced ability of activase to activate Rubisco. We created a more thermostable activase for Arabidopsis by replacing the Rubisco recognition domain in the more thermostable tobacco activase with that from Arabidopsis. We then transformed the Arabidopsis rca mutant, which does not express activase and selected lines with high expression of this chimeric activase. The initial studies indicate that the rate of photosynthesis as measured by gas exchange is higher than the wild type in the transgenic lines at moderately high temperatures and the recovery of photosynthesis is also better in the transgenic lines when the plants are returned to lower temperature. The studies provide support for a �proof of concept� that Rubisco activase is a potential target to improve the temperature tolerance of crop plants. Growth at elevated carbon dioxide enhances the expression of carbohydrate metabolism genes in soybean. Season long photosynthesis under field conditions is stimulated due to elevated carbon dioxide is stimulated more in soybean than most crops presumably due to its nitrogen fixation capacity. How do soybean leaves deal with the increased amount of photosynthetically fixed carbon? Genomic investigations of soybean leaves at different times during crop development revealed that genes coding for many enzymes in carbohydrate metabolism were substantially up- regulated. This work supports the hypothesis that under conditions when reduced nitrogen is non-limiting, leaf metabolism can respond to increased carbon flux by synthesizing more the biological machinery necessary to process carbohydrates. 14-3-3 proteins are essential, positive regulators of brassinosteroid signaling. Brassinosteroids (BRs) are plant hormones that control growth and stress tolerance, but it is not clear how this pathway could be manipulated to increase crop productivity. A first step is to identify the protein components that are essential for BR signaling. We determined that 14-3-3 proteins, which are highly conserved eukaryotic phosphoserine- specific binding proteins, interact with the BR receptor (known as BRI1), both in vivo and in vitro. A knockout mutant lacking five of the twelve Arabidopsis 14-3-3 isoforms was characterized and shown to be specifically impaired in several BR-dependent growth responses, including a novel root growth modification. These results indicate for the first time that 14-3-3s are essential, positive regulators of BR signaling. The results identify a new component of an important plant hormone signaling pathway, and secondly, identify a possible new approach to control crop growth or stress tolerance. Developed high-throughput assays of antioxidant capacity in plant tissues. There is growing interest in antioxidant metabolism in plants, and increasing demand for high-throughput assays of antioxidant metabolism. We adapted and published three general assays of antioxidant status: an oxygen radical absorbance capacity assay, an assay of total phenolic content using Folin�Ciocalteu reagent, and a colorimetric ascorbate assay. All assays were adapted to a microplate reader for high throughput. The assays are rapid and simple and allow a general diagnostic of the antioxidant status of plant tissues. Since publishing these assays early in 2007, I have received over 30 requests for the methods from other scientists. This work is critical to our third objective and specific sub-objective 3.1 Perform metabolite analysis of actively growing leaves under elevated CO2 and O3 to identify key components that may be involved in controlling growth. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 43 Number of Newspaper Articles,Presentations for NonScience Audiences: 7
Impacts
(N/A)
Publications
- Bernacchi, C.J., Leakey, A.B., Heady, L.E., Morgan, P., Dohleman, F.G., Mcgrath, J.M., Gillespie, K.M., Wittig, V.E., Rogers, A., Long, S.P., Ort, D.R. 2006. Hourly and seasonal variation in photosynthesis and stomatal conductance of soybean grown at future CO2 and ozone concentrations for three years under fully open air conditions. Plant Cell and Environment. 29:2077-2090.
- Grennan, A.K., Ort, D.R. 2007. Cool temperatures interfere with D1 synthesis in tomato by causing ribosomal pausing. Photosynthesis Research. Available at http://www.springerlink.com/content/100325/? Content+Status=Accepted&sort=p_OnlineDate&sortorder=desc&v=expanded&o=30.
- Wittig, V.E., Ainsworth, E.A., Long, S.P. 2007. To what extent do current and projected increases in surface ozone affect photosynthesis and stomatal conductance of trees? A meta-analytic review of the last three decades of experiments. Plant Cell and Environment. 30(9):1150-1162.
- Ainsworth, E.A., Rogers, A. 2007. The Response of Photosynthesis and Stomatal Conductance to Rising [CO2]: Molecular Mechanisms and Environmental Interactions. Plant Cell and Environment. 30(3): 258�270.
- Ainsworth, E.A., Rogers, A., Leakey, A.B., Heady, L.E., Gibon, Y., Stitt, M., Schurr, U. 2007. Does elevated atmospheric [CO2] alter diurnal C uptake and the balance of C and N metabolites in growing and fully expanded soybean leaves? Journal of Experimental Botany. 58(3):679-591.
- Ainsworth, E.A., Gillespie, K.M. 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin- Ciocalteu reagent. Nature Protocols. 2:875-877.
- Gillespie, K.M., Ainsworth, E.A. 2007. Measurement of reduced, oxidized and total ascorbate content in plants. Nature Protocols. 2:871-874.
- Gillespie, K.M., Chae, J.M., Ainsworth, E.A. 2007. Rapid Measurement of Total Antioxidant Capacity in Plants. Nature Protocols. 2:867-870.
- Long, S.P., Ainsworth, E.A., Leakey, A.B., Nosberger, J., Ort, D.R. 2006. Food for thought: Open-air field experiments suggest lower than expected crop yield stimulation with rising [CO2]. Science. 312:1918-1921.
- Chung, D.W., Pruzinska, A., Hortensteiner, S., Ort, D.R. 2006. The role of pheophorbide a oxygenase expression and activity in the canola green seed problem. Plant Physiology. 142:88-97.
- Ainsworth, E.A., Rogers, A., Vodkin, L.O., Walter, A., Schurr, U. 2006. The effects of elevated [CO2] on soybean gene expression. An analysis of growing and mature leaves. Plant Physiology. 142:135-147.
- Chalivendra, S.C., Huber, S.C., Sachs, M.M., Rhoads, D.M. 2007. Sucrose Synthase: Expanding Protein Function. Plant Signaling and Behavior. 2(1) :28-29.
- Bernacchi, C.J., Kimball, B.A., Quarles, D.R., Long, S.P., Ort, D.R. 2007. Decreases in Stomatal Conductance of Soybean under Open-Air Elevation of [CO2] Are Closely Coupled with Decreases in Ecosystem Evapotranspiration. Plant Physiology. 143:134-144.
- Wang, D., Portis Jr, A.R. 2007. A novel nucleus-encoded chloroplast protein, PIFI, is involved in NAD(P)H dehydrogenase complex mediated chlororespiratory and possibly cyclic electron transport in Arabidopsis. Plant Physiology. 144:1742-1752.
- Qiu, Q., Hardin, S., Mace, J., Brutnell, T.P., Huber, S.C. 2007. Metabolic signals control the selective degradation of sucrose synthase in maize leaves during de-etiolation. Plant Physiology. 144(1):468-478.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is aligned with National Program 302, Plant Biological and Molecular Processes and National Program 204, Global Change. The goal of this research is to identify molecular, biochemical and genetic determinants of photosynthate production and distribution in crop plants and to utilize this new information to address specific agricultural problems of national importance including those associated with atmospheric change. Research is focused in three major areas: 1) Define the key regulatory elements controlling photosynthate production and partitioning and determining sink strength. A major goal is to identify processes that control assimilate partitioning and sink strength with a particular emphasis on the expression and deployment of carbohydrate and amino acid transporters. A
second goal is determining steps and mechanisms that limit photosynthate production with specific focus on Rubisco activase, photoregulation/protection of PSII, and redox regulation of chloroplast enzymes. 2) Determine the mechanistic basis for limitations on photosynthetic performance/productivity imposed by agriculturally significant stresses. The major focus is on the underlying mechanism for deleterious effects of temperature extremes on growth, production and market value of crops. Research concentrates on three nationally important problems: the chilling sensitivity of photosynthesis in warm climate crops, the interference of Rubisco activation by high leaf temperature, and the disruption by early frost of chlorophyll clearing from oil seeds. 3) Establish the major features limiting the response of photosynthetic productivity of soybean at elevated atmospheric CO2 and test potential transgenic amelioration strategies. Funding for establishing a soybean FACE project in Urbana,
IL has recently been secured. Our research will feature gas exchange and chlorophyll fluorescence analyses of the metabolic basis for acclimation of soybean photosynthesis to elevated CO2 in the FACE facility as well as development of transgenic model plants with CO2 adapted Rubisco and over- expressed and retargeted sucrose and amino acid transporters. A major challenge facing agricultural scientists is to protect the high productivity of U.S. crops against changing climatic conditions while improving nutritional value and developing crops that are less dependent on chemical applications. Since photosynthesis underlies each of these goals, our work contributes directly to this effort by identifying gene products that contribute to more productive, stress tolerant, resource efficient, and nutritious crops. For example, one main thrust of our research is to identify genes that regulate resource allocation within crop plants and then develop strategies to improve the nutritional
value of harvested tissue by modifying the activity of those genes. Another research thrust is to identify the physiological and molecular basis for the chilling sensitivity of photosynthesis in commercially significant crops in temperature North America (e.g., corn, soybean, cotton, and others). An improvement of even one-degree Celsius in the low temperature tolerance would both expand the geographical range where these crops are grown as well as improve the economic success of these crops grown at the northern border of their cultivation. We are in the business of developing strategies to change partitioning patterns, improve the ability of crops to be productive under stress, improve the ability of crops to take advantage of increasing atmospheric CO2 levels and be more tolerant to increasing ozone levels, and enhance the nutritional and market value of U.S. crops. 2. List by year the currently approved milestones (indicators of research progress) Define the key regulatory
elements controlling photosynthate partitioning and nitrate assimilation and their interactions; develop and begin to test strategies to modify those processes for agricultural purposes. FY07 A. Produce first 2-DE map of plant 14-3-3 proteins B. Determine membrane binding of the SUS isoforms C. Determine biochemical basis for light regulation of activase on tobacco by establishing expression levels D. Elucidate the mechanism of interaction between activase and Rubisco by validating His-tag assay procedure for binding and verifying differences between native and recombinant activase FY08 A. Develop antibodies to nitrate reductase and identify 14-3-3 proteins B. Identify membrane binding sites on SUS1 C. Determine activity of recombinant tobacco activase D. Screen 10 mutants for activase crystal formation E. Clone and compare actvities of tobacco activase isoforms FY09 A. Determine if mutant 14-3-3s with increased affinity to Arabidopsis NR can be produced B. Explore site-directed
mutagenesis to alter SUS1 membrane binding C. Determine phenotypes of activase knock-out lines D. Characterize factors influencing activase/Rubisco binding E. Finish characterization of native activase FY10 A. Attempt to produce transgenic plants over expressing mutant 14-3-3s B. Identify factors affecting SUS membrane binding. C. Validate or reject activase/Rubisco binding hypothesis D. Extend crystallization attempts to the activase/rubisco complex FY11 A. Determine impact of 14-3-3 binding on NR protein stability B. Produce transgenic Arabidopsis plants to test in vivo function of m- SUS. C. Obtain structure of activase or activase/Rubisco complexes Determine the mechanistic basis for limitations on photosynthetic performance including those imposed by agriculturally significant stresses. FY07 A. Use high-resolution spatial and temporal analysis of leaf growth to identify specific areas by completing the microarray analysis of samples taken from ambient and elevated CO2 during the
2004 field season B. Determine factors causing lower activation state of Rubisco and test amelioration strategies by verifying separation protocol for inhibitor formation C. Determine the biochemical basis for the "Green Seed Problem" of canola by completing protein kinase/phosphatase inhibitor work FY08 A. Determine feasibility of using spatial and temporal in the field B. Identify and quantitate Rubisco inhibitors C. Verify "coverage" of PaO phosphorylation sites in cleavage fragments FY09 A. Identify environmental factors that alter leaf growth dynamics, including day length, temperature and vapor pressure deficit B. Compare effects of short duration/high temperature regimes on photosynthesis and growth C. MALDI-ToF identifications of PaO phosphopeptides FY10 A. Perform cytological analysis of young leaves and identify how temporal and spatial patterns of growth are altered under elevated [O3] in the field B. Compare effects of long duration/moderate temperatures regimes C. Produce
site directed PaO mutants of phosphorylation sites FY11 A. Complete microarray analysis of growing soybean leaves exposed to elevated [CO2] and elevated [O3] in the field. B. Determine metabolic fate of inhibitors formed C. Produce PaO transgenics and determine response in freezing seed and senescing leaf Establish the major features controlling the response of photosynthetic productivity in soybean to elevated atmospheric CO2, tropospheric ozone, and their interactions with drought, explore the basis for genetic variability in responses, and test potential transgenic amelioration strategies. FY07 A. Perform metabolite analysis of actively growing leaves under elevated CO2 and O3 to identify key components that may be involved in controlling growth by developing techniques to measure soluble and insoluble pools of carbohydrates. B. Complete analysis of diel carbohydrate metabolism of soybeans grown under controlled conditions C. Determine factors that lower the activation state of
Rubisco under sink- and/or N-limited conditions by identifying protocols that can induce a TPU limitation in Arabidopsis D. Explore the interaction of elevated O3 and drought on soybean performance by completing 1st season of physiological and evapotranspiration measurements and leaf sampling for transcripts and metabolites E. Explore elevated CO2 and protein oxidation by determining whether short-term exposure to high CO2 increases protein carbonylation in soybean FY08 A. Optimize microplate assays for hexose phosphates, 3-phosphoglycerate, inorganic phosphate, ATP, ADP, UDP and UDP-glucose B. Complete gas-exchange experiments comparing WT and transgenic lines C. Complete 1st season microarray and metabolite profiling on O3/drought D. Investigate metabolic adjustment after long-term exposure to high CO2 FY09 A. Complete diel photosynthesis and carbohydrate analysis of growing leaves exposed to elevated CO2 and O3 at SoyFACE B. Measure photosynthesis, protein, activation state and
RuBP levels after transitions to growth at high CO2 C. Complete 1st season microarray and metabolite profiling on O3/drought D. Identify proteins most affected by CO2 in terms of abundance and/or carbonylation FY10 A. Measure respiration and respiratory metabolites of growing leaves at SoyFACE B. Formulate new activation hypothesis if necessary C. Complete 2nd season microarray and metabolite profiling D. Determine whether metabolic adjustment involves increased antioxidants FY11 A. Determine feasibility of combining non-invasive growth measurements with respiratory flux. B. Identify molecular responses that may predict or explain cultivar differences in yield at high CO2. 4a List the single most significant research accomplishment during FY 2006. This is a new project. No major accomplishments to report at this time. 5. Describe the major accomplishments to date and their predicted or actual impact. This is a new project. No major accomplishments to report at this time.
Impacts
(N/A)
Publications
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