Progress 04/09/01 to 04/08/06
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, Crop Protection and Quarantine. This is the final report. 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) Determination of the biochemical, molecular and genetic factors controlling tolerance and susceptibility of crop photosynthetic performance to drought, temperature extremes, and ozone; 2) Defining the key regulatory elements that control photosynthate production and
partitioning and that determine sink strength; 3) Establishing the major features limiting the response of photosynthetic productivity of soybean and corn at elevated atmospheric CO2 and test potential transgenic amelioration strategies. The experimental approaches are diverse combining biophysics, biochemistry, physiology, molecular biology and genomics. 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) Objective 1: Define the key
regulatory elements controlling photosynthate production and partitioning and determining sink strength. FY01 A. Generate transgenic sugar beet and tobacco with ectopic hyperactive sucrose symporter. B. Define role of transcription and protein turnover in sucrose symporter activity. C. Screen for sucrose-signaling/partitioning mutants. D. Clone gene-trapped tagged phloem genes. E. Complete map based cloning of cfs ATPsynthase mutant. FY02 A. Characterize sucrose signaling and partitioning mutants. B. Screen mutagenized promoter & reporter gene transgenic plants for mutants in nitrate-response C. Determine role of phosphorylation in regulating sucrose transporter activity. D. Determine function of cloned phloem genes. E. Create and complement random mutants in b559. F. Determine protein expression level of site-directed -subunit constructs. FY03 A. Clone mutated genes from sucrose signaling and partitioning mutants. B. Characterize growth of transgenic plants with ectopic hyperactive
sucrose symporter. C. Characterize non-responsive nitrate-signaling mutants. D. Determine genetic lesions random mutants in b559 E. Create b559 heme-binding mutants. F. Develop in vivo assay for chloroplast redox poise of thioredoxin- regulated enzymes and proteins. FY04 A. Clone mutated genes from sucrose signaling and partitioning mutants. B. Characterize growth of transgenic plants with ectopic hyperactive sucrose symporter. C. Characterize non-responsive nitrate-signaling mutants. D. Determine genetic lesions random mutants in b559 E. Create b559 heme-binding mutants. F. Develop in vivo assay for chloroplast redox poise of thioredoxin- regulated enzymes and proteins FY05 A. Determine whether the content of amino acids in developing soybean seeds is correlated with seed protein content at maturity in indeterminate cultivars. B. Identify the membrane-binding domain on sucrose synthase. Objective 2: Determine the mechanistic basis for limitations on photosynthetic
performance/productivity imposed by agriculturally significant stresses. FY01 A. Cross Arabidopsis lines with defects in lipid metabolism with the rca- minus line; select plants retaining both mutations. B. Measure PaO activity in control and frozen seed. C. Determine if there is a circadian rhythm in NR phosphorylation FY02 A. Complete analysis of the temperature dependence of photosynthetic properties for Arabidopsis with wild type, antisense and null levels of rca. B. Analysis microarrays for candidate PaO genes. C. Determine if chill induced ribosomal pausing is site-specific. D. Measure in vivo lifetime of NR in tomato FY03 A. Complete analysis of the temperature dependence of photosynthesis in the Arabidopsis lines with defects in lipid metabolism. B. Identify & clone PaO gene; select stay-green mutants. C. Determine if chloroplast redox change drives polysome pausing FY04 A. Complete analysis of the temperature dependence of photosynthesis in the Arabidopsis lines with defects
in lipid metabolism and rca content. B. Complete investigation of possible chlorophyll degradation operon in Synechocystis. FY05 A. Identify role of pheophorbide oxygenase in green seed problem Objective 3: Establish the major features limiting the response of photosynthetic productivity of soybean at elevated atmospheric CO2 and test potential transgenic amelioration strategies. FY01 A. Perform baseline growth studies at 350 and 750 ppm CO2 on Arabidopsis plants with wild type and modified activase proteins. B. Measure effect of elevated CO2 on TPU limitation in soybean in FACE experiment. FY02 A. Complete feasibility analysis of relocating the small subunit gene to the chloroplast with the small subunit antisense tobacco line. B. Determine the effects of the of light intensity regulation on starch/ sucrose partitioning in the Arabidopsis plants with modified rca proteins at 350 and 750 ppm CO2. C. Clone sucrose-symporter from soybean. D. Determine effect of elevated CO2 on
temperature dependence of TPU limitation in soybean in FACE. E. Determine effect of elevated CO2 on leaf temperature and transpiration in soybean in FACE. F. Over-expression of the hyperactive symporter in sink tissues of sugar beet FY03 A. Measure sucrose symporter message abundance and transport activity in soybean grown at elevated CO2. B. Determine if elevated CO2 enhances or protects high temperature photoinhibition in soybean in FACE. C. Evaluate effect of over-expression of the hyperactive sucrose symporter in sink tissues on assimilate partitioning at elevated CO2 in growth chamber. FY04 A. Complete analysis of mRNA and protein levels in multiply transformed tobacco lines. If needed, eliminate native small subunit expression with additional tobacco antisense constructs. B. Evaluate effect of over-expression of the hyperactive sucrose symporter in sink tissues on assimilate partitioning at elevated CO2 in FACE facility. FY05 A. Analyze if RNAi can be used to specifically
limit native RbcS gene expression while allowing the expression of a foreign RbcS gene. B. Analyze genetic variation in photoprotection in soybean under FACE treatments. C. Analyze interaction between drought and elevated ozone and CO2 on stomatal conductance and evapotranspiration in soybean. D. Determine whether soybean exposure to high O3 or CO2 leads to increased protein oxidation, and if so, begin to identify specific protein targets. 4a List the single most significant research accomplishment during FY 2006. Lower than expected crop yield stimulation with rising CO2 concentrations. This accomplishment is aligned with NP204 Global Change, Component III Agricultural Ecosystem Impacts (2. Cropping Systems: Measure and predict plant responses (above and below-ground) to multiple interactions of abiotic and biotic stresses with rising carbon dioxide). Model projections suggest that while increased temperature and decreased soil moisture will act to reduce global crop yields in 2050,
the direct fertilization effect of rising carbon dioxide concentration ([CO2]) will more than offset these losses. The CO2-fertilization factors used in models to project future yields were derived from enclosure studies conducted approximately 20 years ago. Free-Air Concentration Enrichment (FACE) technology has now facilitated large scale trials of the major grain crops at elevated [CO2] under fully open air field conditions. In FACE trials, elevated [CO2] enhanced yield approximately 50% less than in enclosure studies, casting doubt on projections that rising [CO2] will offset losses due to climate change. 4b List other significant research accomplishment(s), if any. A. Growth at elevated CO2 results in protein oxidation. This accomplishment is aligned with NP204 Global Change, Component III Agricultural Ecosystem Impacts (2. Cropping Systems: Measure and predict plant responses (above and below-ground) to multiple interactions of abiotic and biotic stresses with rising carbon
dioxide). Plants are continually producing reactive oxygen species (ROS) that can irreversibly modify cellular components including proteins, resulting in DNPH-reactive carbonyl formation (hereafter referred to as protein oxidation). In both growth chamber grown Arabidopsis and field-grown soybean leaves, numerous proteins were found to contain DNPH-reactive carbonyls, including Rubisco (LSU and SSU), Rubisco activase, and a 25-kDa protein. In leaves of Arabidopsis plants grown at ambient CO2, protein oxidation levels remained relatively constant during vegetative and reproductive growth but increased late in reproductive development. Growth at elevated CO2 (1000 ppm) resulted in a dramatic increase in protein oxidation early in vegetative growth that was slowly reversed as plants developed until the overall level of oxidation was dramatically lower compared to plants grown at ambient CO2. Soybean leaves, grown in the field at SoyFACE, also had an overall lower level of protein
oxidation when grown at elevated CO2. A new technique was developed to rapidly separate fraction I (Rubisco) from fraction II (non-Rubisco) proteins for 2-DE analysis that greatly enhances the ability to resolve and characterize leaf proteins, including those modified by oxidative stress. We are currently using MALDI-TOF mass spectrometry to identify proteins differing in abundance and/or level of oxidation from leaves grown at elevated CO2 compared to ambient CO2. Our working model is that growth at elevated CO2 results in an initial oxidation of proteins, and that with time plants acclimate, perhaps by increasing antioxidant capacity. This may explain why plants at elevated CO2 are more resistant to O3 injury. B. Meta-analytic analysis of 15 years of FACE outcomes. This accomplishment is aligned with NP204 Global Change, Component III Agricultural Ecosystem Impacts (2. Cropping Systems: Develop physiological criteria for improvement of crop quality (including leaf/forage/residue
quality) of plants grown under elevated carbon dioxide). Data from 120 primary, peer-reviewed articles describing physiology and production in the 12 large-scale FACE experiments (with elevated [CO2] treatments between 475 and 600 ppm) were collected and summarized using meta-analytic techniques. The results confirmed some results from chamber experiments: light-saturated carbon uptake, diurnal C assimilation, growth and above-ground production increased, while specific leaf area and stomatal conductance decreased in elevated [CO2]. There were differences in FACE as well, namely, trees were more responsive to elevated [CO2] than herbaceous species and grain crop yields increased less than anticipated. The reduction in plant nitrogen was small and largely accounted for by decreased Rubisco. These results provide a best estimate of plant responses to elevated [CO2]. C. Defining a role of sucrose synthase in sugar sensing. This accomplishment is aligned with NP302 Plant Biological and
Molecular Processes, Component II Biological Processes that Improve Crop Productivity and Quality (Problem Statement 2A: Understanding growth and development). Studies have focused on the enzyme sucrose synthase, which is recognized to play an important role in the metabolism of sucrose in seeds and tubers. Zea mays has three known sucrose synthase isoforms: SUS1, SH1, and SUS3. The SUS3 isoform was only recently characterized at the transcript level. Recent evidence using a peptide antibody that specifically recognizes the SUS3 isoform suggests that it is a ubiquitous protein. Abundant SUS3 protein is found in developing kernels, leaf midvein, internode cortex tissue, and etiolated roots, and thus, it will be important to characterize its properties and such experiments are underway with recombinant SUS3 protein. SUS is a soluble protein but is also partially associated with plant membranes, but the mechanisms involved in this interaction are unknown. We are investigating the
structural requirements for interaction of maize SUS1 with membranes using site-directed mutants of full length SUS1 and a series of truncation mutants, fused at the N-terminus to maltose binding protein (MBP). The truncations indicated that the N-terminus of SUS1 (residues 1 to 360) contained a membrane-binding domain. In addition, site-directed alanine substitution mutants indicated that residues within the catalytic domain may also play a role. These residues are located within a 100 amino acid region that has sequence similarity to the C-terminal pleckstrin homology domain in human pleckstrin, which is known to interact with membranes. Finally, removal of the C-terminal non- catalytic domain (74 amino acids) of SUS1 resulted in increased membrane binding relative to the full-length construct, implying a negative regulatory role for this region. We have also examined effectors of SUS binding to membranes. Binding of full-length SUS1 to microsomes showed positive cooperativity
with increasing protein concentration and was stimulated by sugars, including those that are not recognized at the catalytic site, and acidic pH. These observations suggest that SUS may play a role in 'sugar-sensing' that allows for diversion of sucrose towards cellulose synthesis only when excess photosynthate is available. Our goal is to identify SUS1 mutants that affect membrane binding (either promote or disrupt) but not enzymatic activity, in order to potentially influence the channeling of carbon to cell wall glucans. D. Role of activase look alike protein This acocmplishment is aligned with NP302 Plant Biological and Molecular Processes, Component II Biological Processes that Improve Crop Productivity and Quality (Problem Statement 2B: Understanding plant interactions with their environment). Arabidopsis plants (T-DNA insertions) that do not express a chloroplast located, but nuclear encoded protein called "MSJ". The C-terminus of this protein has high similarity to the
C-terminus of the large isoform of activase and it could be redox regulated. The function of this protein is currently unknown. The plants grew similarly to the wild type under numerous control and stress regimes and photosynthesis is about the same as the wild type. However the mutants have significantly lower maximum photochemical quantum efficiency of PSII and lower non-photochemical quenching at higher light intensities. The most distinguishing feature of the mutants discovered thus far is that the kinetics and magnitude of a transient increase in Fo fluorescence after light-"dark" transitions differ quite markedly from the wild type. 5. Describe the major accomplishments to date and their predicted or actual impact. A. This accomplishment is aligned with NP204 Global Change, Component III Agricultural Ecosystem Impacts NP204 Global Change, Component III Agricultural Ecosystem Impacts (2. Cropping Systems: Measure and predict plant responses (above and below-ground) to multiple
interactions of abiotic and biotic stresses with rising carbon dioxide). Carbon dioxide in the atmosphere is rising globally by 0.4% per year. The rate of increase that is expected to accelerate with 2050 concentrations projected be 50% higher than today. In many areas, tropospheric ozone is increasing even more rapidly than CO2. Adapting the crop to these changes will require accurate information on exactly how increasing CO2 and ozone will affect crops in the field. A new facility at Urbana, IL allows the controlled enrichment of large plots within a field crop without any enclosure. The technology consists of rings, about 20 m in diameter, of pipes that inject CO2 and/or ozone into the air at the height of the crop and according to wind-speed and direction, maintaining the concentration within the ring at a pre-set elevated concentration through computer feed- back control. Important trends emerging from the initial 5 years of our experiments with soybean show that CO2
fertilization caused an average: 25% increase in photosynthesis 15% increase in yield 18% increase in above ground biomass 22% decrease in mid-day stomatal conductance 12% decrease in season long water use 17% decrease in harvest index increase in canopy temperature Three years of data on the effects of ozone on soybean reveal both susceptibility in current cultivars and untapped tolerance in soybean germplasm. Experiments with soybean show that elevating ozone to levels predicted for the US Corn Belt in 2050 caused on average: 11% decrease in above ground biomass 14% decrease in seed yield 16% decrease in season long water use increase in canopy temperature The corn soybean rotation is the largest ecosystem type in North America. In consequence, these effects of elevated CO2 under agronomically realistic conditions are of significant importance in understanding mechanism, in designing cultivars better suited to future atmospheres, to predicting impact of atmospheric change on crop
production, and in providing quantitative information on the how photosynthesis will directly feedback on climate change. B. This accomplishment is aligned with NP302 Plant Biological and Molecular Processes, Component II Biological Processes that Improve Crop Productivity and Quality (Problem Statement 2B: Understanding plant interactions with their environment). The activity of Rubisco, the enzyme responsible for capturing atmospheric carbon dioxide during photosynthesis, is a major factor limiting photosynthesis in crop plants. The activity of Rubisco is down-regulated under limiting light conditions, thereby further limiting the rate of photosynthesis. We created transgenic plants in which Rubisco remains fully active over a wide range of light intensities by using genetic transformation to express modified forms of the regulatory protein, Rubisco activase. These unique plants are being used to study the basis for high temperature inhibition of photosynthesis and the limitations
of photosynthesis in dynamic light environments. Thus these plants are providing a powerful means to delineate the significance of light regulation of Rubisco in defining plant productivity under different environmental conditions. C. This accomplishment is aligned with NP302 Plant Biological and Molecular Processes, Component II Biological Processes that Improve Crop Productivity and Quality (Problem Statement 2B: Understanding plant interactions with their environment). The major harvested tissues in plants are composed of cells that must import sugars and amino acids produced in photosynthesis to support growth and development. Although we understand the basic mechanics of how sugars are moved in the plant's vascular system, the regulation of sugar allocation is not well understood because of the complicated interplay between leaves and harvested organs. Using biochemical and recombinant DNA methods, we discovered a sucrose-sensing pathway that regulates the activity and
expression level of the sucrose transport protein that mediates long- distance sugar transport in crop plants. This discovery was a major advance in this field and determining how this control pathway works could have a major impact on our ability to enhance crop yield and nutritional value. D. This accomplishment is aligned with NP204 Global Change, Component III Agricultural Ecosystem Impacts NP204 Global Change, Component III Agricultural Ecosystem Impacts (2. Cropping Systems: Measure and predict plant responses (above and below-ground) to multiple interactions of abiotic and biotic stresses with rising carbon dioxide). The expression of over 18,000 transcripts in soybeans exposed to elevated [CO2] was analyzed using cDNA microarrays. In an experiment using fully expanded leaves sampled at approx. 2 a.m. when growth was maximum, 1129 transcripts were consistently expressed across three experimental blocks of the Soybean Free Air CO2 Enrichment (SoyFACE) experiment. In a second
experiment using growing leaves, 1332 transcripts were consistently expressed. There were 181 genes in common between the experiments, representing a best estimate of "CO2 response genes." Fifteen genes were down-regulated in elevated [CO2] by 1.2 fold or more, including genes involved in protein-protein interaction and protein degradation. Ninety genes were up-regulated by 1.2 fold or more, including putative sucrose and starch synthase genes and other genes involved in carbohydrate metabolism. This is the first analysis of transcript responses in a crop grown under elevated [CO2] in the field and provides a molecular basis for understanding soybean response to an important global atmospheric change. E. This accomplishment is aligned with NP302 Plant Biological and Molecular Processes, Component II Biological Processes that Improve Crop Productivity and Quality (Problem Statement 2A: Understanding growth and development). The calcium-dependent protein kinase (CDPK) superfamily is
a large and important family of protein kinases that are unique to plants and are thought to control numerous aspects of growth and development in response to calcium signals. However, it is not entirely understood how protein kinases, such as the CDPKs, target specific serine or threonine residues on their substrates. We demonstrated that CDPKs will recognize an unusual sequence of amino acids characterized by a specific arrangement of hydrophobic and basic amino acids located distal (C- terminal) to the phosphorylated serine. Recognition of this new motif explains phosphorylation of the enzyme ACC synthase, which catalyzes the rate-limiting step in ethylene biosynthesis. These results suggest that calcium signaling may be involved in the control of ethylene biosynthesis. The results also have application to a broad range of other cellular proteins, as database searches suggest that many additional proteins contain sequences that conform to the new motif and thus may be
phosphoproteins. Being able to predict phosphorylation sites in proteins based on primary sequence is not only an important genomics tool but also provides the basis for using molecular genetic approaches to control post- translational modification of proteins. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Research progress about the possible role of amino acids as not only substrates but also signals controlling the composition of developing soybean seeds was shared and discussed with other researchers at a soybean commodity workshop (USB) and annual meeting of a regional research project (NC-1142). Transferred information about the molecular details of the interaction between Rubisco and rubisco activase to academic and industrial scientists via
an invited presentation at a national meeting (ASPB). Transferred information about the genomic basis of crop response to CO2 at national symposium. Transferred information about the control of primary carbon and nitrogen metabolism by reversible protein phosphorylation and potential impacts on enzyme parameters such as activity, protein-protein interactions, localization and degradation at annual Plant Biology meetings. Transferred information about effects of atmospheric change and FACE research as invited speaker to several Universities. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Presented research tour of SoyFACE facility and project at the University of Illinois Agronomy Day. The publication of our Science paper on the lower than expected enhancement of yield by increasing CO2 in the atmosphere was covered in more than 50 newspapers and
2 international radio interviews.
Impacts
(N/A)
Publications
- Ort, D.R., Ainsworth, E.A., Aldea, M., Allen, D.J., Bernacchi, C.J., Berenbaum, M.R., Bollero, G.A., Cornic, G., Davey, P.A., Dermody, O., Dohleman, F.G., Hamilton, J.G., Heaton, E.A., Leaky, A.B., Mahoney, J., Morgan, P.B., Nelson, R.L., O'Neal, B., Rogers, A., Zanger, A.R., Zhu, X.G. , Delucia, E.H., Long, S.P. 2006. SoyFACE: The effects and interactions of Elevated [CO2] and [O3] on soybean. In: Nosberger, J., Long, S.P., Stitt, G.R., Hendrey, G.R., Blum H., editors. Managed Ecosystems and CO2. Heidelberg, Germany. Springer-Verlag. p. 71-86.
- Gong, P., Wu, G., Ort, D.R. 2006. Slow dark deactivation of Arabidopsis chloroplast ATP synthase caused by a mutation in a nonplastic SAC domain protein. Photosynthesis Research. 88:133-142.
- Leakey, A.B., Uribelarrea, M., Ainsworth, E.A., Naidu, S.L., Rogers, A., Long, S.P., Ort, D.R. 2006. Photosynthesis, productivity and yield of maize are not affected by open-air elevation of CO2 concentration in the absence of drought. Plant Physiology. 140:779-790.
- Long, S.P., Naidu, S.L., Ort, D.R. 2006. Can improvement in photosynthesis increase crop yields? Plant Cell and Environment. 29:315-330.
- Rogers, A., Gibon, Y., Morgan, P.B., Bernacchi, C.J., Ort, D.R., Long, S.P. 2006. Increased carbon availability at elevated carbon dioxide concentration improves nitrogen assimilation in legumes. Plant Cell and Environment. 29:1651-1658.
- Leakey, A.B., Bernacchi, C.J., Ort, D.R., Long, S.P. 2006. Growth of soybean under free-air [CO2]enrichment (FACE) does not cause stomatal acclimation. Plant Cell and Environment. 29:1794-1800.
- Zhu, X., Govindjee, Baker, N., De Sturler, E., Ort, D.R., Long, S.P. 2005. Chlorophyll a fluorescence induction kinetics in leaves predicted from a model describing each discrete step of excitation energy and electron transfer associated with photosystem II. Planta. 223:114-133.
- Christ, M.M., Ainsworth, E.A., Nelson, R.L., Schurr, U., Walter, A. 2006. Putative yield loss in field-grown soybean under elevated ozone can be avoided at the expense of leaf growth during early reproductive growth stages in favorable environmental conditions. Journal of Experimental Botany. 57:2267-2275.
- Ainsworth, E.A., Achim, W., Schurr, U. 2005. Glycine max leaves lack a base-tip gradient in growth rate. Journal of Plant Research. 118:343-346.
- Long, S.P., Ainsworth, E.A., Leakey, A.B., Morgan, P.B. 2005. Global food insecurity. Treatment of major food crops with either elevated carbon dioxide or ozone under large-scale fully open-air conditions suggest recent models may have over-estimate future yields. Philosophical Transactions of the Royal Society. 360:2011-2020.
- Long, S.P., Ainsworth, E.A., Bernacchi, C.J., Davey, P.A., Hymus, G.J., Leakey, A.B., Morgan, P.B., Osborne, C.P. 2006. Long term responses of photosynthesis and stomata to elevated [CO2] in managed systems. In: Nosberger, J., Long, S.P., Norby, R.J., Stitt, G.R., Hendrey, G.R. Blum H., editors. Heidelberg, Germany: Springer. p. 253-270.
- Rogers, A., Ainsworth, E.A. 2006. The response of foliar carbohydrates to elevated carbon dioxide concentration. In: Springer. Managed Ecosystems and CO2, Nosberger, J. Long, S.P., Stitt, G.R., Hendrey, G.R. Blum H., editors. Heidelberg, Germany: Springer. p.296-310.
- Rogers, A., Ainsworth, E.A., Kammann, C. 2006. FACE value. Perspectives on the future of free air CO2 enrichment studies. In: Nosberger, J. Long, S.P. , Norby, R.J, Stitt, G.R., Hendrey, G.R. Blum H., editors. Managed ecosystems and CO2: Case studies, processes, and perspectives. Heidelberg, Germany: Springer. p. 431-450.
- Ehsan, H., Ray, W.K., Phinney, B., Wang, X., Huber, S.C., Clouse, S.D. 2005. Interaction of Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase with a homolog of mammalian TGF receptor interacting protein. Plant Journal. 43:251-261.
- Kim, K., Portis Jr, A.R. 2005. Kinetic analysis of the slow inactivation of rubisco during catalysis: Effects of temperature, O2 and Mg++. Photosynthesis Research. 87:195-204.
- Wang, D., Portis Jr, A.R. 2006. Two conserved tryptophan residues are responsible for intrinsic fluorescence enhancement of Rubisco activase upon ATP binding. Photosynthesis Research. 88:185-193.
- Li, C., Wang, D., Portis Jr, A.R. 2006. Identification of critical arginine residues in the functioning of Rubisco activase. Archives Of Biochemistry and Biophysics. 450:176-182.
- Long, S.P., Zhu, X., Naidu, S.L., Raines, C.A., Ort, D.R. Limits to efficiencies of primary production, constraints and opportunities. In: Sylvester, B.R., Wiseman, J. Yields of Farmed Species: Constraints and Opportunities In the 21st Century. Nottingham, United Kingdom. Nottingham University Press. p. 319-333.
- Ainsworth, E.A., Long, S.P. 2005. What have we learned from fifteen years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2. New Phytologist. 165:351-372.
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Progress 10/01/04 to 09/30/05
Outputs
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? What does it matter? 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) Determination of the biochemical, molecular and genetic factors controlling tolerance and susceptibility of crop photosynthetic performance to drought, temperature extremes, and ozone; 2) Defining the key regulatory elements that control photosynthate production and partitioning and that determine sink strength; 3) Establishing the major features limiting the response of photosynthetic productivity of soybean and corn at elevated atmospheric CO2 and test
potential transgenic amelioration strategies. The experimental approaches are diverse combining biophysics, biochemistry, physiology, molecular biology and genomics. 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 the milestones (indicators of progress) from your Project Plan. Objective 1: Define the key regulatory elements controlling photosynthate production and partitioning and determining sink strength. FY01 - Generate transgenic sugar beet and tobacco with ectopic hyperactive sucrose symporter. -
Define role of transcription and protein turnover in sucrose symporter activity. - Screen for sucrose-signaling/partitioning mutants. - Clone gene-trapped tagged phloem genes. - Complete map based cloning of cfs ATPsynthase mutant. FY02 - Characterize sucrose signaling and partitioning mutants. - Screen mutagenized promoter & reporter gene transgenic plants for mutants in nitrate-response - Determine role of phosphorylation in regulating sucrose transporter activity. - Determine function of cloned phloem genes. - Create and complement random mutants in b559. - Determine protein expression level of site-directed -subunit constructs. FY03 - Clone mutated genes from sucrose signaling and partitioning mutants. - Characterize growth of transgenic plants with ectopic hyperactive sucrose symporter. - Characterize non-responsive nitrate-signaling mutants. - Determine genetic lesions random mutants in b559 - Create b559 heme-binding mutants. - Develop in vivo assay for chloroplast redox
poise of thioredoxin- regulated enzymes and proteins. FY04 - Clone mutated genes from sucrose signaling and partitioning mutants. - Characterize growth of transgenic plants with ectopic hyperactive sucrose symporter. - Characterize non-responsive nitrate-signaling mutants. - Determine genetic lesions random mutants in b559 - Create b559 heme-binding mutants. - Develop in vivo assay for chloroplast redox poise of thioredoxin- regulated enzymes and proteins FY05 - Determine whether the content of amino acids in developing soybean seeds is correlated with seed protein content at maturity in indeterminate cultivars. - Identify the membrane-binding domain on sucrose synthase. Objective 2: Determine the mechanistic basis for limitations on photosynthetic performance/productivity imposed by agriculturally significant stresses. FY01 -Cross Arabidopsis lines with defects in lipid metabolism with the rca- minus line; select plants retaining both mutations. - Measure PaO activity in control
and frozen seed. - Determine if there is a circadian rhythm in NR phosphorylation FY02 - Complete analysis of the temperature dependence of photosynthetic properties for Arabidopsis with wild type, antisense and null levels of rca. - Analysis microarrays for candidate PaO genes. - Determine if chill induced ribosomal pausing is site-specific. - Measure in vivo lifetime of NR in tomato FY03 - Complete analysis of the temperature dependence of photosynthesis in the Arabidopsis lines with defects in lipid metabolism. - Identify & clone PaO gene; select stay-green mutants. - Determine if chloroplast redox change drives polysome pausing FY04 - Complete analysis of the temperature dependence of photosynthesis in the Arabidopsis lines with defects in lipid metabolism and rca content. - Complete investigation of possible chlorophyll degradation operon in Synechocystis. FY05 - Identify role of pheophorbide oxygenase in green seed problem Objective 3: Establish the major features limiting
the response of photosynthetic productivity of soybean at elevated atmospheric CO2 and test potential transgenic amelioration strategies. FY01 - Perform baseline growth studies at 350 and 750 ppm CO2 on Arabidopsis plants with wild type and modified activase proteins. - Measure effect of elevated CO2 on TPU limitation in soybean in FACE experiment. FY02 - Complete feasibility analysis of relocating the small subunit gene to the chloroplast with the small subunit antisense tobacco line. - Determine the effects of the of light intensity regulation on starch/ sucrose partitioning in the Arabidopsis plants with modified rca proteins at 350 and 750 ppm CO2. - Clone sucrose-symporter from soybean. - Determine effect of elevated CO2 on temperature dependence of TPU limitation in soybean in FACE. - Determine effect of elevated CO2 on leaf temperature and transpiration in soybean in FACE. - Over-expression of the hyperactive symporter in sink tissues of sugar beet FY03 - Measure sucrose
symporter message abundance and transport activity in soybean grown at elevated CO2. - Determine if elevated CO2 enhances or protects high temperature photoinhibition in soybean in FACE. - Evaluate effect of over-expression of the hyperactive sucrose symporter in sink tissues on assimilate partitioning at elevated CO2 in growth chamber. FY04 -Complete analysis of mRNA and protein levels in multiply transformed tobacco lines. If needed, eliminate native small subunit expression with additional tobacco antisense constructs. -Evaluate effect of over-expression of the hyperactive sucrose symporter in sink tissues on assimilate partitioning at elevated CO2 in FACE facility. FY05 - Analyze if RNAi can be used to specifically limit native RbcS gene expression while allowing the expression of a foreign RbcS gene. - Analyze genetic variation in photoprotection in soybean under FACE treatments. - Analyze interaction between drought and elevated ozone and CO2 on stomatal conductance and
evapotranspiration in soybean. - Determine whether soybean exposure to high O3 or CO2 leads to increased protein oxidation, and if so, begin to identify specific protein targets. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Determine whether the content of amino acids in developing soybean seeds is correlated with seed protein content at maturity in indeterminate cultivars. Milestone Substantially Met 2. Identify role of pheophorbide a oxygenase in green seed problem. Milestone Substantially Met 3. Analyze if RNAi can be used to specifically limit native RbcS gene expression while allowing the expression of a foreign RbcS gene. Milestone Substantially Met 4. Analyze genetic variation in photoprotection in soybean under FACE treatments. Milestone Substantially Met 5. Analyze interaction between drought and elevated ozone and CO2 on stomatal conductance and
evapotranspiration in soybean. Milestone Not Met Other 6. Determine whether soybean exposure to high O3 or CO2 leads to increased protein oxidation, and if so, begin to identify specific protein targets. Milestone Not Met Other 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? New Project Plan under development. 4a What was the single most significant accomplishment this past year? Identifying genes involved in the response of soybean to elevated atmospheric [CO2] The expression of over 18,000 transcripts in soybeans exposed to elevated [CO2] was analyzed using cDNA microarrays. In an experiment using fully expanded leaves sampled at 2 a.m. when growth was maximum, 1129 transcripts were consistently expressed across three experimental blocks of the Soybean Free Air CO2 Enrichment (SoyFACE) experiment. In a second experiment using growing
leaves, 1332 transcripts were consistently expressed. There were 181 genes in common between the experiments, representing a best estimate of "CO2 response genes." Fifteen genes were down-regulated in elevated [CO2] by 1.2 fold or more, including genes involved in protein-protein interactions and protein degradation. Ninety genes were up-regulated by 1.2 fold or more, including putative sucrose and starch synthase genes and other genes involved in carbohydrate metabolism. This is the first analysis of transcript responses in a crop grown under elevated [CO2] in the field and provides a molecular basis for understanding soybean response to an important global atmospheric change. 4b List other significant accomplishments, if any. Finding a better Rubisco and activase for the changing atmosphere. Rubisco activity must be sustained by the activity of Rubisco activase and this requires specific interactions between the proteins. High but physiologically relevant temperatures appear to
decrease the activation state of Rubisco and the activity of Rubisco activase. Rubisco activity might be better retained at high temperatures by altering its interaction with Rubisco activase, but only some molecular details of the interaction are currently known. Past work showed that neither Rubisco nor activase from one family of plants, the Solanaceae, worked well in combination with the other protein from other families. This peculiar specificity preference was shown to be determined by two amino acids on the surface of Rubisco. In the current work by using chimeric activase proteins and site directed mutagenesis, we identified a substrate recognition region in activase in which two amino acids may directly interact with the two previously identified amino acids in Rubisco. This research may provide new and rational approaches for improving the interaction and activation of Rubisco by activase at high temperatures. Elevated CO2 does not stimulate C4 photosynthesis directly, but
impacts water relations and indirectly enhances carbon gain during drought stress in maize (Zea mays) grown under free-air CO2 enrichment (FACE). The potential for, and mechanism of, [CO2] effects on C4 plants has received considerable interest but remains poorly understood. In 2002 and 2004, a rainfed-field experiment utilizing FACE technology was undertaken, in the U.S. Corn Belt, to determine the effects of elevated [CO2] on Zea mays. FACE allows experimental treatments to be imposed on an undisturbed soil-plant-atmosphere continuum without the effects of experimental enclosures on plant microclimate. Each year, crop performance was compared at ambient [CO2] (370 ppm) and the elevated [CO2] (550 ppm) predicted for 2050, within a fully replicated design. The diurnal course of gas exchange of upper canopy leaves was measured across the growing season of 2002. This was repeated in 2004 along with analysis of carbon and nitrogen metabolism, water relations, growth and yield. This
tested if elevated [CO2] would directly: (1) stimulate C4 photosynthesis, and (2) reduce stomatal conductance and, therefore, crop water use. The experiments also tested if altered water relations under elevated [CO2] could feedback to enhance carbon gain during water stress. 2004 was unusual climatically in that at no time in the growing season was there any soil water deficit. In this year, there was no [CO2] effect on photosynthesis, carbon metabolism, growth or yield. Nevertheless, elevated [CO2] reduced stomatal conductance, crop evapo-transpiration and soil moisture depletion. 2002 was a "typical" year in which plants experienced episodic water stress. During these dry periods, photosynthesis was greater under elevated [CO2]. We conclude, elevated CO2 can only indirectly enhance carbon gain during drought. The green seed problem in canola: The role of pheophorbide a oxygenase (PaO) Brassica napus, canola, is an important oil seed crop grown extensively in North America and
northern Europe with annual yields exceeding seven million metric tons. Currently, canola is the worlds third most important vegetable oil, mainly due to its low levels of erucic acid and glucosinolates. In canola oil, the level of chlorophyll (Chl) content is significantly higher than that found in other vegetable oils and this is the biggest quality impediment in the canola oil industry. Under normal field conditions, Chl is degraded into colorless catabolites as the seed matures, resulting in seeds without Chl. Although canola is in general a cold hardy plant, an early frost can disrupt the normal pattern of Chl degradation, resulting in green seed at harvest and significantly devaluing the crop. PaO appears to be a key regulatory step in overall Chl degradation and specifically responsible for the loss of green color during senescence. PaO is known to play a prominent role in Chl degradation in both normal leaf aging and induced leaf senescence. The major objective of this
study was to identify candidate steps in Chl degradation in canola seeds that are disrupted by exposure to freezing temperatures early in seed development. The results show that freezing interferes with the induction of PaO activity that normally occurs in the later phases of canola seed development. Moreover, it is demonstrated that the regulation of PaO activity is largely posttranslational and it is at this level where freezing interferes with PaO activation in canola seeds. Identification and resolution of sucrose synthase (SUS) isoforms SUS is the major enzyme in the cytoplasm for the initial metabolism of sucrose leading to energy production or synthesis of structural components and storage compounds. One of our major goals is to manipulate SUS as a new approach to control how plant cells partition C among different pathways during growth and development. Essential to this project is a fundamental understanding of which isoforms of SUS are expressed in different plant organs
and to identify any endogenous posttranslational modifications. There are three known members of the SUS gene family in maize, which encode the proteins SUS1, Sh1, and SUS3. To date, SUS3 has only been identified at the transcript level, and it is not known to what extent this particular isoform contributes to sucrose metabolism. To resolve some of these issues, we produced isoform- specific antibodies to bolster the utility of existing antibodies that detect all isoforms, including SUS1. The results establish that SUS3 is an abundant protein and is expressed in both vegetative and reproductive organs. In addition, we have spent considerable effort examining various protocols to resolve SUS forms on 2-dimensional electrophoresis (2-DE). The most important result is that the Sh1 isoform seems to be selectively lost in all cases and hence is not observed on 2-DE. Other approaches are currently under evaluation. However, with the SUS1 isoform, several distinct spots can be resolved
by 2-DE and all are phosphorylated at the known serine-15 phosphorylation site. The results highlight the need to critically evaluate results obtained by 2-DE, and suggest that SUS may be subject to presently unrecognized posttranslational modification(s). Control of selective degradation of SUS protein De-etiolation of maize seedlings was investigated as a possible system to study the selective degradation of SUS protein. In contrast to green seedlings, the maize leaf blade that emerges in continuous darkness still retains a substantial amount of SUS protein and isoform-specific antibodies indicated the presence of all three isoforms (SUS1, Sh1 and SUS3). Illumination of the etiolated leaf blade triggered the loss of SUS protein and the appearance of the sucrose biosynthetic protein, sucrose-phosphate synthase (SPS) and other photosynthetic components. The nature of the light signal was further investigated by using light of different wavelengths, and also the elm-1 phytochrome
mutant. The loss of SUS protein in wild type leaves was induced by red, blue or white light, and also occurred in the phytochrome mutant, suggesting that blue light receptors (cryptochromes) might play a major role in perception of the light signal. De-etiolation also occurred with detached leaf blades (cut end in solution), which provided a convenient system to provide assimilates and inhibitors to the leaf blade. Using this system, we found that the loss of SUS protein was blocked by cycloheximide and also the proteasome inhibitor, MG132. Consistent with this, recovery of proteasomes by differential centrifugation indicated that proteasomes contained SUS polypeptides but the entrapped polypeptide fragments were not highly phosphorylated on either Ser15 or Ser170. This is in contrast to results obtained for degradation of SUS during elongation of "green" seedlings, where results suggested that phosphoSer170 was a trigger for degradation. Thus, it is possible that there are
multiple "triggers" that initiate SUS degradation and that the de-etiolation system is different than the green seedling. However, we have identified one metabolic factor that may influence the rate of SUS degradation. We observed that the light-induced loss of SUS protein occurred more rapidly in detached compared to attached leaves. Feeding assimilates to the detached leaves indicated that SUS degradation was controlled by the supply of N-assimilates (e.g., amino acids) and not sugars. Our current working model is that light stimulates SUS degradation indirectly, by enhancing the synthesis of proteins such as SPS. The control of SUS degradation by free amino acids provides an important new insight that should help further elucidate the overall mechanism(s) involved. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Carbon dioxide in the atmosphere is rising globally by 0.4% per year. The rate of increase that is
expected to accelerate with 2050 concentrations projected be 50% higher than today. In many areas, tropospheric ozone is increasing even more rapidly than CO2. Adapting the crop to these changes will require accurate information on exactly how increasing CO2 and ozone will affect crops in the field. A new facility at Urbana, IL allows the controlled enrichment of large plots within a field crop without any enclosure. The technology consists of rings, about 20 m in diameter, of pipes that inject CO2 and/or ozone into the air at the height of the crop and according to wind-speed and direction, maintaining the concentration within the ring at a pre-set elevated concentration through computer feed-back control. Important trends emerging from the initial 5 years of our experiments with soybean show that CO2 fertilization caused an average: 25% increase in photosynthesis 15% increase in yield 18% increase in above ground biomass 22% decrease in mid-day stomatal conductance 12% decrease
in season long water use 17% decrease in harvest index increase in canopy temperature Three years of data on the effects of ozone on soybean reveal both susceptibility in current cultivars and untapped tolerance in soybean germplasm. Experiments with soybean show that elevating ozone to levels predicted for the US Corn Belt in 2050 caused on average: 11% decrease in above ground biomass 14% decrease in seed yield 16% decrease in season long water use increase in canopy temperature The corn soybean rotation is the largest ecosystem type in North America. In consequence, these effects of elevated CO2 under agronomically realistic conditions are of significant importance in understanding mechanism, in designing cultivars better suited to future atmospheres, to predicting impact of atmospheric change on crop production, and in providing quantitative information on the how photosynthesis will directly feedback on climate change. The activity of Rubisco, the enzyme responsible for capturing
atmospheric carbon dioxide during photosynthesis, is a major factor limiting photosynthesis in crop plants. The activity of Rubisco is down- regulated under limiting light conditions, thereby further limiting the rate of photosynthesis. We created transgenic plants in which Rubisco remains fully active over a wide range of light intensities by using genetic transformation to express modified forms of the regulatory protein, Rubisco activase. These unique plants are being used to study the basis for high temperature inhibition of photosynthesis and the limitations of photosynthesis in dynamic light environments. Thus these plants are providing a powerful means to delineate the significance of light regulation of Rubisco in defining plant productivity under different environmental conditions. The major harvested tissues in plants are composed of cells that must import sugars and amino acids produced in photosynthesis to support growth and development. Although we understand the basic
mechanics of how sugars are moved in the plant's vascular system, the regulation of sugar allocation is not well understood because of the complicated interplay between leaves and harvested organs. Using biochemical and recombinant DNA methods, we discovered a sucrose-sensing pathway that regulates the activity and expression level of the sucrose transport protein that mediates long-distance sugar transport in crop plants. This discovery was a major advance in this field and determining how this control pathway works could have a major impact on our ability to enhance crop yield and nutritional value. The expression of over 18,000 transcripts in soybeans exposed to elevated [CO2] was analyzed using cDNA microarrays. In an experiment using fully expanded leaves sampled at 2 a.m. when growth was maximum, 1129 transcripts were consistently expressed across three experimental blocks of the Soybean Free Air CO2 Enrichment (SoyFACE) experiment. In a second experiment using growing
leaves, 1332 transcripts were consistently expressed. There were 181 genes in common between the experiments, representing a best estimate of "CO2 response genes." Fifteen genes were down-regulated in elevated [CO2] by 1.2 fold or more, including genes involved in protein-protein interaction and protein degradation. Ninety genes were up-regulated by 1.2 fold or more, including putative sucrose and starch synthase genes and other genes involved in carbohydrate metabolism. This is the first analysis of transcript responses in a crop grown under elevated [CO2] in the field and provides a molecular basis for understanding soybean response to an important global atmospheric change. The calcium-dependent protein kinase (CDPK) superfamily is a large and important family of protein kinases that are unique to plants and are thought to control numerous aspects of growth and development in response to calcium signals. However, it is not entirely understood how protein kinases, such as the
CDPKs, target specific serine or threonine residues on their substrates. We demonstrated that CDPKs will recognize an unusual sequence of amino acids characterized by a specific arrangement of hydrophobic and basic amino acids located distal (C-terminal) to the phosphorylated serine. Recognition of this new motif explains phosphorylation of the enzyme ACC synthase, which catalyzes the rate- limiting step in ethylene biosynthesis. These results suggest that calcium signaling may be involved in the control of ethylene biosynthesis. The results also have application to a broad range of other cellular proteins, as database searches suggest that many additional proteins contain sequences that conform to the new motif and thus may be phosphoproteins. Being able to predict phosphorylation sites in proteins based on primary sequence is not only an important genomics tool but also provides the basis for using molecular genetic approaches to control post- translational modification of
proteins. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Transferred information about the control of primary carbon and nitrogen metabolism by reversible protein phosphorylation and potential impacts on enzyme parameters such as activity, protein-protein interactions, localization and degradation at annual Plant Biology meetings. PATENT APPLICATION: International patent application (PCT/US 04/34996) was filed Oct 20, 2004. "Synthetic peptides that cause F-actin bundling and block actin depolymerization." Inventors: H. Winter, S.C. Huber and C. Larabell. Research progress about the possible role of amino acids as not only substrates but also signals controlling the composition of developing soybean seeds was shared and discussed with other
researchers at a soybean commodity workshop (USB) and annual meeting of a regional research project (NC-1142). Transferred information about proper usage of portable infrared gas analyzers for measuring level photosynthesis and fluorescence to Mexican research scientists at a Photosynthesis Workshop in Monterrey, Mexico. Transferred information about the molecular details of the interaction between Rubisco and rubisco activase to academic and industrial scientists via an invited presentation at a national meeting (ASPB). Transferred information about the effects of temperature on Rubisco and Rubisco activase in vitro and in transgenic Arabidopsis plants as an invited speaker at an international meeting (Gordon Research Conference). Transferred information about effects of atmospheric change and FACE research as invited speaker at international workshop in Canberra, Australia and an international meeting in Barcelona Spain. 7. List your most important publications in the popular press
and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Presented research tour of SoyFACE facility and project at the University of Illinois Agronomy Day.
Impacts
(N/A)
Publications
- Duncan, K.A., Hardin, S.C., Huber, S.C. The third sucrose synthase in <1>zea mays(sus3) is a ubiquitous protein that is phosphorylated and membrane associated [abstract]. Plant Biology Annual Meeting. Available: http://abstracts.aspb.org/pb2005/public/P56/7486.html.
- Hardin, S.C., Duncan, K.A., Huber, S.C. 2005. The association of sucrose synthase with intracellular membranes may involve a region with similarity to a pleckstrin homology domain [abstract]. Plant Biology Annual Meeting. Available: http://abstracts.aspb.org/pb2005/public/P56/7488.html.
- Qiu, Q., Huber, J.L., Huber, S.C. 2005. Identification of the major leaf proteins subject to o-glcnac posstranslational modification [abstract]. Plant Biology Annual Meeting. Available: http://abstracts.aspb. org/pb2005/public/P69/7476.html.
- Chalivendra, S.C., Palaniappan, A., Duncan, K., Huber, S.C., Sachs, M.M. 2005. New intracellular locations and putative novel functions for maize sucrose synthases [abstract]. Plant Biology Annual Meeting. Available: http://abstracts.aspb.org/pb2005/public/P51/7674.html.
- Wang, X., Goshe, M.B., Soderblom, E.J., Phinney, B.S., Kuchar, J.A., Li, J. , Asami, T., Yoshida, S., Huber, S.C., Clouse, S.D. 2005. Molecular mechanisms of early events in brassinosteroid signal transduction [abstract]. Plant Biology Annual Meeting. Available: http://abstracts/aspb.org/pb2005/public/P66/7861.html.
- Sebastia, C.H., Hardin, S.C., Clouse, S.C., Kieber, J.J., Huber, S.C. 2004. Identification of A New Motif for CDPK Phosphorylation In Vitro That Suggests ACC Synthase May Be A CDPK Substrate. Archives Of Biochemistry and Biophysics. 428:81-91.
- Zhu, X., Portis Jr, A.R., Ort, D.R., Long, S.P. 2005. Can Manipulation of The C2 Pathway Enzyme Activities Increase Photosynthetic Rate? An In Silico Analysis [abstract]. Plant Biology Annual Meeting. Paper No. 266. Available: http://abstracts.aspb.org/pb2005/public/P43/7516.html.
- Li, C., Salvucci, M.E., Portis Jr, A.R. 2005. Two residues of rubisco activase involved in recognition of the rubisco substrate. Journal of Biological Chemistry. 280(26):24864-9.
- Zuniga-Feest, A., Ort, D.R., Gutierrezc, A., Gidekel, M., Bravo, L.A., Corcuera, L.J. 2005. Light regulation of sucrose-phosphate synthase activity in the grass Deschampsia antarctica. Photosynthesis Research. 83:75-86.
- Zhu, X., Ort, D.R., Whitmarsh, C.J., Long, S.P. 2004. The Slow Reversibility of Photosystem II Thermal Energy Dissipation On Transfer From High To low Light May Cause Large Losses In Carbon Gain By Crop Canopies: A Theoretical Analysis. Journal of Experimental Botany. 55:1167- 1175.
- Gong, P., Wu, G., Ort, D.R. 2004. Cloning of A Chloroplast Atp Synthase Activation Mutation In Arabidopsis [abstract]. Plant Biology. Available: http://abstracts.aspb.org/pb2004/public/P39/7963.html.
- Wang, X., Goshe, M.B., Soderblom, E.J., Phinney, B.S., Kuchar, J., Li, J., Asami, T., Huber, S.C., Clouse, S.D. 2005. Identification and functional analysis of in vivo phosphorylation sites of the arabidopsis brassinosteroid insensitive 1 receptor kinase. The Plant Cell. 17:1685- 1703.
- Huber, S.C., Hardin, S.C. 2004. Numerous post-translational modifications provide opportunites for the intricate regulation of metabolic enzymes at multiple levels. Plant Biology. 7:318-322.
- Hardin, S., Huber, S.C. 2004. Proteasome activity and the post- translational control of sucrose synthase stability in maize leaves. Plant Physiology and Biochemistry. 42:197-208.
- Hardin, S.C., Winter, H., Huber, S.C. 2004. Phosphorylation of the amino- terminus of maize sucrose synthase in relation to membrane association and enzyme activity. Plant Physiology. 134:1427-1438.
- Qiu, Q., Huber, J.L., Portis Jr, A.R., Huber, S.C. 2005. Protein oxidation in leaves of arabidopsis and soybean: implications for plant response to elevated CO2. Plant Biology Annual Meeting. Available: http://abstracts/aspb.org/pb2005/public/P36/7475.html.
- Morgan, P.B., Bernacchi, C.J., Ort, D.R., Long, S.P. 2004. An In Vivo Analysis of The Effect of Season-Long, Open-Air Elevation of Ozone to Anticipated 2050 Levels On Photosynthesis in Soybean. Plant Physiology. 135:2348-2357.
- Leakey, A., Bernacchi, C.J., Dohleman, F., Ort, D.R., Long, S. 2004. How Will Photosynthesis of Maize In The U.S. Corn Belt respond To Future CO2 Rich Atmospheres? Soybean Biotechnology Meeting. p. 951-962.
- Prior, S.A., Torbert III, H.A., Runion, G.B., Rogers Jr, H.H., Ort, D.R., Nelson, R.L. 2005. Elevated atmospheric CO2 effects on residue decomposition of different soybean varieties [abstract]. Third USDA Symposium on Greenhouse Gases & Carbon Sequestration in Agriculture and Forestry, Program and Abstracts. p. 202.
- Leakey, A., Davey, P., Allen, D., Rogers, A., Delucia, E., Drake, B., Murthy, R., Ort, D.R., Long, S.P. 2004. How Will Leaf Respiration Respond To Growth Under Elevated Carbon Dioxide Concentration In Three Diverse Tree Canopies [abstract]? Ecological Society of America Abstracts. Available: http://abstracts.co.allenpress.com/pweb/esa2003/document/? ID=25515
- Leakey, A., Bernacchi, C.J., Below, F., Bordigno, R., Mies, T., Morgan, P., Nelson, R.L., Ort, D.R., Uribellarea, M., Long, S. 2004. The heart project: determining how the major agricultural ecosystem of the U.S. will respond to atmospheric change in 2050 [abstract]. Plant Biology Annual Meeting.
- Bernacchi, C.J., Ort, D.R., Morgan, P.B., Long, S.P. 2004. The Growth of Soybean Under Free Air Concentration Enrichment (FACE) Stimulates Photosynthesis While Decreasing In Vivo Rubisco Capacity [abstract]. Planta (2005) 220:434-446.
- Wall, G.W., Kimball, B.A., Ort, D.R., Bernacchi, C.J., Olivieri, L.M., Long, S.P., Harrison, M. 2003. Univariate Effect of Elevated CO2 or O3 On Water Relations of Soybean and Corn Leaves. ASA-CSSA-SSSA Proceedings.
- Prior, S.A., Torbert III, H.A., Runion, G.B., Rogers Jr, H.H., Ort, D.R. 2004. Free-air carbon dioxide enrichment of soybean: influence of crop variety on residue decomposition [abstract]. American Society of Agronomy Branch Meeting.CD-ROM
- Kangmin, K., Portis Jr., A.R. 2003. Rubisco Deactivation Caused By Altered Activase Activity Drives The Early Inhibition of Photosynthesis At Moderately High Temperature [abstract]. American Society of Plant Biologists. Available: http://abstracts.aspb.org/pb2003/public/P37/1056. html.
- Li, C., Portis Jr, A.R. 2004. Identification of an amino acid residue involved in the species specificity of Rubisco activase [abstract]. American Society of Plant Biologists Annual Meeting. Available: http://abstracts.aspb.org/pb2004/public/P40/7257.html.
- Kim, K., Portis Jr, A.R. 2004. Oxygen-dependent H2O2 production by Rubisco. Federation of European Biochemical Societies Letters. 571:124-128.
- Huber, S.C. 2003. Phosphorylation of metabolic enzymes: effects on activity, localization and degradation [abstract]. Molecular and Cellular Proteomics. 2:679.
- HUBER, S.C., TANG, G.Q., HARDIN, S.C. 2003. Protein Phosphorylation As A Possible Trigger For Degradation of Metabolic Enzymes [abstract]. Current Topics In Plant Biochemistry, Physiology and Molecular Biology. Plant Protein Phosphorylation-Dephosphorylation. 21:1415.
- Hardin, S.C., Huber, S.C. 2004. Proteasome activity and the post- translational control of sucrose synthase stability in maize leaves [abstract]. Plant Physiology and Biochemistry. 42:197-208.
- Ehsan, H., Ray, W.K., Huber, S.C., Clouse, S.D. 2003. An arabidopsis protein with sequence similarity to animal tgf-beta receptor interacting protein is phosphorylated by the br11 receptor kinase in vitro [abstract]. American Society of Plant Biologists. Available: http://abstracts.aspb. org/pb2003/public/M12/5068.html
- Shen, W., Clark, C., Huber, S.C. 2003. The c-terminal tail of arabidopsis 14-3 functions as an autoinhibitor and may contain a tenth a-helix. Plant Journal. 34:473-484.
- Tang, G., Hardin, S.C., Dewey, R., Huber, S.C. 2003. A novel c-terminal proteolytic processing of cytosolic pyruvate kinase, its phosphorylation and degradation by the proteasome in developing soybean seeds. Plant Journal. 34:77-95.
- Hardin, S.C., Tang, G.Q., Scholz, A., Holtgraewe, D., Winter, H., Huber, S. C. 2003. "Phosphorylation of Sucrose Synthase at Serine 170: Occurrence and Possible Role As a Signal for Proteolysis" [abstract]. Plant Journal. 35:588-603.
- Wang, D., Portis Jr, A.R. 2004. Site-directed mutagenesis of negatively charged residues in the carboxyl terminus of the 46-kDa isoform of Arabidopsis Rubisco activase [abstract]. American Society of Plant Biologists Annual Meeting. Available: http://abstracts.aspb. org/pb2004/public/P40/7654.html.
- Hernandez, C., Saravitz, C., Zhang, P., Israel, D., Dewey, R., Huber, S.C. 2003. Do amino acids regulate transcription of lipogenic genes in developing soybean seeds? [abstract]. International Congress of Plant Molecular Biology. Paper No. 3620.
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Progress 10/01/03 to 09/30/04
Outputs
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? What does it matter? 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) Determination of the biochemical, molecular and genetic factors controlling tolerance and susceptibility of crop photosynthetic performance to drought, temperature extremes, and ozone; 2) Defining the key regulatory elements that control photosynthate production and partitioning and that determine sink strength; 3) Establishing the major features limiting the response of photosynthetic productivity of soybean and corn at elevated atmospheric CO2 and test
potential transgenic amelioration strategies. The experimental approaches are diverse combining biophysics, biochemistry, physiology, molecular biology and genomics. 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 the milestones (indicators of progress) from your Project Plan. Objective 1: Define the key regulatory elements controlling photosynthate production and partitioning and determining sink strength. FY01 - Generate transgenic sugar beet and tobacco with ectopic hyperactive sucrose symporter. -
Define role of transcription and protein turnover in sucrose symporter activity. - Screen for sucrose-signaling/partitioning mutants. - Clone gene-trapped tagged phloem genes. - Complete map based cloning of cfs ATPsynthase mutant. FY02 - Characterize sucrose signaling and partitioning mutants. - Screen mutagenized promoter & reporter gene transgenic plants for mutants in nitrate- response - Determine role of phosphorylation in regulating sucrose transporter activity. - Determine function of cloned phloem genes. - Create and complement random mutants in b559. - Determine protein expression level of site-directed gamma-subunit constructs. FY03 - Clone mutated genes from sucrose signaling and partitioning mutants. - Characterize growth of transgenic plants with ectopic hyperactive sucrose symporter. - Characterize non-responsive nitrate-signaling mutants. - Determine genetic lesions random mutants in b559 - Create b559 heme-binding mutants. - Develop in vivo assay for chloroplast
redox poise of thioredoxin- regulated enzymes and proteins. FY04 - Clone mutated genes from sucrose signaling and partitioning mutants. - Characterize growth of transgenic plants with ectopic hyperactive sucrose symporter. - Characterize non-responsive nitrate-signaling mutants. - Determine genetic lesions random mutants in b559 - Create b559 heme-binding mutants. - Develop in vivo assay for chloroplast redox poise of thioredoxin- regulated enzymes and proteins FY05 - Complete biochemical and molecular analysis of phloem-specific gene in assimilate partitioning. - Complete biochemical and molecular dissection if sucrose signaling pathway Objective 2: Determine the mechanistic basis for limitations on photosynthetic performance/productivity imposed by agriculturally significant stresses. FY01 -Cross Arabidopsis lines with defects in lipid metabolism with the rca- minus line; select plants retaining both mutations. - Measure PaO activity in control and frozen seed. - Determine if
there is a circadian rhythm in NR phosphorylation FY02 - Complete analysis of the temperature dependence of photosynthetic properties for Arabidopsis with wild type, antisense and null levels of rca. - Analysis microarrays for candidate PaO genes. - Determine if chill induced ribosomal pausing is site-specific. - Measure in vivo lifetime of NR in tomato FY03 - Complete analysis of the temperature dependence of photosynthesis in the Arabidopsis lines with defects in lipid metabolism. - Identify & clone PaO gene; select stay-green mutants. - Determine if chloroplast redox change drives polysome pausing FY04 - Complete analysis of the temperature dependence of photosynthesis in the Arabidopsis lines with defects in lipid metabolism and rca content. - Complete investigation of possible chlorophyll degradation operon in Synechocystis. FY05 - Identify mechanism of green seed problem Objective 3: Establish the major features limiting the response of photosynthetic productivity of soybean
at elevated atmospheric CO2 and test potential transgenic amelioration strategies. FY01 - Perform baseline growth studies at 350 and 750 ppm CO2 on Arabidopsis plants with wild type and modified activase proteins. - Measure effect of elevated CO2 on TPU limitation in soybean in FACE experiment. FY02 - Complete feasibility analysis of relocating the small subunit gene to the chloroplast with the small subunit antisense tobacco line. - Determine the effects of the of light intensity regulation on starch/ sucrose partitioning in the Arabidopsis plants with modified rca proteins at 350 and 750 ppm CO2. - Clone sucrose-symporter from soybean. - Determine effect of elevated CO2 on temperature dependence of TPU limitation in soybean in FACE. - Determine effect of elevated CO2 on leaf temperature and transpiration in soybean in FACE. - Over-expression of the hyperactive symporter in sink tissues of sugar beet FY03 - Measure sucrose symporter message abundance and transport activity in soybean
grown at elevated CO2. - Determine if elevated CO2 enhances or protects high temperature photoinhibition in soybean in FACE. - Evaluate effect of over-expression of the hyperactive sucrose symporter in sink tissues on assimilate partitioning at elevated CO2 in growth chamber. FY04 -Complete analysis of mRNA and protein levels in multiply transformed tobacco lines. If needed, eliminate native small subunit expression with additional tobacco antisense constructs. -Evaluate effect of over-expression of the hyperactive sucrose symporter in sink tissues on assimilate partitioning at elevated CO2 in FACE facility. FY05 - Analyze the photosynthetic properties of tobacco lines expressing the Chlamydomonas Rubisco genes under high CO2 in growth chamber and FACE. - Complete analysis of genetic variation in photoprotection in soybean under FACE treatments. 3. Milestones: A. List the milestones (from the list in Question #2) that were scheduled to be addressed in FY2004. How many
milestones did you fully or substantially meet in FY2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. Objective 1 - FY04 - Clone mutated genes from sucrose signaling and partitioning mutants. Not fully or substantially met because SY on this project left ARS in FY03. - Characterize growth of transgenic plants with ectopic hyperactive sucrose symporter. Not fully or substantially met because SY on this project left ARS in FY03. - Characterize non-responsive nitrate-signaling mutants. Substantially completed. - Determine genetic lesions random mutants in b559 Not fully or substantially met because SY on this project left ARS in FY02. - Create b559 heme-binding mutants. Not fully or substantially met because SY on this project left ARS in FY02. - Develop in vivo assay for chloroplast redox poise of thioredoxin- regulated enzymes and proteins. Substantially completed Objective 2 - FY04 - Complete analysis of the temperature
dependence of photosynthesis in the Arabidopsis lines with defects in lipid metabolism and rca content. Substantially completed - Complete investigation of possible chlorophyll degradation operon in Synechocystis. Not fully or substantially met because SY on this project left ARS in FY02. Objective 3 -FY04 -Complete analysis of mRNA and protein levels in multiply transformed tobacco lines. If needed, eliminate native small subunit expression with additional tobacco antisense constructs. Substantially completed -Evaluate effect of over-expression of the hyperactive sucrose symporter in sink tissues on assimilate partitioning at elevated CO2 in FACE facility. Not fully or substantially met because SY on this project left ARS in FY03. B. List the milestones that your expect to address over the next 3 years. What do you expect to accomplish, year by year, over the next 3 years under each milestone? Objective 1 - FY05 (last year of Project Plan) - Determine whether the content of amino
acids in developing soybean seeds is correlated with seed protein content at maturity in indeterminate cultivars. - Identify the membrane-binding domain on sucrose synthase. Objective 2 - FY05 (last year of Project Plan) - Identify role of pheophorbide oxygenase in green seed problem Objective 3 - FY05 (last year of Project Plan) - Analyze if RNAi can be used to specifically limit native RbcS gene expression while allowing the expression of a foreign RbcS gene. - Analyze genetic variation in photoprotection in soybean under FACE treatments. - Analyze interaction between drought and elevated ozone and CO2 on stomatal conductance and evapotranspiration in soybean. - Determine whether soybean exposure to high O3 or CO2 leads to increased protein oxidation, and if so, begin to identify specific protein targets. 4. What were the most significant accomplishments this past year? A. Single most significant FY04 accomplishment Identification of a new phosphorylation motif: Understanding how
protein kinases target their substrates. The calcium-dependent protein kinase (CDPK) superfamily is a large and important family of protein kinases that are unique to plants and are thought to control numerous aspects of growth and development in response to calcium signals. However, it is not entirely understood how protein kinases, such as the CDPKs, target specific serine or threonine residues on their substrates. We demonstrated that CDPKs will recognize an unusual sequence of amino acids characterized by a specific arrangement of hydrophobic and basic amino acids located distal (C-terminal) to the phosphorylated serine. Recognition of this new motif explains phosphorylation of the enzyme ACC synthase, which catalyzes the rate- limiting step in ethylene biosynthesis. These results suggest that calcium signaling may be involved in the control of ethylene biosynthesis. The results also have application to a broad range of other cellular proteins, as database searches suggest
that many additional proteins contain sequences that conform to the new motif and thus may be phosphoproteins. Being able to predict phosphorylation sites in proteins based on primary sequence is not only an important genomics tool but also provides the basis for using molecular genetic approaches to control post- translational modification of proteins. B. Other significant accomplishments Direct warming of the atmosphere not caused by the greenhouse effect. Using free-air concentration enrichment (FACE) technology soybean was grown at predicted 2050 concentrations of ozone ([O3]) and carbon dioxide ([CO2]) under fully open-air conditions. Evapotranspiration and surface temperatures were measured by near-ground remote micrometeorological methods. Elevated [O3] caused a season long 16% decrease in transpired water and elevated [CO2] a 12% decrease consistent with mean mid-day increases in canopy temperatures. The results provide experimental validation of model predictions that
under open-air conditions the direct effects of atmospheric change on stomata, even in the absence of greenhouse warming, have the potential to substantially reduce summer moisture supply to the atmosphere and surface temperature in temperate continental interiors. This work has provided the first evidence that at a field scale the effects of rising tropospheric ozone and CO2 on photosynthesis and stomata cause a warming of vegetated surfaces which will raise surface temperatures independently of any greenhouse effect and have the potential to substantially reduce summer moisture supply to the atmosphere. This is a rare experimentally determined example of how photosynthesis will directly feedback on climate change. Selecting a better Rubisco for the changing atmosphere. Genetic modification of Rubisco to increase its activity and/or the specificity for carbon dioxide relative to oxygen to decrease photorespiration should in principle increase crop productivity. In this study with
University of Illinois cooperators, a biochemical model for leaf photosynthesis was coupled to a canopy biophysical microclimate model to quantify 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. The outcome of this research provides specific guidance for selecting foreign Rubisco proteins to replace the existing proteins in crop species. Phosphorylation of sucrose synthase affects properties. Sucrose synthase is an important enzyme of sucrose metabolism in growing plant organs, such as developing seeds, and channels carbon from sucrose into various metabolic and biosynthetic pathways. The activity of sucrose synthase in a growing organ is thought to be a marker for, or possible determinant of, growth. Consequently, there is great interest in identifying mechanisms, such as protein phosphorylation, that control the activity and function of this critical plant
enzyme. Sucrose synthase is known to be phosphorylated on a specific amino acid near the amino terminus of the protein (serine-15). However, the effects of phosphorylation on the enzyme parameters are still not clear. We used modification-specific antibodies to show that phosphorylation affects the conformation of the amino terminus of the protein, and that this conformational change may be responsible for increased enzyme activity and changes in binding to membranes that are observed. Elucidating the basis for control of plant metabolism by protein phosphorylation may provide rational approaches to control specific aspects of metabolism. Selective degradation of sucrose synthase. Plants produce and feed themselves sucrose. The agricultural productivity of most crops such as maize, wheat and soybean, is dependent upon the ability of leaves to produce sucrose, as an end product of photosynthesis, and the ability of growing parts such as roots, stems and seeds, to utilize the sugar
in growth and development. Recent evidence indicates that sucrose is not only a primary nutrient but also an important "signal compound" that can control gene expression. Consequently, understanding the mechanisms that control the utilization of sucrose in crop plants is very important. The enzyme sucrose synthase is recognized to play an important role in the metabolism of sucrose in seeds and tubers, and the enzymatic activity of this enzyme is often correlated closely with organ growth. Hence, factors that influence the activity of this enzyme may impact plant growth and development. Recent results suggest that degradation of the sucrose synthase protein molecule is tightly controlled and may involve a large multiprotein complex known as the "proteasome." Furthermore, phosphorylation of the sucrose synthase protein at a specific serine residue (serine-170) may be the trigger that initiates its degradation via this selective process. Understanding the complex mechanisms that
control sucrose metabolism may produce new approaches to regulate crop growth. C. Significant Activities that Support Special Target Populations: None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Carbon dioxide in the atmosphere is rising globally by 0.4% per year. The rate of increase that is expected to accelerate with 2050 concentrations projected be 50% higher than today. In many areas, tropospheric ozone is increasing even more rapidly than CO2. Adapting the crop to these changes will require accurate information on exactly how increasing CO2 and ozone will affect crops in the field. A new facility at Urbana, IL allows the controlled enrichment of large plots within a field crop without any enclosure. The technology consists of rings, about 20 m in diameter, of pipes that inject CO2 and/or ozone into the air at the height of the crop and according to wind-speed and direction, maintaining the concentration
within the ring at a pre-set elevated concentration through computer feed-back control. Important trends emerging from the initial 3 years of our experiments with soybean show that CO2 fertilization caused an average: 25% increase in photosynthesis 15% increase in yield 18% increase in above ground biomass 22% decrease in mid-day stomatal conductance 12% decrease in season long water use 17% decrease in harvest index increase in canopy temperature Two years of data on the effects of ozone on soybean reveal both susceptibility in current cultivars and untapped tolerance in soybean germplasm. Experiments with soybean show that elevating ozone to levels predicted for the US Corn Belt in 2050 caused on average: 11% decrease in above ground biomass 14% decrease in seed yield 16% decrease in season long water use increase in canopy temperature The corn soybean rotation is the largest ecosystem type in North America. In consequence, these effects of elevated CO2 under agronomically
realistic conditions are of significant importance in understanding mechanism, in designing cultivars better suited to future atmospheres, to predicting impact of atmospheric change on crop production, and in providing quantitative information on the how photosynthesis will directly feedback on climate change. The activity of Rubisco, the enzyme responsible for capturing atmospheric carbon dioxide during photosynthesis, is a major factor limiting photosynthesis in crop plants. The activity of Rubisco is down- regulated under limiting light conditions, thereby further limiting the rate of photosynthesis. We created transgenic plants in which Rubisco remains fully active over a wide range of light intensities by using genetic transformation to express modified forms of the regulatory protein, Rubisco activase. These unique plants are being used to study the basis for high temperature inhibition of photosynthesis and the limitations of photosynthesis in dynamic light environments. Thus
these plants are providing a powerful means to delineate the significance of light regulation of Rubisco in defining plant productivity under different environmental conditions. The major harvested tissues in plants are composed of cells that must import sugars and amino acids produced in photosynthesis to support growth and development. Although we understand the basic mechanics of how sugars are moved in the plant's vascular system, the regulation of sugar allocation is not well understood because of the complicated interplay between leaves and harvested organs. Using biochemical and recombinant DNA methods, we discovered a sucrose-sensing pathway that regulates the activity and expression level of the sucrose transport protein that mediates long-distance sugar transport in crop plants. This discovery was a major advance in this field and determining how this control pathway works could have a major impact on our ability to enhance crop yield and nutritional value. The
calcium-dependent protein kinase (CDPK) superfamily is a large and important family of protein kinases that are unique to plants and are thought to control numerous aspects of growth and development in response to calcium signals. However, it is not entirely understood how protein kinases, such as the CDPKs, target specific serine or threonine residues on their substrates. We demonstrated that CDPKs will recognize an unusual sequence of amino acids characterized by a specific arrangement of hydrophobic and basic amino acids located distal (C-terminal) to the phosphorylated serine. Recognition of this new motif explains phosphorylation of the enzyme ACC synthase, which catalyzes the rate- limiting step in ethylene biosynthesis. These results suggest that calcium signaling may be involved in the control of ethylene biosynthesis. The results also have application to a broad range of other cellular proteins, as database searches suggest that many additional proteins contain sequences
that conform to the new motif and thus may be phosphoproteins. Being able to predict phosphorylation sites in proteins based on primary sequence is not only an important genomics tool but also provides the basis for using molecular genetic approaches to control post- translational modification of proteins. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Transferred information about the control of primary carbon and nitrogen metabolism by reversible protein phosphorylation and potential impacts on enzyme parameters such as activity, protein-protein interactions, localization and degradation as an invited speaker at 2 international meetings (IUBMB Congress; GRC), one symposium (Danforth), and 3 seminars at US Universities (UIUC; UFL; UT). Research
progress about the possible role of amino acids as not only substrates but also signals controlling the composition of developing soybean seeds was shared and discussed with other researchers at a soybean commodity workshop (USB) and annual meeting of a regional research project (NC-1142). Research progress report on the role of Rubisco activase in the reversible inhibition of photosynthesis at high temperatures, NC-1142 Regulation of Photosynthetic Processes, November 22, 2003, Reno NV NC (researchers from 17 state Agric. Expt. Stations and two USDA locations). Transferred information about resource allocation, sucrose sensing, and nitrogen regulation of amino acid transporter gene expression to academic and industrial scientists as an invited speaker at international meetings or at US universities or companies (2 presentations). Could be applied to commercial systems within 2 to 3 years if successful. It will be a critical enabling accomplishment to understand the regulation of
transporter expression by the products of photosynthesis. Transferred information about effects of atmospheric change and FACE research as invited speaker at 2 international meetings (ISPR International Congress, Monte Verita FACE Workshop), a national meeting (ASA), and to Argonne National Lab. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Presented research tour of SoyFACE facility and project at the University of Illinois Agronomy Day. News release by University of Illinois on our work with state collaborators on DOE funded project investigating the genomics of atmospheric change. News release by ARS on a synthetic protein fashioned after a natural plant enzyme that has surprised researchers with its Taxol-like ability to curtail abnormal cell growth in test tubes.
Impacts
(N/A)
Publications
- Rogers, A., Allen, D.J., Davey, P.A., Morgan, P.B., Ainsworth, E.A., Bernacchi, C.J., Cornic, G., Dermody, O., Heaton, E.A., Mahoney, J., Zhu, X., Delucia, E.H., Ort, D.R., Long, S.P. 2004. Leaf photosynthesis and carbohydrate dynamics of soybeans grown throughout their life-cycle under free-air carbon dioxide enrichment. Plant Cell and Environment. 27:449-458.
- Schrader, S.M., Wise, R.R., Wacholtz, W.F., Ort, D.R., Sharkey, T.D. 2004. High leaf temperature limits photosynthesis in Pima cotton II. thylakoid membrane responses to moderate heat stress. Plant Cell and Environment. 27:725-735.
- Singsaas, E.L., Ort, D.R., Delucia, E.H. 2003. Elevated CO2 effects on mesophyll conductance and its consequences for interpreting photosynthetic physiology. Plant Cell and Environment. 27:41-50.
- Tucker, D., Allen, D.J., Ort, D.R. 2004. Control of nitrate reductase by circadian and durral rhythm in tomato. Planta. 219:277-285.
- Portis Jr., A.R. 2004. Rubisco activase. In: Goodman, R.M., editor. Encyclopedia of Plant and Crop Science. New York, NY: Marcel Dekker, Inc. p. 1117-1119.
- Leakey, A.D., Bernacchi, C.J., Dohleman, F.G., Ort, D.R. 2004. Will photosynthesis of maize (zea mays) in the U.S. corn belt increase in future [CO2] rich atmospheres? an analysis of diurnal courses of CO2 uptake under free-air concentration enrichment (face). Global Change Biology. 10:951-962.
- Long, S.P., Ainsworth, E.A., Rogers, A., Ort, D.R. 2004. Rising atmospheric carbon dioxide: plants face the future. Annual Reviews of Plant Biology. 55:591-628.
- Zhu, X-G., Portis Jr., A.R., Long, S.P. 2004. Would transformation of C3 crop plants with foreign Rubisco increase productivity? A computational analysis extrapolating from kinetic properties to canopy photosynthesis. Plant Cell and Environment. 27:155-165.
- HARDIN, S.C., HUBER, S.C. 2004. Proteasome activity and the post- translational control of sucrose synthase stability in maize leaves. Plant Physiology and Biochemistry. 42:197-208.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? 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) Determination of the biochemical, molecular, and genetic factors controlling tolerance and susceptibility of crop photosynthetic performance to drought, temperature extremes, and ozone; 2) Defining the key regulatory elements that control photosynthate production and partitioning and that determine sink strength; 3) Establishing the major features limiting the response of photosynthetic productivity of soybean and corn at elevated atmospheric CO2 and test potential transgenic amelioration strategies. The experimental approaches are diverse
combining biophysics, biochemistry, physiology, molecular biology, and genomics. 2. How serious is the problem? Why does it matter? 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 proposed 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 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 302, Plant Biological and Molecular Processes (70%) Photosynthesis research is the foundation for many applications in biotechnology, environmental safety, and agronomic practices. By
determining the molecular and physiological basis for limiting constraints, which are often altered and exacerbated by diverse and changing environmental factors, the research is intended to develop new methodologies for alleviating such limitations. The research thrusts have the potential to ameliorate important agricultural problems such as chilling and drought inhibition of photosynthesis and to provide novel molecular strategies for altering nutrient distribution to harvested tissues. National Program 204, Global Change (30%) Our objective to establish the major limiting features and to test potential transgenic amelioration strategies of the response of photosynthetic productivity of soybean at elevated atmospheric CO2 and the interaction with ozone pollution and drought is centrally relevant to the goals of Cropping Systems in Component III of National Program 204 concerning stresses interacting with rising carbon dioxide. 4. What were the most significant accomplishments this
past year? Photosynthesis is reversibly inhibited at moderately high temperature, which often limits crop productivity. To gain insight into the possible biochemical mechanisms, we examined the effects of high temperatures on the photosynthetic characteristics of a series of transgenic and mutant Arabidopsis plants with known alterations in either the regulation of a key photosynthetic enzyme or membrane fluidity. From the results, we conclude that increased thermal stability of the photosynthetic membranes caused no apparent changes on heat inhibition of photosynthesis below 40DGC, while the reduced activity of a key photosynthetic enzyme, potentially caused by the reduced activity of its regulatory protein, accounts for the early stages of inhibition. Identifying the primary mechanisms responsible for the inhibition of photosynthesis at higher temperatures will provide specific targets to genetically engineer in order to improve crop productivity in stressful temperature
environments. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Carbon dioxide in the atmosphere is rising globally by 0.4 percent per year. The rate of increase is expected to accelerate with 2050 concentrations projected to be 50% higher than today. Adapting the crop to these changes will require accurate information on exactly how this increase in CO2 will affect crops in the field. A new facility at Urbana, IL allows the controlled enrichment of large plots within a field crop without any enclosure. The technology consists of rings of pipes, about 20 m in diameter, that inject CO2 into the air at the height of the crop and according to wind-speed and direction, maintaining the concentration within the ring at a pre-set elevated concentration through computer feed- back control. Important trends are already emerging from our experiments showing that carbon dioxide fertilization caused a greater than 40% increase in
net carbon gain by soybean. We are also beginning to see greater above ground biomass (i.e., size of the plants) under the elevated carbon dioxide conditions and that these plants are using water more efficiently. To improve photosynthetic performance we conducted experiments designed to reveal how photosynthesis is regulated by studying the performance of algal cells during development. The results show that photosystem II is a key regulatory element during a significant period of cell development, and that electron transport is a limiting factor in photosynthesis prior to and during cell division. The results show that the factors that control photosynthetic performance switch from one rate limiting site to another during cell division. This information will help researchers understand the complex regulation of photosynthesis during plant growth. The activity of Rubisco, the enzyme responsible for capturing atmospheric carbon dioxide during photosynthesis, is a major factor
limiting photosynthesis in crop plants. The activity of Rubisco is down- regulated under limiting light conditions, thereby further limiting the rate of photosynthesis. We created transgenic plants in which Rubisco remains fully active over a wide range of light intensities by using genetic transformation to express modified forms of the regulatory protein, Rubisco activase. These unique plants provide a powerful means to delineate the significance of light regulation of Rubisco in defining plant productivity under different environmental conditions. The major harvested tissues in plants are composed of cells that must import sugars and amino acids produced by photosynthesis to support growth and development. Although we understand the basic mechanics of how sugars are moved in the plant's vascular system, the regulation of sugar allocation is not well understood because of the complicated interplay between leaves and harvested organs. Using biochemical and recombinant DNA methods, we
discovered a sucrose-sensing pathway that regulates the activity and expression level of the sucrose transport protein that mediates long-distance sugar transport in crop plants. This discovery was a major advance in this field and determining how this control pathway works could have a major impact on our ability to enhance crop yield and nutritional value. 6. What do you expect to accomplish, year by year, over the next 3 years? FY2004 Use microarrays to identify genes coordinately regulated by the sucrose-sensing regulatory pathway. Use microarrays to identify nitrogen-sensing regulatory pathways that alter nitrogen allocation to harvested organs. Analyze mutants of transgenic plants that no longer respond to high sucrose in the phloem. Develop amino acid promoter:: reporter gene constructs for genetic dissection of nitrogen signaling pathway. Determine the effects of under- and over-expressing activase and altered lipid composition in Arabidopsis mutants on the regulation of
Rubisco and its relationship to the sensitivity of photosynthesis to high temperature. Obtain the quantitative information needed to predict the likely effects of future elevated levels of CO2 and O3 on the stomatal physiology, leaf water status, canopy temperature, energy balance, and water use of soybean. Use microarrays to clone the gene for pheophorbide a oxygenase in canola. FY2005 Identify key steps in sugar and nitrogen regulatory pathways that control resource allocation. Generate transgenic plants that over-express putative regulatory genes to test impact on plant growth. Establish which domains in Rubisco activase interact with Rubisco and obtain a structure for Rubisco activase. Investigate the interaction of drought, ozone and CO2 fertilization on soybean. FY2006 Complete biochemical and molecular analysis of phloem-specific gene in assimilate partitioning. Complete biochemical and molecular dissection of sucrose signaling pathway. Identify mechanism of green seed problem.
Analyze the photosynthetic properties of tobacco lines expressing the Chlamydomonas Rubisco genes under high CO2 in growth chamber and Free-Air Concentration Enrichment (FACE). Complete analysis of genetic variation in photoprotection in soybean under FACE treatments. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Transferred information about resource allocation, sucrose sensing, and nitrogen regulation of amino acid transporter gene expression to academic and industrial scientists as an invited speaker at international meetings or at U.S. universities or companies (2 presentations). Could be applied to commercial systems within 2 to 3 years if successful. It will be a critical enabling accomplishment to understand the regulation of transporter
expression by the products of photosynthesis. Research progress report about constraints in improving photosynthesis and plant productivity by altering Rubisco and its regulation of Rubisco are lack of facile technology to genetically alter or replace the protein in agronomic species and lack of comprehensive knowledge of the mechanism of regulation. NC-142 Regulation of Photosynthetic Processes (researchers from 17 State Agricultural Experiment Stations and two USDA locations). 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). News Gazette front page article on our research concerning the challenges and opportunities of crop production in our rapidly changing atmosphere. Presented research tour of SoyFACE facility and project at the University of Illinois Agronomy Day. Broadcast of an interview for NPR affiliate public radio
station WFPL in Louisville "Home Grown" program about our work on the effect of cool tempertures on biological clocks in tomato. The program aired June 28 and again on the 29th and is is posted on their website (http://www.wfpl. org/homegrown.htm)
Impacts
(N/A)
Publications
- BERNACCHI, C.J., PORTIS JR, A.R., NAKANO, H., VON CAEMMERER, S., LONG, S.P. TEMPERATURE RESPONSE OF MESOPHYLL CONDUCTANCE, IMPLICATIONS FOR THE DETERMINATION OF RUBISCO ENZYME KINETICS AND FOR LIMITATIONS TO PHOTOSYNTHESIS IN VIVO. PLANT PHYSIOLOGY. 2002. v. 130. p. 1992-1998.
- TUCKER, D.E., ORT, D.R. LOW TEMPERATURE INDUCES EXPRESSION OF NITRATE REDUCTASE THAT TEMPORARILY OVERRIDES CIRCADIAN REGULATION OF NITRATE REDUCTASE ACTIVITY IN TOMATO. PHOTOSYNTHESIS RESEARCH. 2002. v 74. p. 1-9.
- ORT, D.R. CHILLING-INDUCED LIMITATIONS OF PHOTOSYNTHESIS IN WARM CLIMATE PLANTS: CONSTRASTING MECHANISMS. PLANT PHYSIOLOGY AND BIOCHEMISTRY. 2003. v. 40. p. 7-18.
- PORTIS JR, A.R., SALVUCCI, M.E. THE DISCOVERY OF RUBISCO ACTIVASE - YET ANOTHER STORY OF SERENDIPITY. PHOTOSYNTHESIS RESEARCH. 2002. v. 73. p. 257- 264.
- ZHANG, X., EWY, R.G., WIDHOLM, J.M., PORTIS JR, A.R. COMPLEMENTATION OF THE NUCLEAR ANTISENSE RBCS GENE INTO THE TOBACCO PLASTID GENOME. PLANT AND CELL PHYSIOLOGY. 2002. V. 43. P. 1302-1313.
- PORTIS JR, A.R. RUBISCO ACTIVASE --- RUBISCO'S CATALYTIC CHAPERONE. PHOTOSYNTHESIS RESEARCH. 2003. v. 75. p. 11-27.
- Houtz, R.L., Portis Jr., A.R. 2003. The life of ribulose 1,5-biphosphate carboxylase/oxygenase - posttranslational facts and mysteries. Archives of Biochemistry and Biophysics. 414:150-158.
- RANSOM-HODGKINS, W., VAUGHN, M., BUSH, D.R. PROTEIN PHOSPHORYLATION MEDIATES A KEY STEP IN SUCROSE-REGULATION OF A PROTON-SUCROSE SYMPORTER. PLANT PHYSIOLOGY SUPPLEMENT. 2003. v. 218. p. 121-130.
- WU, G., ORT, D.R. MUTATION IN THE -SUBUNIT OF CHLOROPLAST ATP SYNTHASE IN ARABIDOPSIS ALTERS THE REDOX REGULATORY PROCESS AND PHOTOSYNTHESIS UNDER THE LIGHT. PLANT PHYSIOLOGY SUPPLEMENT. 2003. Abstract. p. 103.
- BERNACCHI, C.J., MORGAN, P.B., ORT, D.R., LONG, S.P. PHOTOSYNTHETIC RESPONSES OF POPLAR AND SOYBEAN TO FREE AIR ENRICHMENT OF CO2. PLANT PHYSIOLOGY SUPPLEMENT. 2003. Abstract p. 92.
- LEAKEY, A.D., BERNACCHI, C.J., LONG, S.P., ORT, D.R. WILL PHOTOSYNTHESIS OF MAIZE (ZEA MAYS) IN THE U.S. CORN BELT INCREASE IN FUTURE [CO2] RICH ATMOSPHERES? AN ANALYSIS OF DIURNAL COURSES OF CO2 UPTAKE UNDER FREE-AIR CONCENTRATION ENRICHMENT (FACE). PLANT PHYSIOLOGY SUPPLEMENT. 2003. Abstract. p. 45.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? 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) Determination of the biochemical, molecular and genetic factors controlling tolerance and susceptibility of crop photosynthetic performance to drought, temperature extremes, and ozone; 2) Defining the key regulatory elements that control photosynthate production and partitioning and that determine sink strength; 3) Establishing the major features limiting the response of photosynthetic productivity of soybean and corn at elevated atmospheric CO2 and test potential transgenic amelioration strategies. The experimental approaches are diverse combining
biophysics, biochemistry, physiology, molecular biology and genomics. 2. How serious is the problem? Why does it matter? 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 depend on chemical applications. Since photosynthesis underlies each of these goals, our proposed 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. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program 302, Improving Plant Biological and Molecular Processes Photosynthesis research is the foundation for many applications in biotechnology, environmental safety, and agronomic practices. By determining the molecular
and physiological basis for limiting constraints, that are often altered and exacerbated by diverse and changing environmental factors, the research is intended to develop new methodologies for alleviating such limitations. The research thrusts have the potential to ameliorate important agricultural problems such as chilling and drought inhibition of photosynthesis and to provide novel molecular strategies for altering nutrient distribution to harvested tissues. National Program 204, Global Change Our objective to establish the major limiting features and to test potential transgenic amelioration strategies of the response of photosynthetic productivity of soybean at elevated atmospheric CO2 and the interaction with ozone pollution and drought is centrally relevant to the goals of Cropping Systems in Component III of NP 204 concerning stresses interacting with rising carbon dioxide. 4. What was your most significant accomplishment this past year? A. Single most significant
accomplishment during FY 2002: Determining the factors that control photosynthetic performance is an important goal in plant research. We are engaged in a project to model photosynthesis from the molecular to the canopy level designed to provide a tool to identify metabolic processes and proteins that limit photosynthetic performance under diverse environmental conditions. This year we developed a model of canopy photosynthesis that predicts the impact of photoinhibition on canopy photosynthesis. This model shows that the exposure of leaves to alternating high and low light in a canopy reduces carbon uptake and provides researchers a means for the intelligent design of genetically modified crops that is beyond experimentally based technologies because of the complexity of canopy dynamics. B. Other significant accomplishment(s), if any: none. C. Significant activities that support special target populations: none. 5. Describe your major accomplishments over the life of the project,
including their predicted or actual impact? Increasing the activity of Rubisco by replacement of the endogenous protein with a foreign one via genetic engineering has great potential to increase the productivity of many agricultural crops. In collaboration with Jack Widholm (Univ. of Illinois) we incorporated a gene encoding one of the Rubisco subunits, which is located in the nuclear genome, into the chloroplast DNA of tobacco plants defective in the expression of this subunit and exhibiting reduced photosynthesis. Transgenic plants were obtained in which messenger RNA for this subunit was increased many-fold, but increased expression of the protein itself was not obtained. The results demonstrate that unknown factors are limiting translation/assembly of Rubisco using this RNA and thus relocation of the gene encoding this subunit to the chloroplast where the other subunit gene is located, may not be a feasible way to express a foreign Rubisco in a simple manner. We recently
discovered that sucrose regulates the transport activity of the sucrose transport protein that moves sugar from the leaves to harvested organs. This year we started two long-term genetic experiments to determine how this system works. We isolated mutant Arabidopsis plants that no longer respond to sucrose analogues, and we also created a promoter::reporter transgenic plant that we can use to identify critical steps in the sucrose-signaling pathway. This pathway appears to be a major control point in assimilate partitioning and dissecting how it works will allow plant scientists to develop methods to control carbon allocation in crops which could have a dramatic impact on yield. Other major accomplishments over the life of the project, including their predicted or actual impact are: Carbon dioxide in the atmosphere is rising globally by 0.4% per year. The rate of increase that is expected to accelerate, with 2050 concentrations projected be 50% higher than today. Adapting the crop
to these changes will require accurate information on exactly how this increase in CO2 will affect crops in the field. A new facility at Urbana, IL allows the controlled enrichment of large plots within a field crop without any enclosure. The technology consists of rings, about 20 m in diameter, of pipes that inject CO2 into the air at the height of the crop and according to wind-speed and direction, maintaining the concentration within the ring at a pre-set elevated concentration through computer feed-back control. Important trends that are already emerging from our experiments show carbon dioxide fertilization caused a greater 40% increase in net carbon gain by soybean. We are also beginning to see greater above ground biomass (i.e., size of the plants) under the elevated carbon dioxide conditions and that these plants are using water more efficiently. To improve photosynthetic performance we performend experiments designed to reveal how photosynthesis is regulated by studying
the performance of algal cells during development. The results show that photosystem II is a key regulatory element during a significant period of cell development, and that electron transport is a limiting factor in photosynthesis prior to and during cell division. The results show that the factors that control photosynthetic performance switch from one rate limiting site to another during cell division. This information will help researchers understand the complex regulation of photosynthesis during plant growth. The activity of Rubisco, the enzyme responsible for capturing atmospheric carbon dioxide during photosynthesis, is a major factor limiting photosynthesis in crop plants. The activity of Rubisco is down- regulated under limiting light conditions, thereby further limiting the rate of photosynthesis. We created transgenic plants in which Rubisco remains fully active over a wide range of light intensities by using genetic transformation to express modified forms of the
regulatory protein, Rubisco activase. These unique plants provide a powerful means to delineate the significance of light regulation of Rubisco in defining plant productivity under different environmental conditions. The major harvested tissues in plants are composed of cells that must import sugars and amino acids produced in photosynthesis to support growth and development. Although we understand the basic mechanics of how sugars are moved in the plant's vascular system, the regulation of sugar allocation is not well understood because of the complicated interplay between leaves and harvested organs. Using biochemical and recombinant DNA methods, we discovered a sucrose-sensing pathway that regulates the activity and expression level of the sucrose transport protein that mediates long-distance sugar transport in crop plants. This discovery was a major advance in this field and determining how this control pathway works could have a major impact on our ability to enhance crop yield
and nutritional value. 6. What do you expect to accomplish, year by year, over the next 3 years? FY2003 Determine how Rubisco is regulated in response to light in plants like tobacco, which contain only a redox-insensitive activase isoform. Characterize sucrose-symporter promoter and reporter gene transgenic plants to use in genetic dissection of sucrose signaling pathway. Identify all genes that respond to changing levels of nitrogen metabolites (expected to be around 2000 genes). Determine growth characteristics of gene "knock-outs" of previously un- described members of the sucrose transporter gene family to understand their role in plant growth. Identify protein phosphatase that controls chill-sensitive circadian pattern of nitrate reductase activity in tomato. Use microarrays to clone the gene for pheophorbide a oxygenase in canola. FY2004 Use microarrays to identify genes coordinately regulated by the sucrose- sensing regulatory pathway. Use microarrays to identify
nitrogen-sensing regulatory pathways that alter nitrogen allocation to harvested organs. Analyze mutants of transgenic plants that no longer respond to high sucrose in the phloem. Develop amino acid promoter:: reporter gene constructs for genetic dissection of nitrogen signaling pathway. Determine the effects of under- and over-expressing activase and altered lipid composition in Arabidopsis mutants on the regulation of Rubisco and its relationship to the sensitivity of photosynthesis to high temperature. Obtain the quantitative information needed to predict the likely effects of future elevated levels of CO2 and O3 on the stomatal physiology, leaf water status, canopy temperature, energy balance, and water use of soybean. FY2005 Identify key steps in sugar and nitrogen regulatory pathways that control resource allocation. Generate transgenic plants that over-express putative regulatory genes to test impact on plant growth. Establish which domains in Rubisco activase interact with
Rubisco and obtain a structure for Rubisco activase. Investigate the interaction of drought, ozone and CO2 fertilization on soybean. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Transferred information about resource allocation, sucrose sensing, and nitrogen regulation of amino acid transporter gene expression to academic and industrial scientists as an invited speaker at international meetings or at US universities or companies (6 presentations). Could be applied to commercial systems within 3 years if successful. It will be a critical enabling accomplishment to understand the regulation of transporter expression by the products of photosynthesis. Research progress report about constraints in improving photosynthesis and plant productivity by altering Rubisco and its regulation of Rubisco
are lack of facile technology to genetically alter or replace the protein in agronomic species and lack of comprehensive knowledge of the mechanism of regulation., NC-142 Regulation of Photosynthetic Processes, November 17-18, 2001, Raleigh NC (researchers from 17 state Ag. Exp. Stations and two USDA locations). 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) News Gazette front page article on "Modeling Photosynthesis". Chloroplast transformation work with a key enzyme in the synthesis of tryptophan (reported last year) featured at the ScienceNOW (September 12, 2001) and AgBiotechNet web sites and reported in New&Comments, TRENDS in Genetics, Vol 12, December 2001.. Presented reseearch tour of SoyFACE facility and project to Illinois Soybean Association.
Impacts
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Publications
- Zhang, X-H., Portis, A.R., Jr., Wildhold, J.M. Plastid transformation of soybean suspension cultures, Journal of Plant Biotechnology. 2001. v. 3 p. 39-44.
- Zhang, N., Kallis, R.P., Ewy, R.G., Portis, A.R., Jr. Light modulation of Rubisco in Arabidopsis requires redox regulation of the larger Rubisco activase isoform. Proceedings of the National Academy of Sciences USA. 2002. v. 99. p. 3330-3334.
- Wang, D., Portis, A.R., Jr. Light modulation of Rubisco activation in species without a larger activase isoform - existence of an activase regulatory protein? American Society of Plant Biologists Annual Meeting Abstract. 2002. Available from http://abstracts.aspb. org/pb2002/public/[2002].
- Kim, K., Portis, A.R., Jr. Oligomerization state of Rubisco activase revealed by dynamic light scattering. Plant Biology 2002 Program. American Society of Plant Biologists Annual Meeting Abstract. 2002. Available from http://abstracts.saspb.org/pb.org/pb2002/public/[2002].
- Tucker, D.E., Ort, D.R. Low temperature induces expression of nitrte reductase in tomato that temporarily overrides circadian regulation of activity. Photosynthesis Research. 2002. v. 74. p. 1-9
- Vaughn, M.W., Harrington, G.N., Bush, D.R. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem. Proceedings of the National Academy of Science USA. 2002. v. 99. p. 10876- 10880.
- Zhang, N., Schurmann, P., Portis, A.R., Jr. Characterization of the regulatory function of the 46-kDa isoform of Rubisco activase from Arabidopsis. Photosynthesis Research. 2001. v. 68. p. 29-37.
- Ort, D.R., Baker, N.R. A photoprotective role for O2 as an alternative electorn sink inphotosynthesis? Current Opinion Plant Biology. 2002. v. 5. p. 193-198.
- Wang, Q., Whitmarsh, C.J. Investigating oxygen evolution in the absence of photosystem I in the cyanobacterium Synechocystis 6803I. PS2001 Proceedings: 12th International Congress on Photosynthesis. 2001. CSIRO Publishing: Melbourne, Australia, 2001. Available from httpp://www.publish. csiro.au/ps 2001.
- Kashino, K., Lauber, W.M., Carroll, J.A., Wang, Q., Whitmarsh, C.J., Satoh, K., Pakrasi, H.B. Characterization of purified His-tagged CP47-containing photosystem II complexes from a cyanobacterium (Synechocystis 6803). PS2001 Proceedings: 12th International Congress on Photosynthesis. 2001. CSIRO Publishing: Melbourne, Australia, 2001. Available from http://www. publish.csiro.au/ps2001.
- Soukupoval, J., Smatannova, S., Jergorov, A., Ferimazova, H., Kupper, H., Setlik, S., Maldener, Whitmarsh, C.J., Trtilek, M., Melichar, M., Nedbal, L. Early detection of biotic and abiotic stress by kinetic imaging of chlorophyll flourescence. I PS2001 Proceedings: 12th international Congress on Photosynthesis.2001. CSIRO Publishing: Melbourne, Australia, 2001. Available from http://www.publish.csiro.qu/ps2001.
- Kashino, Y., Lauber, W.M., Carroll, J.A., Wang, Q., Whitmarsh, C.J., Satoh, K., Pakrasi, H.B. Proteomic analysis of a highly active photosystem II preparation from the cyanobacterium Synechocystis sp. PCC 5803 reveals the presence of novel polypeptides. Biochemistry. 2002. v. 41. p. 8004-8012.
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