Progress 10/01/12 to 09/30/17
Outputs
Target Audience:The NC-1200 regional project is composed of 25 scientists from 16 land grant institutions. Funds from this project support the travel of one WSU scientist to attend the annual meeting, which was hosted by Virginia Tech. University at Arlington, VA on November 10-12, 2017. At the meeting, scientists described their latest research developments in the general area of photosynthesis and arrange collaborative research efforts for the following year. The opportunity for WSU scientists to participate at the annual meeting enables them to learn the most recent advances in photosynthesis research, which enables them to be competitive in extramural grant funding from federal agencies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Although not supported by the NC1200 project, postdocs, graduate students and visiting scientists are supported by the research activities of the three WSU participating laboratories. How have the results been disseminated to communities of interest?Information disseminated to the scientific community is via publications of research articles, book chapters, and reviews, seminar presentations at universities and private companies, and invited addresses at major conferences and symposia. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Accomplishments - Helmut Kirchhoff A new method for quantification of surface charges of thylakoid membranes was developed. Surface charges are critical for the vertical stacking of thylakoid membranes to grana and lateral protein-protein interactions in membranes. Furthermore, the surface charges in thylakoid membranes are variable and change by reversible protein phosphorylation. Based on our quantification, we now are able to predict how protein phosphorylation affects the interaction of thylakoid membrane proteins and grana stacking. Many photosynthetic protein complexes distribute laterally inhomogeneous between stacked and unstacked thylakoid membrane regions. This spatial separation of protein complexes controls essential functions of energy transformation and was likely a main driving force for the evolution of stacked grana. However, the lateral distribution of central cytochrome b6f (cyt b6f) complex is unclear. Our new ultrastructural data of the thylakoid membrane architecture derived from state-of-the-art electron microscopy revealed small changes in grana stacking induced by light. Based on this structural information, we developed a model that explains modulation of cyt b6f complex accessibility to stacked membrane regions caused by steric constraints. This model forms a basis to understand the dynamic reorganization of the cyt b6f complex in thylakoid membranes that controls electron transport processes (e.g. linear and cyclic electron transport). We have established electron microscopic analysis on Arabidopsis leaf discs by using high-pressure freezing and freeze substitution. This procedure preserves the structural features with high confidence (minimizing fixation artifacts). It will be used to study how recently discovered ion transporters/channels determine architectural thylakoid membrane dynamics induced by light. Therefore, we have also produced higher order ion transporter/channel mutants to study how individual and different combinations of transporters/channels effect structural thylakoid membrane alterations. Accomplishments - Tom Okita RNAs are transported and localized to specific subdomains of the cortical ER by their interaction with multi-protein complexes containing RNA binding proteins and accessory proteins involved in membrane vesicular trafficking. The protein interacting network for three RNA binding proteins, required for RNA localization, was generated. The network consisted of three overlapping nodes consisting of about 30 RNA binding proteins and accessory proteins. Rice lines containing mutations in RBP-P, an RNA binding protein required for glutelin RNA localization, were grown for several generations and shown to exhibit altered localization of storage protein mRNAs on the cortical ER by in situ RT-PCR. Past studies have demonstrated an unexpected interaction between the phosphorylase enzyme, Pho1, and PsaC, a major component of photosystem I. The interaction of Pho1 to PsaC suggests that Pho1 may control PSI activity and, in turn, plant growth. To obtain direct evidence for such a relationship, we employed the LED-based flash spectrophotometer to measure the in vivo redox changes in P700 to study PSI performance of wildtype, BMF136 pho1 mutant and transgenic BMF136 pho1 rice plants expressing wildtype Pho1 and Pho1ΔL80 gene sequences. Preliminary results indicate that the Pho1ΔL80 plant lines exhibit enhanced PSI quantum yield activity (YI) than the BMF mutant line, BMF expressing wildtype Pho1 or wildtype. Accomplishments - Asaph Cousins We have used leaf carbon isotope composition (δ13C) to determine how water use efficiency differs in two C4 species of Setaria species. This research helped to better understand the relationship of leaf carbon isotopes and the influence intrinsic water use efficiency to whole plant water use efficiency. We also used ?13C and maize genetic diversity to explore biochemical and post-photosynthetic factors that may influence ?13C. Our findings indicate that the observed variation in ?13C across diverse maize lines is likely driven by differences in CO2 availability and not photosynthetic or respiratory metabolism. Little is know about intraspecific variation mesophyll conductance (gm), which describes the movement of CO2 from the intercellular air spaces to the site of initial carboxylation in the mesophyll, about in C4 plants. To address these questions, gm was measured on five maize lines in response to CO2, employing three different estimates of gm. Our results provide strong support for a CO2 response of gm in Zea mays, and indicate that gm in maize is likely driven by anatomical constraints rather than biochemical limitations. The CO2 response of gm indicates a potential role for CO2-transporting aquaporins in C4-gm. These results also suggest that water-use efficiency could be enhanced in C4 species such as maize by targeting gm. If gm were to limit C4 photosynthesis, it would likely be at low CO2 concentrations (pCO2); however, data on C4-gm across pCO2 are scarce. We describe the response of C4-gm to short-term variation in pCO2, at three temperatures in Setaria viridis, and at 25 ºC in Zea mays. Additionally, we quantify across pCO2 the potential limitations to photosynthesis imposed by stomata, mesophyll and carbonic anhydrase (CA) and the effect of finite gm calculations of leakiness. In both species, gm increased with decreasing pCO2. At pCO2 below ambient, photosynthetic rate was limited by CO2 availability. In this case, the limitation imposed by mesophyll was similar or slightly lower than stomata limitation. At very low pCO2, CA further constrained photosynthesis. High gm could increase CO2 assimilation at low pCO2 and improve photosynthetic efficiency under situations when CO2 is limited, such as drought. Finite gm increased estimates of leakiness over values derived with gm infinite in Setaria but not in Zea.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2017
Citation:
Koochak, M. Li, H. Kirchhoff (2017) Thylakoid membrane dynamics in higher plants. In: Photosynthesis and Bioenergtics. (Editors, A.V. Ruban, J. Barber) World Scientific Publishing Co, Singapore, 221-242
- Type:
Book Chapters
Status:
Accepted
Year Published:
2017
Citation:
H. Kirchhoff, R. Yarbrough, Evaluation of lipids and membranes for the study of photosynthetic superorganization. In: Methods in Molecular Biology. (Editor, S. Covshoff) Springer Press, accepted
- Type:
Other
Status:
Published
Year Published:
2017
Citation:
H. Kirchhoff, M. Li, S. Puthiyaveetil (2017) Sublocalization of cytochrome b6f complexes in photosynthetic membranes. Trends Plants Science 22, 574-582
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
M. Gargouri, P.D. Bates, J.J. Park, H. Kirchhoff, D.R. Gang (2017) Functional photosystem I maintains proper energy balance during nitrogen depletion in Chlamydomonas reinhardtii, promoting triacylglycerol accumulation. Biotechnology for Biofuels 10, 89
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
S. Puthiyaveetil, B. von Oort, H. Kirchhoff (2017) Surface charge dynamics in photosynthetic membranes and its structural consequences. Nature Plants 3, 17020
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Chou, H.-L., L. Tian, T. Kumamaru, and T.W. Okita (2017) Multifunctional RNA binding protein OsTudor-SN in storage protein mRNA transport and localization. Plant Physiology 175:1608-1623.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Ellsworth P.Z., Ellsworth P.V., Cousins A.B. (2017) Measurements of Leaf Oxygen and Carbon Isotopic Signatures to estimate Transpiration Efficiency in the C4 Grasses Setaria viridis and Setaria italica. Journal of Exp Botany 68, 3513-3528
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Kolbe A.R., Studer A.J., Cousins A.B. (2017) Mesophyll conductance in Zea mays responds transiently to CO2 availability: implications for transpiration efficiency in C4 crops. New Phytologist DOI: 10.1111/nph.14942
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Kolbe A.R., Studer A.J., Cousins A.B. (2017) Biochemical and transcriptomic analysis of maize diversity to elucidate drivers of leaf carbon isotope composition Functional Plant Biology https://doi.org/10.1071/FP17265
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Ubierna N., Gandin A., Boyd R.A., Cousins A.B. (2017) Temperature response of mesophyll conductance in three C4 species calculated with two methods: 18O discrimination and in-vitro Vpmax New Phytologist 214, 66-80
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The NC-1200 regional project is composed of 25 scientists from 16 land grant institutions. Funds from this project support the travel of one WSU scientist to attend the annual meeting, which was held in Bozeman Montana on September 1st 2016. At the meeting, scientists described their latest research developments in the general area of photosynthesis and arrange collaborative research efforts for the following year. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?From the Cousins lab 4 postdocs, 5 graduate students and one undergraduate research intern were trained in this research project. How have the results been disseminated to communities of interest?Research products were disseminated at the NC-1200 annual meeting and by publications of research articles and book chapters. What do you plan to do during the next reporting period to accomplish the goals? Cousins Lab: Drought is a major worldwide agricultural problem. Therefore, selection for increased water use efficiency (WUE) in crop species is an important trait for plant breeding programs. For example, increased CO2 diffusion from the leaf intercellular air space to site of carboxylation (gm) is a potential trait for increasing net rates of CO2 assimilation (Anet), WUE and crop productivity. Unfortunately, traditional phenotyping for WUE requires laborious and costly measurements that are not feasible on a large numbers of plants. However, WUE and leaf carbon isotopic composition (δ13C) are correlated through their relationships with intercellular to ambient CO2 partial pressures (Ci/Ca) and using δ13C as a proxy for high throughput phenotyping for WUE in both C3 and C4 species. To validate this method the Cousins laboratory is testing the relationship between δ13Cleaf and gas exchange measurements of WUE in C3 species such as wheat and rice as well as C4 species such as maize and sorghum. Additionally, they are using measurements of leaf CO2 isotope exchange to determine the biochemical and leaf anatomical traits influencing gm in both C3 and C4 plants.
Impacts
What was accomplished under these goals?
Cousins Lab: For a Setaria RIL density and drought experiments we finished processing samples from three years of prior field seasons for analysis of δ13C and leaf nitrogen content. There are a total of 839 and 757 samples for drought and density field experiments, respectively. We also analyzed 358 leaf samples from the RIL drought experiment and another 507 samples from the S. viridis accession drought experiment that were conducted by the Baxter lab in the Danforth phenotyper. Additionally, water from 150 soil cores, 90 root crowns and 90 leaves from the drought experiment have been analyzed for δ18O. QTLs that contribute to variation in foliar δ13C and δ18O have been mapped to compare QTLs under high/low density and well-watered/drought conditions using QTL pipeline. QTL were identified for 13C that aligned with WUE and soon 13C GWAS data for accessions coming soon. We were able to identify QTLs for carbon isotope composition and water use efficiency in the C4 grass Setaria. This information will help determine and be able to modify the biochemical and regulatory factors that impact the photosynthetic capture and photorespiratory release of C02. We published 9 peer-reviewed manuscripts in 2016 related to our major goals of the project. The PI, students and postdocs presented 24 oral or poster presentations during 2016.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Uberna N., Gandin A., Boyd R.A., Cousins A.B. (2016) Temperature response of mesophyll conductance in three C4 species calculated with two methods: 18O discrimination and in-vitro Vpmax New Phytologist Doi: 10.1111/nph.14359
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Shengyang J., Sun J., Wunder T., Tang D., Cousins A.B., Sze S.K., Mueller-Cajar O., Gao Y.G. (2016) Structural insights into the LCIB protein family reveals a new group of ?-carbonic anhydrases PNAS 113, 14716-14721
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Studer A.J., Schnable J.C., Weissmann S., Kolbe A.R., McKain M.R., Shao Y., Cousins A.B., Kellogg E.A. & Brutnell T.P. (2016) The draft genome of the C3 panicoid grass species Dichanthelium oligosanthes. Genome Biol, 17, 223.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Ma J.Y., Sun W., Koteyeva N.K., Voznesenskaya E., Stutz S.S., Gandin A., Smith-Moritz A.M., Heazlewood J.L. & Cousins A.B. (2016) Influence of light and nitrogen on the photosynthetic efficiency in the C4 plant Miscanthus x giganteus. Photosynth Res. DOI:10.1007/s11120-016-0281-7
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Zia A., Walker B.J., Oung H.M., Charuvi D., Jahns P., Cousins A.B., Farrant J.M., Reich Z., Kirchhoff H. (2016) Protection of the photosynthetic apparatus against dehydration stress in the resurrection plant Craterostigma pumilum. Plant Journal, 87, 664-680.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Ellsworth P.Z., Cousins A.B. (2016) Carbon isotopes and water use efficiency in C4 plants. Curr Opin Plant Biol, 31, 155-161.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Wang M., Ellsworth P.Z., Zhou J., Cousins A.B., Sankaran S. (2016) Evaluation of water-use efficiency in foxtail millet (Setaria italica) using visible-near infrared and thermal spectral sensing techniques. Talanta, 152, 531-539.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Koteyeva N.K., Voznesenskaya E.V., Berry J.O., Cousins A.B., Edwards G.E. (2016) The unique structural and biochemical development of single cell C4 photosynthesis along longitudinal leaf gradients in Bienertia sinuspersici and Suaeda aralocaspica (Chenopodiaceae). J Exp Bot, 67, 2587-2601.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Lin H., Karki S., Coe R.A., Bagha S., Khoshravesh R., Balahadia C.P., Ver Sagun J., Tapia R., Israel W.K., Montecillo F., de Luna A., Danila F.R., Lazaro A., Realubit C.M., Acoba M.G., Sage T.L., von Caemmerer S., Furbank R.T., Cousins A.B., Hibberd J.M., Quick W.P. & Covshoff S. (2016) Targeted Knockdown of GDCH in Rice Leads to a Photorespiratory-Deficient Phenotype Useful as a Building Block for C4 Rice. Plant Cell Physiol, 57, 919-932.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The NC-1200 regional project is composed of 25 scientists from 16 land grant institutions. Funds from this project support the travel of one WSU scientist to attend the annual meeting, which was held in St. Louis Missouri on November 7, 2015. At the meeting, scientists described their latest research developments in the general area of photosynthesis and arrange collaborative research efforts for the following year. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?2postdocs, 3 graduate students and one undergraduate research intern were trained in this research project. How have the results been disseminated to communities of interest?Research products were disseminated at the NC-1200 annual meeting and by publications of research articles and book chapters. What do you plan to do during the next reporting period to accomplish the goals? Determine factors that control dynamic swelling/shrinkage of the thylakoid lumen. This includes studies on channel mutants, kinase/phosphatase mutants, and the newly discovered CURT protein family (required for bending grana margins). Measuring detailed light dependence of swelling and shrinkage and determine the kinetics of these processes Determine the role of dynamics of the lipid matrix composition on supramolecular protein organization in grana. Continue studies to identify the constraints that limit maximum carbon flow into starch in the rice lines over-producing ADPglucose Continue studies to identify proteins involved in RNA transport and localization and the sets of RNAs that are targeted by these proteins.
Impacts
What was accomplished under these goals?
For Objective 1 (from CO-PI Helmut Kirchhoff): The structure of stacked grana thylakoid membranes in plants is not static but highly dynamic, changing its shape in response to environmental cues. These architectural dynamics of the grana thylakoids are involved in regulating and maintaining photosynthetic performance. Therefore, the knowledge of how thylakoid membranes change their shape and identification of functional consequences of structural alterations is required for an in-depth understanding of photosynthetic energy transformation. We have found that light causes swelling of the thylakoid lumen. This is a paradigm shift since previous studies postulated light-induced shrinkage of the lumen. The swelling of the lumen activates plastocyanin-dependent electron transport by facilitating its diffusion through the crowded luminal space. We identified that under high light, only the margins of the grana stacks swell but the grana core remains tightly stacked. This preferential swelling of the grana periphery is important for protein repair because it allows better access of proteases to damaged PSII. These proteases catalyze degradation of damaged D1, i.e. a critical step in the PSII repair. The proteins in grana (PSII, LHCII) can sometimes rearrange from disordered to highly ordered, semicrystalline structures. The role of this supramolecular reorganization is not known. By using a model plant that constitutively has a high abundance of semiscrystalline protein arrays, we found that protein ordering facilitates diffusion of small lipophilic metabolites (plastoquinone, xanthophylls) but impairs mobility of larger protein supercomplexes. Thus, dynamic reorganization of protein order in grana fine-tunes diffusion dependent electron transport and photoprotection or protein repair. For Objective 3 (from PI Thomas Okita): The sink strength of developing cereal grains is dictated by the conversion of transported photoassimilates into starch and protein, the major storage reserves. Current efforts have been directed at understanding how starch and proteins are synthesized and stored in developing rice seeds. Specifically, we are interested in elucidating the role of the regulatory enzymes, ADPglucose pyrophosphorylase (AGPase) and starch phosphorylase 1 (Pho1) in starch biosynthesis and how storage protein synthesis is controlled by the localization of RNAs on distinct subdomains of the cortical endoplasmic reticulum. Using isothermal titration calorimetry, the wildtype heterotetrameric (SWTLWT) AGPase was shown to possess two distinct ATP binding sites, whereas the homotetrameric LS and SS variant forms only exhibited properties of one of the two binding sites. EM1093 rice line which produced shrunken seeds was found to contain a mutation in the Brittle1 gene, which codes for the ADPglucose transporter. Analysis of wildtype and ADPglucose-overproducing rice lines expressing OsBt1 indicate that BT1 transport activity does not limit starch synthesis. RNAseq analysis of transgenic rice lines over-producing ADPglucose and, in turn, increase starch levels, show significant re-programming of gene expression with a significant increase in RNA levels of an uncharacterized starch binding domain protein (SBDP) and reduction in RNA levels for starch synthase III. SBDP was demonstrated to interact with SSIII. The RNA binding protein, RBP-P, which interacts with the glutelin zipcode sequence was found to bind directly to NSF and three other RNA binding proteins and indirectly to Rab5. This evidence provides direct evidence for the involvement of membrane trafficking events in RNA localization.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Puthiyaveetil, S., Tsabari, O., Lowry, T., Lenhert, S., Lewis, R.R., Reich, Z., Kirchhoff, H. (2014). Compartmentalization of the Protein Repair Machinery in Photosynthetic Membranes. Proc. Natl. Acad. Sci. USA 111, 15839-15844.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Puthiyaveetil, S., Woodiwiss, T., Knoerdel, R., Zia, A., Wood, M., Hoehner, R., Kirchhoff, H. (2014). Significance of the Photosystem II Core Phosphatase PBCP for Plant Viability and Protein Repair in Thylakoid Membranes. Plant Cell Physiology 55, 1245-1254.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Kirchhoff H. (2014). The mechanisms of repair. Research Media (UK), Nov 2014, 17-19.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Kirchhoff H. (2014). Diffusion of molecules and macromolecules in thylakoid membranes. Special issue on Dynamics and Ultrastructure of Bioenergetic Membranes and their Components. Biochimica et Biophysica Acta-Bioenergetics 1837, 495-502.
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
Cakir, B., Tuncel, A., Hwang, S.-K., and Okita, T.W. (2015a). Increase of grain yields by manipulating starch synthesis. In Starch: Metabolism and Structure, Y. Nakamura, ed (Japan: Springer), pp. 371-395.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Cakir, B., Tuncel, A., Green, A.R., Koper, K., Hwang, S.-K., Okita, T.W., and Kang, C.-H. (2015b). Substrate binding properties of ADP-glucose pyrophosphorylase determined by isothermal titration calorimetry. FEBS Lett. 589, 1444-1449.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Cakir, B., Shiraishi, S., Tuncel, A., Matsusaka, H., Satoh, R., Singh, S., Hwang, S.-K., Satoh, H., and Okita, T.W. (2015c). Analysis of the rice ADPglucose transporter (OsBT1) indicates the presence of regulatory processes in the amyloplast stroma that control ADPglucose flux into starch. submitted.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Wensel, P., Helms, G., Hiscox, B., Davis, W.C., Kirchhoff, H., Bule, M., Yu, L., Chen, S. (2014). Isolation, characterization, and validation of oleaginous, multi-trophic, and haloalkaline-tolerant microalgae for two-stage cultivation. Algal Research 4, 2-11.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Kirchhoff H. (2014). Structural changes of the thylakoid membrane network induced by high-light stress in plant chloroplasts. Philosophical Transactions B 369, 20130225.
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Kirchhoff H. (2014). Dynamic architecture of plant photosynthetic membranes. In: Advances in Plant Biology: Plastid Biology (Editors: F.-A. Wollman and S. Theg), Springer Press, 129-154.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Hwang, S.-K., Singh, S., Satoh, H., and Okita, T.W. (2015). Plastidial starch phosphorylase from the starch storage tissue of rice is highly active at low temperature. Submitted.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Offermann, S., Frisco, G., Doroshenk, K.A., Sun, Q., Sharpe, R.M., Okita, T.W., Edwards, G.E., and van Wijk, K.J. (2015). Development and subcellular organization of single-cell C4 photosynthesis in Bienertia sinuspersici determined by large scale proteomics and cDNA assembly from 454 DNA sequencing. J. Proteome Res. 14, 2090-2108.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The NC-1200 regional project is composed of 25 scientists from 16 land grant institutions. Funds from this project support the travel of one WSU scientist to attend the annual meeting, which was held in Kansas City on November 22, 2014. At the meeting, scientists described their latest research developments in the general area of photosynthesis and arrange collaborative research efforts for the following year. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Five graduate students were trained in this research project. How have the results been disseminated to communities of interest? Research products were disseminated at the NC-1200 annual meeting and by publications of research articles and book chapters What do you plan to do during the next reporting period to accomplish the goals? Characterize the biochemical and structural transitions along a longitudinal leaf gradient in single cell C4 species, which leads to the development of C4 photosynthesis. • Complete a proteomics study on the single cell C4 system in Bienertia sinuspersici to identify proteins recruited for function of an NAD-ME type C4 cycle during development, and in mature leaves. • Complete a study on the consequences of photorespiratory release of CO2 in rice by studying the relationship between the kinetic properties of Rubisco, the CO2 compensation point and refixation of photorespired CO2. • Identify proteins that interact with the starch binding domain protein and elucidate their role in starch biosynthesis. • Characterize rice transgenic lines expressing wildtype Pho1 and Pho1Δ80. • Identify and characterize rice RBP mutants and assess their potential role in RNA localization.
Impacts
What was accomplished under these goals?
Accomplishments: Objective 2: Photosynthetic Capture and Photorespiratory Release of CO2 1. The cell specific expression of biochemistry to form C4 photosynthesis was found to occur very early during leaf development in C4 species in the eudicot family Cleomaceae prior to structural differentiation. In Cleome angustifolia and C. gynandra development of the C4 system occurs similarly, irrespective of these species having very different types of Kranz anatomy, different ontogenetic origins of BS and M, and independent evolutionary origins of C4 photosynthesis. 2. Besides C4 plants having high levels of phosphoenolpyruvate carboxylase (PEPC), the C4 type enzyme has altered kinetic properties. There is evidence for this in C4 monocots, with positive selection occurring for certain amino acid residues. In a study of PEPC among species in the eudicot family Chenopodiaceae, we find differences. There is divergence in amino acid substitutions indicating the same substitutions found in monocots are not required in PEPC for function of C4 photosynthesis. 3. A unique structural form of C4 photosynthesis occurs in some species in the family Chenopodiaceae, not by the dual-cell Kranz system, but rather within individual photosynthetic cells which contain two types of chloroplasts. One chloroplast type has pyruvate, Pi dikinase for function in the carboxylation phase of the C4 cycle, the other has Rubisco, which functions in the C3 cycle, but how this differentiation occurs is unknown. In the single cell C4 species, Bienertia sinuspersici, transient expression analysis using GFP fusion constructs containing various lengths of Rubisco small subunit (RbcS) gene and the transit peptide of PPDK revealed that their import was not specific to either chloroplast type, indicating lack of selectivity in import. Post-transcription regulation is proposed to control selective expression of Rubisco. A post-transcriptional regulator factor RLSB (Rubisco large subunit mRNA binding factor) was found to be selectively localized in the Rubisco containing chloroplast. RLSB, which also is selectively localized in Rubisco containing chloroplasts in Kranz type C4 species, is proposed to mediate selective accumulation of Rubisco in chloroplasts in C4 systems, including single cell C4 species. 4. In the single-cell C4 Bienertia species, decarboxylation of C4 acids in the C4 cycle occurs via NAD-malic enzyme in mitochondria which are surrounded by Rubisco containing chloroplasts. There is interest in introducing C4 traits into C3 crops, e.g. rice. Rice mesophyll cells have chloroplasts in lobes at the cell periphery and mitochondria located internally. In a recent review we discuss features of terrestrial and aquatic plants having single cell C4 photosynthesis. Incorporation of an NAD-ME type C4 cycle in rice mesophyll cells was proposed, in the interest of providing supplemental CO2 to Rubisco to increase photosynthesis and reduce photorespiration. Objective 3: Mechanisms Regulating Photosynthate Partitioning. 1. In rice, the endosperm cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, which code for the small (S2b) and large (L2) subunits of the heterotetrameric enzyme, respectively. Several missense and null OsAGPL2 mutants were identified, which showed comparable seed weights and starch content. Kinetic analysis showed that the catalytic and regulatory properties of recombinant heterotetrameric enzymes containing the missense L2 mutations and S2b homotetramer enzyme were significantly impaired. Overall, these results showed that the L2 subunit is essential for the enzyme's catalytic and allosteric regulatory properties. 2. The catalytic activity of the major rice endosperm AGPase is controlled by redox potential. Site-directed mutagenesis studies showed that, unlike the leaf and tuber enzymes whose catalytic activity is altered by the interchain disulfide bond formation between the two small subunits, the catalytic activity of the rice endosperm cytoplasmic enzyme was regulated by the redox status of the large subunits. Site directed mutagenesis of the conserved cysteine residues at the N-terminal of the large subunit showed that C47 and C58, but not C12, are essential for proper redox-response of the enzyme. Overall, the catalytic activity of the rice endosperm cytoplasmic AGPase is regulated by allosterism and redox potential. 3. RNAseq of transgenic rice lines over-expressing a bacterial AGPase showed a pronounced elevation of transcripts for a starch binding domain protein while RNAs coding for other known starch biosynthetic enzymes were up-regulated or down-regulated depending on the RNA species. Immunoblot analysis using available antibodies showed that these changes in gene expression at the RNA level were extended to the protein level as well. 4. A unique structural feature of Pho1 that is not present in non-plant eucaryotic phosphorylases is that the higher plant enzyme contains a extra 80 amino acid peptide located near the middle of the primary sequence. Kinetic analysis of the wildtype Pho1 and Pho1 lacking the L80 peptide (PPho1Δ80) showed no significant differences in catalytic properties suggesting a potential regulatory role for L80 peptide. To elucidate the potential role of the L80 peptide, Pho1 null rice plants transformed with wildtype Pho1 and Pho1Δ80 were generated and are presently under study. 5. RNAs that code for the rice prolamine storage protein are transported and localized on the ER membrane that delimit the prolamine protein body. Previous studies have shown that a complex collection of RNA binding proteins (RBPs) specifically recognized the RNA-cis-determinants that govern RNA localization. By using a variety of techniques, we demonstrated that five of these RBPs assemble in at least three multi-protein complexes. The complexity of RBPs and their organization into different multi-protein complexes is consistent with the multiple steps required for RNA localization, a process that initiates in the nucleus and terminates by translation at the ER destination site.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Kirchhoff, H., Sharpe, R.M., Herbstova, M., Yarbrough, R., and Edwards, G.E. 2013 Differential mobility of pigment-protein complexes in granal and agranal thylakoid membranes of C3 and C4 plants. Plant Physiol 161, 497-507.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Voznesenskaya, E.V., Koteyeva, N.K., Akhani, H., Roalson, E.H., and Edwards, G.E. Structural and physiological analyses in Salsoleae (Chenopodiaceae) indicate multiple transitions among C3, intermediate and C4 photosynthesis. J Exp Bot 64, 3583-3604.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Giuliani, R., Koteyeva, N., Voznesenskaya, E., Evans, M.A., Cousins, A.B., and Edwards, G.E. 2013 Coordination of leaf photosynthesis, transpiration and structural traits in rice and wild relatives (genus Oryza). Plant Physiol 162, 1632-1651.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Suleyman, A., Shen, J-R., and Edwards, G.E. 2013 Guest Editorial. Special issues on Photosynthesis Education honoring Govindjee. Photosyn Res 116,107-110.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ocampo, G., Koteyeva, N.K., Voznesenskaya, E.V., Edwards, G.E. Sage, T.L., Sage, R.F. and Columbus, J.T. 2013. Evolution of leaf anatomy and photosynthetic pathways in Portulacaceae. Am J Bot, 100(12):2388-402.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Stutz, S.S., Edwards, G.E., Cousins, A.B. 2014 Single cell C4 photosynthesis: efficiency and acclimation of Bienertia sinuspersici to growth under low light. New Phyto 202, 220-232.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Koteyeva, N.K., Voznesenskaya, E.V., Cousins, A.B. and Edwards, G.E. 2014 Differentiation of C4 photosynthesis along a leaf developmental gradient in two Cleome species having different forms of Kranz anatomy. J Exp Bot 65, 3525-3541 doi:10.1093/jxb/eru042
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Rosnow, J., Edwards, G.E. Roalson, E.H. 2014 Evolutionary Positive selection of Kranz and nonKranz C4 phosphoenolpyruvate carboxylase amino acids in Suaedoideae (Chenopodiaceae). J Exp Bot 65, 3595-3607 doi:10.1093/jxb/eru053
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Rosnow, J., Yerramsetty, P.K., Berry, J.O., Okita, T.W. and Edwards, G.E. 2014 Exploring mechanisms linked to differentiation and function of dimorphic chloroplasts in the single cell C4 species Bienertia sinuspersici. BMC Plant Biology 14, 34 doi:10.1186/1471-2229-14-34
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Gandin, A., Koteyeva, N.K., Voznesenskaya, E.V., Edwards, G.E., and Cousins, A.B. 2014 The acclimation of photosynthesis and respiration to temperature in the C3-C4 intermediate Salsola divaricata: Induction of high respiratory CO2 release under low temperature. Plant Cell and Environment 37:2601-2612. DOI: 10.1111/pce.12345
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Von Caemmerer, S., Edwards, G.E., Koteyeva, N., and Cousins, A. 2014 Single cell C4 photosynthesis in aquatic and terrestrial plants: a gas exchange perspective. Aquatic Botany. 118, 71-80.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Tuncel, A, J. Kawaguchi, Y. Ihara, Y. Yoshida, H. Matsusaka, A. Nishi, T. Nakamura S. Kuhara, H. Hirakawa, Y. Nakamura, B. Cakir, A. Nagamine, T.W. Okita, S.-K. Hwang, and H. Satoh (2014) The rice endosperm ADP-glucose pyrophosphorylase large subunit is essential for optimal catalysis and allosteric regulation of the heteotetrameric enzyme. Plant Cell Physiol. 55(6):1169-83. doi: 10.1093/pcp/pcu057
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Tuncel, A., B. Cakir, S.-K. Hwang and T.W. Okita (2014) Redox-regulation of the rice endosperm ADP-glucose pyrophosphorylase occurs through disulfide formation in the large subunit and not the small subunit as evident for the leaf and tuber enzymes. FEBS J. 281:4951-4963 doi:10.1111/febs.13041
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2014
Citation:
Cakir, B. A. Tuncel, S.-K. Hwang and T.W. Okita (2014) Increase of grain yields by manipulating starch synthesis. In: Starch: Metabolism, Structure and Function, Nakamura, Y. ed., Springer
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Yang, Y., A. J. Crofts, N. Crofts and T.W. Okita (2014) Multiple RNA binding protein complexes interact with the cis-localization RNA zipcode sequences of prolamine RNAs. Plant Physiol. 164:1271-1282. www.plantphysiol.org/cgi/doi/10.1104/pp.113.234187
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Doroshenk, K.A., L. Tian, A.J. Crofts, T. Kumamaru, T.W. Okita (2014) Identification and characterization of a novel RNA-binding protein involved in glutelin mRNA processing and localization. Plant Mol. Biol. 85:381-394. DOI 10.1007/s11103-014-0191-
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Tian, L. and T.W. Okita (2014) mRNA-based protein targeting to the endoplasmic reticulum and chloroplasts in plant cells. Curr. Op. Plant Biol. 22:77-85. DOI: 10.1016/j.pbi.2014.09.007 (http://www.sciencedirect.com/science/article/pii/S1369526614001289)
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Products generated by this project are directed to participants of the NC1200 project on Photosynthesis. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Three graduate students received training in this project. How have the results been disseminated to communities of interest? Results were disseminated to the participants of NC1200 who attended the annual meeting at the University of Nebraska, Lincoln, Nebraska, November 22-23, 2013. What do you plan to do during the next reporting period to accomplish the goals? 1. Characterize the biochemical and structural transitions along a longitudinal leaf gradient which lead to the development of functional single-cell C4 photosynthesis (family Chenopodiaceae). 2. Characterize the biochemical and structural transitions along a longitudinal leaf gradient in eudicots which leads to the development of C4 photosynthesis having different forms of Kranz anatomy. 3. Determine the consequences of photorespiratory release of CO2 in rice by studying the relationship between the kinetic properties of Rubisco, the CO2 compensation point and refixation of photorespired CO2. 4. Continue transcriptome analysis of over-producing starch plants and validate changes in gene expression. 5. Identify the cysteine residues involved in redox control of the rice AGPase.
Impacts
What was accomplished under these goals?
1. In a study on the structural and functional traits of rice, we provided insight into modifications which will be required to install C4 photosynthesis; and, information on structural and functional traits in wild relatives which will be useful in employing strategies for increasing photosynthesis and water use efficiency in rice. 2. In a review, we discuss features of terrestrial and aquatic plants having single cell C4 photosynthesis, consider the structure of rice mesophyll cells, and propose a form of C4 cycle which could provide supplemental CO2 to Rubisco in rice mesophyll chloroplasts. 3. In family Chenopodiaceae we identified structural transitions from C3, to C3-C4 intermediates, to C4 which shows how some succulent species develop a form of Kranz anatomy around the periphery of the leaf which surrounds all the water storage and vascular tissue. 4. The catalytic activity of the major rice endosperm AGPase is controlled by redox potential via oxidation of cysteine residues on the large subunit. 5. Missense mutations in the large subunit of the major rice AGPase resulted in a 3- to 4-fold decrease in catalytic activity and a 4- to 6-fold decrease in the affinity for the activator 3-PGA. 6. Transcriptome analysis of transgenic rice plants over-producing starch indicate major changes in starch metabolism and a re-programming of metabolism.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Wakuta, S., Nishimura, Y, Obana, Y., Saburi, W., Hamada, S., Ito, H., Hwang, S.-K., Okita, T.W., and Matsui, H. (2013) Modulation of allosteric regulation by E38K and G101N mutations in the potato tuber ADP-glucose pyrophosphorylase. Bioscience, Biotechnology and Biochemistry, 77:1854-1859.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Kirchhoff, H., Sharpe, R.M., Herbstova, M., Yarbrough, R., and Edwards, G.E. 2013 Differential mobility of pigment-protein complexes in granal and agranal thylakoid membranes of C3 and C4 plants. Plant Physiol 161, 497-507.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Voznesenskaya, E.V., Koteyeva, N.K., Akhani, H., Roalson, E.H., and Edwards, G.E. Structural and physiological analyses in Salsoleae (Chenopodiaceae) indicate multiple transitions among C3, intermediate and C4 photosynthesis. J Exp Bot 64, 3583-3604.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Giuliani, R., Koteyeva, N., Voznesenskaya, E., Evans, M.A., Cousins, A.B., and Edwards, G.E. Coordination of leaf photosynthesis, transpiration and structural traits in rice and wild relatives (genus Oryza). Plant Physiol 162, 1632-1651.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Suleyman, A., Shen, J-R., and Edwards, G.E. 2013 Guest Editorial. Special issues on Photosynthesis Education honoring Govindjee. Photosyn Res 116,107-110.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ocampo, G., Koteyeva, N.K., Voznesenskaya, E.V., Edwards, G.E. Sage, T.L., Sage, R.F. and Columbus, J.T. Evolution of leaf anatomy and photosynthetic pathways in Portulacaceae. Am J Bot, In press.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Hwang S.-K,. Tuncel, A. and Okita, T.W. 2013. Redesigning starch metabolism to increase rice yields. In: Muralidharan K, Viraktamath BC and Siddiq EA, Eds. 2012. Proceedings of International Dialogue on Designer Rice for Future: Perception and Prospects, July 9-10, 2012. Society for Advancement of Rice Research, Directorate of Rice Research, Hyderabad, India.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Tuncel, A. and Okita, T.W. (2013) Improving starch yield in cereals by over-expression of ADPglucose pyrophosphorylase: Expectations and unanticipated outcomes. Plant Science, 211:52-60.
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Funds from this regional project normally support the travel of one of the P.I.s (Gerald Edwards or Thomas W. Okita) to attend the annual meeting. Neither Dr. Edwards nor Dr. Okita was able to attend the 2012 meeting which was hosted by University of Nevada, Reno. The research outputs of the two P.I.s are: Under Objective 2: C4 plants have a mechanism to reduce photorespiration by concentrating CO2 around Rubisco. Under this objective we are studying species which perform C4 photosynthesis in individual photosynthetic cells (single-cell C4) compared to the dual-cell Kranz type C4 species in family Chenopodiaceae. Under Objective 3: The mechanism by which carbon flow is controlled into starch is poorly understood. Under this objective, we are studying the structure-function relationships of the starch regulatory enzyme ADPglucose pyrophosphorylase. These studies center on the generation of genetic mutations for this enzyme and the effect of these mutations on catalytic and/or allosteric regulatory properties. PARTICIPANTS: Dr. Gerald Edwards and Dr. Thomas W. Okita are the WSU representatives on this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Although Washington State University representatives were unable to participate in the 2012 annual meeting, contributions from this university and participation at the next meeting are expected in 2013. As in previous years, Dr. Edwards or Dr. Okita will attend the annual meeting to discuss their recent developments in characterizing the two structural forms of C4 photosynthesis which occur within individual photosynthetic cells in family Chenopodiaceae and in identifying the molecular and biochemical processes that limit starch synthesis in plants. Specific Outputs under Objective 2: During the last year, analyses using gas exchange and online carbon isotope discrimination measurements showed that the single-cell C4 species are as efficient as their Kranz type relatives in concentrating CO2 and repressing photorespiration. This is significant considering the interest in engineering C3 crops to perform C4 photosynthesis, and the possibility that the C4 system could be introduced without requiring structural changes of leaf anatomy. Specific Outputs under Objective 3: Genetic mutants were obtained for the major ADPglucose pyrophosphorylase expressed during rice seed development. Missense mutations in the large subunit of the enzyme significantly lowered the enzyme's affinity to the activator. By contrast, null mutations in the large subunit gene resulted in the generation of a small subunit homotetrameric enzyme which exhibited properties similar to the wildtype heterotetrameric enzyme. These properties accounted for the lower amounts of starch accumulated by the missense mutants than by the null mutant.
Publications
- Hwang S.K., T.W.Okita 2012. Understanding structure-function relationship of ADP-glucose pyrophosphorylase by deciphering its mutant forms. Essential Reviews In Experimental Biology. Ed. I. Tetlow. London, UK. Society for Experimental Biology. 77-114.
- King, J, GE Edwards, AB Cousins 2012 The efficiency of the CO2 concentrating mechanism during single-cell C4 metabolism. Plant Cell Environment 35: 513-523.
- Kirchhoff, H, RM Sharpe, M Herbstova, R Yarbrough, GE Edwards 2013 Differential mobility of pigment-protein complexes in granal and agranal thylakoid membranes of C3 and C4 plants. Plant Physiology 161:497-507.
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Funds from this regional project supported the travel of one of the P.I.s (Gerald Edwards) to attend the annual meeting. In 2011, the meeting was hosted by Michigan State University where attendees discussed their latest research findings on the regulation of photosynthetic-related processes. The meeting also enabled the members to exchange ideas and experimental approaches and to discuss potential collaborative research projects. A summary of Washington State University's (WSU's) contribution to the meeting is listed in the Outcomes/Impact section. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In the genera Bienertia and Suaeda, two structural forms of C4 photosynthesis occur within individual photosynthetic cells in family Chenopodiaceae in which two cytoplasmic domains develop with dimorphic chloroplasts that are considered to function analogous to mesophyll and bundle sheath cells in Kranz type species. Using a combination of antibodies against enzymes of the C4 cycle, Rubisco, and markers for photorespiration, as well as enzymatic assays and functional studies on the intact chloroplasts, the intracellular compartmentation for function of the C4 cycle was established. A method of regeneration of Bienertia was developed from stem/explant callus culture (3 steps, 9 weeks) which is the first step towards developing a transformation system. Also, the structural and biochemical sequence of events during C4 development in two Kranz type C4 Suaeda species (relatives of the single-cell C4 species in subfamily Suaedoideae) were elucidated. Previous results indicated that the plastidic starch phosphorylase, Pho1, is essential for starch initiation at low temperatures. Pho1 differs from other eukaryotic phosphorylases in possessing a unique peptide domain (L80) rich in polar and charged amino acids. The role of this L80 domain is not known. TILLING studies have resulted in the putative identification of three rice lines with mutations in the L80 peptide domain. The catalytic activity of the leaf and tuber AGPases is suppressed by the formation of an inter-subunit disulfide bond formed between Cys-12 residues of small subunits. Although Cys-12 is not conserved in the endosperm AGPase small subunit, the protein interacts with thioredoxin, the cofactor which reduces the disulfide bond and activates enzyme activity. To study the basis of this behavior, the endosperm AGPase has been successfully expressed in bacteria and the recombinant enzyme isolated for further studies.
Publications
- Offermann, S., T.W.Okita, and G.E.Edwards. 2011. How do single cell C4 species form dimorphic chloroplasts. Plant Signaling and Behavior 6(5):762-765.
- Rosnow, J., S.Offermann, J.Park, T.W.Okita, N.M.Tarlyn, A.Dhingra, and G.E.Edwards. 2011. In vitro cultures and regeneration of Bienertia sinuspersici (Chenopodiaceae) under increasing concentrations of sodium chloride and carbon dioxide. Plant Cell Reports. 30:1541-1553.
- Offermann, S., T.W.Okita, and G.E.Edwards. 2011. Resolving the compartmentation and function of C4 photosynthesis in the single-cell C4 species Bienertia sinuspersici. Plant Physiology. 155:1612-1628.
- Gibson, K., J.Park, Y.Nagai, S.K.Hwang, Y.Cho, K.Roh, S.Lee, D.Kim, S.Choi, H.Ito, G.E.Edwards, and T.W.Okita. 2011. Exploiting leaf starch as a transient sink to increase plant productivity and yields.. Plant Science : An International Journal of Experimental Plant Biology. 181:275-281.
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Funds from this regional project support the travel of one of the P.I.s (Gerald Edwards) to attend the annual meetings. In 2010, the meeting was hosted by Virginia Tech University where about 11 members attended and discussed their latest research findings on the regulation of photosynthetic-related processes. The meeting also enables the members to exchange ideas and experimental approaches and to discuss potential collaborative research projects. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
During the last year, we continued research on a number of different aspects of higher plant photosynthesis. The structural and biochemical diversity in mechanisms of C4 photosynthesis which evolved to concentrate CO2 around Rubisco, enhance photosynthesis, and reduce losses of CO2 due to photorespiration was studied. A second project involved the characterization of the mechanism of C4 in single-cell C4 species of the family Chenopodiaceae, in which two cytoplasmic domains develop with dimorphic chloroplasts considered to function analogous to mesophyll and bundle sheath cells in Kranz type species. A purification protocol for the two different chloroplast types has been developed, and functional studies performed on intact chloroplasts. The results provide evidence that one domain is specialized for supporting fixation of atmospheric CO2 in the C4 cycle, and the other for accepting CO2 from decarboxylation of C4 acids and its assimilation by Rubisco in the C3 cycle. A third project involved the characterization of the plastidic phosphorylase. Previous results indicated that the plastidic starch phosphorylase, Pho1, is essential for in starch initiation at low temperatures. Although previous kinetic studies suggested that Pho1 was not involved in starch synthesis but rather starch degradation, our kinetic analysis of the purified recombinant Pho1 indicates that it strongly favors the synthesis reaction especially when short malto-oligosaccharides are used as the primer. Interestingly, even under degradative conditions in the presence of Pi alone, Pho1 is able to show pronounced extension of malto-oligosaccharides. Our studies will be useful in efforts to modify source-sink relationships to improve crop productivity. There is interest in modifying C3 crops like rice to have C4 traits (through the International Rice Research Institute, IRRI) to increase source activity and secure food supply under CO2 limited conditions predicted with global warming. The single-cell C4 form of photosynthesis presents a different model system for studies on the mechanism of C4 and factors controlling the development of dimorphic chloroplasts in a single cell which must involve post-transcriptional control of targeting of nuclear encoded genes. In the developing sink tissue, genetically modifying rice to increase the capacity for starch synthesis is an important strategy for improving yields of this economically important crop.
Publications
- Hwang, S.K., A.Nishi, H.Satoh, and T.W.Okita. 2010. Rice endosperm-specific plastidial alpha-glucan phosphorylase is important for synthesis of short-chain malto-oligosaccharides. Archives of Biochemistry and Biophysics. 495:82-92.
- Park, J., T.W.Okita, and G.E.Edwards. 2010. Expression profiling and proteomic analysis of isolated photosynthetic cells of the non-Kranz C4 species Bienertia sinuspersici. Functional Plant Biology. 37:1-13.
- Lee, S., and T.W.Okita. 2010. Characterization of the potato upreg1gene, encoding a mutated ADP-glucose pyrophosphorylase large subunit, in transformed rice. Plant Cell, Tissue and Organ Culture. 102:171-179.
- Leisner, C., A.B.Cousins, S.Offermann, T.W.Okita, and G.E.Edwards. 2010. The effects of salinity on photosynthesis and growth of the single-cell C4 species Bienertia sinuspersici (Chenopodiaceae).. Photosynthesis Research. 106:201-214.
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Our goals are to understand and modify source-sink relationships to improve crop productivity. With respect to source we are studying the single-cell C4 system which is very unique in developing two types of chloroplasts within individual photosynthetic cells. Defining the pattern of development of this mechanism, which prevents photorespiration and improve photosynthesis under CO2-limited conditions, is important in designing approaches to genetically modify important C3 crops, like rice, to improve carbon acquisition in source tissue. In the developing sink tissue, genetically modified rice to increase the capacity for starch synthesis is an important strategy for improving yields of this economically important crop. Results from this project will be disseminated to the scientific community by the publication in scientific journals, participation at international and national scientific conferences, and international symposiums. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Funds support the travel of one of the P.I.s to attend the annual meeting of this regional project. This year the project was hosted by Ohio State University where about 13 members attended and discussed their latest research findings on the regulation of photosynthetic-related processes. At the meeting,T.O. presented results on the single C4 rsearch study and starch regulation study. The relevant information on the starch regulation project can be found in the CRIS report WPN00590 while information on the single-cell C4 system is as follows: A study in a natural habitat in Iran showed from analysis of the carbon isotope composition that the single-cell C4 species Bienertia cycloptera performs C4 photosynthesis throughout the growing season, along with 8 other Kranz type C4 species in family Chenopodiaceae. This demonstrates that Kranz anatomy is not required for effective function of C4 under natural conditions. A study on the physiological features of two structural forms of single-cell C4 plants (Bienertia sinuspersici and Suaeda aralocaspica) in comparison to close Kranz type C4 and C3 relatives, showed that they perform C4 similar to Kranz types with respect to photosynthetic rates, light response curves, and level of CO2 required for saturation, CO2 compensation points, carbon isotope composition, and water use efficiency. The single-cell C4 species and close relatives are halophytes. A study on Bienertia sinuspersici identified and defined a number of halophytic features: succulent leaves with centrally located water storage tissues, synthesis of glycine betaine as a compatible solute (including expression of transcripts for enzymes required, and high levels of glycine betaine), accumulation of large amounts of NaCl in leaves, and storage of NaCl in salt glands. An initial study on transcript profiling and proteomics has been been performed on isolated chlorenchyma cells of B. sinuspersici, identifying enzymes involved in C4 photosynthesis which provide the framework for function as an NAD-malic enzyme type C4 plant. Recently, the two chloroplast types were separated and purified from this species, which will be characterized.
Publications
- Lee, S., T.Ryu, S.Kim, T.W.Okita, and D.Kim. 2009. Kinetic and regulatory properties of plant ADP-glucose pyrophosphorylase genetically modified by heterologous expression of potato upreg mutants in vitro and in vivo. Plant Cell, Tissue and Organ Culture. 96:161-170.
- Park, J., T.W.Okita, and G.E.Edwards. 2009. Salt tolerant mechanisms in single-cell C4 species Bienertia sinuspersici and Suaeda aralocaspica (Chenopodiaceae). Plant Science : An International Journal of Experimental Plant Biology. 176:616-626.
- Nagai, Y., C.Sakulsingharoj, G.E.Edwards, T.Greene, H.Sato, B.Blakeslee, and T.W.Okita. 2009. Control of starch synthesis in cereals: metabolite analysis of transgenic rice expressing upregulated cytoplasmic ADP-glucose pyrophosphorylase in developing seeds. Plant and Cell Physiology. 50:635-643.
- Smith, M.E., N.K.Koteyeva, E.V.Voznesenskaya, T.W.Okita, and G.E.Edwards. 2009. Photosynthetic features of non-Kranz C4 species, Bienertia sinuspersici and Suaeda aralocaspica (Chenopodiaceae). Functional Plant Biology. 36:770-782.
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Preliminary characterization of the plastidic phosphorylase: A rice seed starch deficient mutant was identified that was caused by a mutation in the plastidic phosphorylase, Pho1. The starch deficient phenotype was temperature dependent and was readily evident when rice plants were grown at 20 degrees C but not at 30 degrees C. These results indicate that Pho1 is essential for in starch initiation at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures. Although previous kinetic studies suggested that Pho1 was not involved in starch synthesis but rather starch degradation, our analysis showed that even at physiological levels (0.2 mM) of Glc 1-P, Pho1 was able to carry out the net biosynthetic reaction even in the presence of 50-fold excess Pi. These results support a role for Pho1 in starch biosynthesis and not in starch degradation. Structure-function of AGPase subunits: The higher plant AGPase is composed of a pair of large subunits and a pair of small subunits. The small subunit is capable of forming a catalytically active homotetrameric enzyme but with altered allosteric regulatory properties. A mutation S302N in the large subunit increased its solubility, enabling the formation of a homotetrameric large subunit enzyme. The large subunit enzyme possesses very low catalytic enzyme and is not activated by 3-PGA. However, when assembled with the catalytically silenced small subunit containing a D143N mutation, the resulting enzyme shows significantly elevated catalytic activity and is activated by 3-PGA. : We have previously demonstrated that expression of a bacterial AGPase in developing rice seeds resulted in increased starch synthesis and seed weight. Analysis of the major metabolites in these transgenic plants showed that ADPglucose levels were elevated compared to wildtype. In addition, metabolites that lead to ADPglucose formation ( Glc 1-P and UDPglucose) were also elevated indicating that the AGPase catalyzed reaction was no longer limiting and that a downstream process, i.e. transport into amyloplast or utilization by starch synthases, was now the major limitation in optimizing starch synthesis. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our goals are to understand and modify source-sink relationships to improve crop productivity. With respect to source we are studying the single-cell C4 system which is very unique in developing two types of chloroplasts within individual photosynthetic cells. Defining the pattern of development of this mechanism, which prevents photorespiration and improve photosynthesis under CO2-limited conditions, is important in designing approaches to genetically modify important C3 crops, like rice, to improve carbon acquisition in source tissue. In the developing sink tissue, genetically modified rice to increase the capacity for starch synthesis is an important strategy for improving yields of this economically important crop.
Publications
- Hwang, S.K., Y.Nagai, D.Kim, and T.W.Okita. 2008. Direct appraisal of the potato tuber ADP-glucose pyrophosphorylase large subunit in enzyme function by study of a novel mutant form.. The Journal of Biological Chemistry. 283:6640-6647.
- Satoh, H., K.Shibahara, T.Tokunaga, A.Nishi, M.Tasaki, S.Hwang, T.W.Okita, N.Kaneko, N.Fujita, M.Yoshida, Y.Hosaka, A.Sato, Y.Utsumi, and Y.Nakamura. 2008. Plastidic a-Glucan Phosphorylase Mutation Dramatically Affects the Synthesis and Structure of Starch in Rice Endosperm. The Plant Cell. 20:1833-1849.
- Lara, M.A., S.Offermann, S.Smith, T.W.Okita, C.S.Andreo, and G.E.Edwards. 2008. Leaf development in the single-cell C4 system in Bienertia sinuspersici: Expression of genes and peptide levels for C4 metabolism in relation to chlorenchyma structure under different light conditions.. Plant Physiology. 148:593-610.
- Park, J., M.Knoblauch, T.W.Okita, and G.E.Edwards. 2008. Structural changes in the vacuole and cytoskeleton are key to development of the two cytoplasmic domains supporting single. Planta. 229:369-382.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Structure-function of AGPase subunits. Past studies have suggested that the large and small subunits had different roles in AGPase function. The small subunit (SS) was the catalytic subunit while the large subunit (LS) was regulatory. This view was found to be overly simplistic as the LS was found to have the potential to be catalytic when two mutations were introduced (Ballicora et al. J. Biol. Chem. 280, 10189-10195). Results obtained from a site-directed mutagenesis study of the effect of six proline residues found within the first 66 amino acids at its N-terminus on enzyme properties were consistent with the catalytic potential of the LS. Although non-catalytic, the LS was found to bind ATP at the same efficiency as the catalytic SS. More recent results have verified the catalytic potential of the LS. Introduction of a single mutation elevated the solubility of the expressed LS enabling it to form a homotetrameric structure. The LS homotetramer possesses very little
enzyme activity at a level 100-fold less than that seen for the unactivated SS homotetramer. Unlike the SS enzyme, however, the LS homotetramer enzyme is not activated by the effector 3-phosphoglycerate or inhibited by Pi. When combined with the catalytically silenced SS containing a D143N mutation, however, LS shows significantly enhanced enzyme activity and restored 3-PGA activation. Overall, these markedly distinctive properties of the LS when assembled with itself or with the SS provide new insights on its role in modulating enzyme catalysis and regulation. These results together with the kinetic data provide evidence that the LS is not only required to serve as an synergistic partner to the SS in affecting the net allosteric properties of the enzyme but also of catalysis as well. Manipulation of leaf starch metabolism in Arabidopsis. During the last year we continued research on Arabidopsis plants which have been genetically modified to produce more leaf starch. The TL46
Arabidopsis mutant, which lacks the large subunit of AGPase is starch deficient and has lower rates of photosynthesis than wildtype. As reported last year, complementation of this mutant with up-regulated forms of the large subunit results in higher AGPase and higher starch biosynthesis than wildtype. This past year a study with several transgenic lines versus wildtype plants grown under current atmospheric levels of CO2 was completed including measurements of AGPase activity, chlorophyll content, leaf starch content, analysis of photosynthetic capacity and plant biomass. These results, combined with earlier studies indicate a progressive increase in photosynthetic capacity and biomass production from starch deficient mutants, to wildtype, to transgenic plants having increased capacity for starch biosynthesis. This indicates starch synthesis in leaves is an important transient sink for the initial products (triose-P) of photosynthesis.
PARTICIPANTS: Seon-kap Hwang; Yasuko Nagai; Aytug Tuncel
Impacts
Starch metabolism has a profound impact on overall plant productivity and seed yields. In Arabidopsis, there is a direct correlation between leaf starch, photosynthetic capacity and plant growth. Moreover, elevation of leaf starch levels results in alleviating inhibition of photosynthesis by feedback and enhanced growth. Application of this approach to cereal plants, which are low leaf starch accumulators, may increase photosynthesis and growth resulting in improving overall plant productivity. The key regulatory step in starch biosynthesis is controlled by ADPglucose pyrophosphorylase, an enzyme composed of a pair of non-catalytic large subunits and a pair of catalytic small subunits. Information gained from structure-functions studies of this enzyme will provide new insights on how this enzyme operates to control carbon flux into starch.
Publications
- Hwang, S., S.Hamada, and T.W.Okita. 2007. Catalytic implications of the higher plant ADP-glucose pyrophosphorylase large subunit. Phytochemistry. 68:464-477.
- Kim, D., S.Hwang, and T.W.Okita. 2007. Subunit interactions specify the allosteric properties of potato tuber ADP-glucose pyrophosphorylase. FEBS Letters.
- Lee, S., S.Hwang, M.Han, H.Kang, Y.Han, S.Choi, M.Cho, S.Bhoo, G.An, T.Hahn, T.W.Okita, and J.Jeon. 2007. Identification of ADP-glucose pyrophosphorylase isoforms essential for starch synthesis in leaf and seed endosperm of rice (Oryza sativa L.). Plant Molecular Biology. 65:531-546.
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Progress 01/01/06 to 12/31/06
Outputs
(Note: Funds from this project enabled one of the P.I.s to attend the annual meeting of NC-1142 Project on the Regulation of Photosynthetic Processes. At the 2006 meeting, which was held at the University of Kentucky, T.W. Okita presented his research results on the manipulation of the starch regulatory enzyme ADPglucose pyrophosphorylase. A brief summary of his presentation is provided here). Past studies have suggested that the large and small subunits had different roles in AGPase function. The small subunit (SS) is the catalytic subunit while the large subunit (LS) is regulatory. This view was found to be overly simplistic as the LS was found to have the potential to be catalytic when two mutations were introduced. Results obtained from a site-directed mutagenesis study of the first six proline residues found within the N-terminal region of the LS were consistent with the catalytic potential of this subunit. While P17L, P26L, and P55L mutations only moderately
affected kinetic properties, P52L and P66L gave rise to significant and contrasting changes in allosteric properties: P66L enzyme displayed up-regulatory properties toward 3-PGA while the P52L enzyme had down-regulatory properties. Unlike the other mutants, the P44L enzyme showed only moderate changes in regulatory properties, but had severely impaired catalytic activity (less then 3% of wildtype) as well as decreased apparent substrate affinities. Modeling studies showed that P44 was located upstream from the putative ATP binding site of the LS. The latter view was verified by photoaffinity labeling studies with 8-azido-ATP, which mimics ATP in catalysis. Although non-catalytic, the LS was found to bind ATP at the same efficiency as the catalytic SS. Mass spectrometry analysis of photoaffinity-labeled LS tryptic fragments identified a peptide W125-R134. The homologous peptide in the SS corresponds to part of the ATP binding site. Other residues for ATP binding domain were also
predicted from structural model of the LS. Site-directed mutagenesis of selected residues contained within this peptide and others predicted to participate in ATP binding in the LS and subsequent kinetic enzyme analysis showed that G128A, A132V and D157L significantly decreased enzyme activities (5- to 11-fold) while G128A, T129V, and A132V had significantly lowered affinities toward ATP and glucose-1-phosphate. Further mutations at G128, T129, and A132 also led to similar effects on substrate affinities. The catalytic rates of the LS mutant enzymes were highly correlated with the changes in the affinity toward ATP, suggesting that the LS is involved in catalysis. The role of the LS in catalysis was further supported by our photoaffinity labeling of LT129V/A132VSWT which revealed that the double mutant enzyme had decreased binding affinity to the ATP analog compared with wildtype AGPase. These results indicate that the LS, although catalytically disabled, may mimic many of the
catalytic events undertaken by the SS and, thereby, influence net catalysis of the heterotetrameric enzyme.
Impacts
Starch metabolism has a profound impact on overall plant productivity and seed yields. In Arabidopsis, there is a direct correlation between leaf starch, photosynthetic capacity and plant growth. Moreover, elevation of leaf starch levels results in alleviating inhibition of photosynthesis by feedback and enhanced growth. Application of this approach to cereal plants, which are low leaf starch accumulators, may increase photosynthesis and growth resulting in improving overall plant productivity. The key regulatory step in starch biosynthesis is controlled by ADPglucose pyrophosphorylase, an enzyme composed of a pair of non-catalytic large subunits and a pair of catalytic small subunits. Information gained from structure-functions studies of this enzyme will provide new insights on how this enzyme operates to control carbon flux into starch.
Publications
- Obana, Y., D.Omoto, C.Kato, K.Matsumoto, Y.Nagai, I.Kavakli, S.Hamada, G.E.Edwards, T.W.Okita, H.Matsui, and H.Ito. 2006. Enhanced turnover of transitory starch by expression of up-regulated ADP-glucose pyrophosphorylases in Arabidopsis thaliana. Plant Science : An International Journal of Experimental Plant Biology. 170:1-11.
- Hwang, S., S.Hamada, and T.W.Okita. 2006. Catalytic implications of the higher plant ADP-glucose pyrophosphorylase large subunit. Phytochemistry. doi:10.1016/j.phytochem. 2006.11.027.
- Hwang, Seon-Kap, Hamada, Shigeki, and Okita, Thomas W. 2006. ATP binding site in the plant ADP-glucose pyrophosphorylase large subunit. FEBS Letters. 580:6741-6748.
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Progress 01/01/05 to 12/31/05
Outputs
Starch metabolism has a profound impact on overall plant productivity and seed yields. In Arabidopsis, there is a direct correlation between leaf starch, photosynthetic capacity and plant growth. Metabolic engineering studies have demonstrated that restoration of leaf starch metabolism in Arabidopsis plants defective in leaf starch metabolism re-establishes photosynthetic capacity and growth rates to normal levels. Moreover, elevation of leaf starch levels results in alleviating inhibition of photosynthesis by feedback and enhanced growth. Funds from this project enables one of the P.I.s of this project (Dr. Gerald Edwards or Dr. Thomas Okita) to attend the annual meeting of the NC-1142 regional project. The meeting this year was held at the University of Florida, Gainsville, FL. Due to logistic problems in travel, WSU was unable to participate in this year's meeting.
Impacts
Application of this approach to cereal plants, which are low leaf starch accumulators, may increase photosynthesis and growth resulting in improving overall plant productivity.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Funds from this project normally enables one of the P.I.s of this project (Dr. Gerald Edwards or Dr. Thomas Okita) to attend the annual meeting of the NC-1142 regional project. The meeting this year was held at Columbia, South Carolina on November 20-21. The P.I.s decided not to attend this meeting due to the unusually high travel costs between Pullman and Columbia. A description of the P.I.s' progress on this project can be found in the CRIS Project 4589 report.
Impacts
Starch metabolism has a profound impact on overall plant productivity and seed yields. In Arabidopsis, there is a direct correlation between leaf starch, photosynthetic capacity and plant growth. Metabolic engineering studies have demonstrated that restoration of leaf starch metabolism in Arabidopsis plants defective in leaf starch metabolism re-establishes photosynthetic capacity and growth rates to normal levels. Moreover, elevation of leaf starch levels results in alleviating inhibition of photosynthesis by feedback and enhanced growth. Application of this approach to cereal plants, which are low leaf starch accumulators, may increase photosynthesis and growth resulting in improving overall plant productivity.
Publications
- Edwards, G.E., V. Franceschi, V. Voznesenskaya and T.W. Okita. 2004. Stategies in addressing source versus sink limitations in photosynthesis. International Symposium on Crop Functional Genomics 2004, Cheju (Jeju), Korea, April 7-9, 2004.
- Invited symposium presentation, Symposium on Biotechnology in Agriculture, Society for Industrial Microbiology Annual Meeting, Anaheim, CA, July 25-29, 2004.
- Nagai, Y., Hwang, S.-K., Okita, T.W. Genetic modification of ADP-glucose pyrophosphorylase in Arabidopsis: effects on enzyme activity, starch biosynthesis and physiology. Plant Biology 2004, Annual Meeting of the American Society of Plant Biology Lake Buena Vista, FL, Abstract 297.
- Sakulsingharoj, C., S.-B. Choi, S.-K. Hwang, J. Bork, C.R. Meyer, G.E. Edwards, J. Preiss, and T.W. Okita 2004 Engineering starch biosynthesis for enhanced rice yields: the role of the cytoplasmic ADP-glucose pyrophosphorylase. Plant Science 167:1323-1333.
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Progress 01/01/03 to 12/31/03
Outputs
Funds from this project enabled one of the Co-P.I.s of this project (Dr. Gerald Edwards) to attend the annual meeting of the NC-1142 regional project which was held in Reno, Nevada on November 22-23, 2003. At the meeting, Dr. Edwards presented a 30 min summary on recent developments on the effects of starch manipulation on plant growth and development in Arabidopsis and rice and on a collaborative project with Dr. Wayne Loescher (Michigan State University) on the changes in photosynthetic potential in Arabidopsis plants over-expressing mannitol metabolism. Progress on the former study can be found in CRIS Project 4589 report.
Impacts
Starch metabolism has a profound impact on overall plant productivity and seed yields. In Arabidopsis, there is a direct correlation between leaf starch, photosynthetic capacity and plant growth. Metabolic engineering studies have demonstrated that restoration of leaf starch metabolism in Arabidopsis plants defective in leaf starch metabolism re-establishes photosynthetic capacity and growth rates to normal levels. Moreover, elevation of leaf starch levels results in alleviating inhibition of photosynthesis by feedback and enhanced growth. Application of this approach to cereal plants, which are low leaf starch accumulators, may increase photosynthesis and growth resulting in improving overall plant productivity.
Publications
- Gibson, K.M., S. K. Hwang, G. E. Edwards, T. W. Okita, C. Kato, H. Matsui, and H. Ito. 2003 Metabolic engineering of starch for enhanced plant productivity and yields. J. Applied Glycoscience. 50:201-206.
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Progress 01/01/02 to 12/31/02
Outputs
The P.I. attended the annual meeting of the NC-142 (renewed as NC-1142) project which was held in Newport Oregon, November 23-24, 2002. A 30-min ute presentation was given to the attendees on recent developments in the P.I.s' laboratory on modification of the higher plant ADP-glucose pyrophosphorylase, expression of mutant enzyme forms in rice and Arabidopsis, and the consequences of this expression on photosynthetic capacity, starch levels and growth rates of transformed plants. At the meeting, collaborative arrangements were made with several members of the project to carryout joint studies for the coming year.
Impacts
Discussed with the leading experts in the general area of photosynthesis in the nation on the feasibility of modifying plants for increased plant productivity and crop yields by manipulation of primary carbon metabolism. Plants were generated that showed reduced photosynthetic feedback and higher CO2 assimilation rates which translated into higher growth rates and seed yields. At the annual NC-1142 meeting, collaborative arrangements were made to grow these plants in the field under ambient and elevated carbon dioxide.
Publications
- Kavakli, H.I., Kato, C., Choi, S.-B., Kim, K.-H., Salamone, P.R., Ito, H. and Okita, T.W. 2002 Generation, Characterization, and Heterologous Expression of Up-Regulated Arabidopsis thaliana Leaf ADP-Glucose Pyrophosphorylases. Planta 215:430-439.
- Sun, J., Gibson, K.M., Kiirats, O., Okita, T.W. and Edwards, G.E. 2002 Interactions of Nitrate and CO2 Enrichmont on Growth, Carbohydrates, and Rubisco in Arabidopsis Starch Mutants - Significance of Starch and Hexose. Plant Physiol. 130:1573-1583.
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Progress 01/01/01 to 12/31/01
Outputs
Funds from this project were used to support the travel of the P.I.s (G.E. Edwards) to attend the annual meeting of the Hatch NC-142 Regional Project which was held on November 17-18 in Raleigh, NC. A summary of recent developments from the P.I.s laboratories relevant to "Regulation of Photosynthetic Processes" was presented. Ongoing collaborative interactions with other members of the NC-142 project were discussed.
Impacts
This project enables the P.I.s to keep abreast of some of the latest developments in the field of photosynthesis especially with regard to carbon partitioning and enzyme regulation.
Publications
- Okita, T.W., J. Sun, C. Sakulsingharoj, S.-B.Choi, G.E. Edwards, C. Kato, H. Ito, and H. Matsui. 2001. Increasing Rice Productivity and Yield by Manipulation of Starch Synthesis. In "Rice Biotechnology: Improving Yield, Stress Tolerance and Grain Quality (Good, J.A. and Chadwick, D., eds.), John Wiley and Sons, New York, NY, pp. 135-152.
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Progress 01/01/00 to 12/31/00
Outputs
Funds from this project were used to support the travel of one of the P.I.s (T. Okita) to attend the annual meeting of the Hatch NC-142 project which was held on November 18-19, 2000, at the University of Illinois. A summary of recent developments of the CRIS Project 3590, sponsored by the USDA-NRICGP, was presented. In addition, potential collaborative interactions with other members of the NC-142 project were discussed. These included collaborative studies on the effect of single amino acid replacements on the protein structure of ADP-glucose pyrophosphorylase, the role of starch degradative enzymes during cereal seed development, and in vitro methods for cereal seed culturing.
Impacts
The travel support provided by this project enables one of the P.I.s to participate in the annual meeting of the North Central (NC) 142 Project. This meeting, which brings together many of the leading scientists in photosynthetic carbon processes from throughout the United States, as well as Guam, provides a forum to discuss the latest developments in photosynthesis and unique opportunities to collaborate on projects of mutual interest.
Publications
- Kavakli, I.H., Slattery, C.J., Ito, H. and Okita, T.W. 2000. The Conversion of Carbon and Nitrogen into Starch and Storage Proteins in Developing Storage Organs: An Overview. Australian J. Plant Physiol. 27:561-570.
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Progress 01/01/99 to 12/31/99
Outputs
Funds from this project support the travel costs for either Gerald Edwards or Thomas Okita to attend and participate at the annual NC-142 meeting. Neither of the WSU P.I.s was able to attend the meeting in 1999.
Impacts
(N/A)
Publications
- Ito, H., Sokolov, L.N., Kleczkowski, L.A. and Okita, T.W. 1999 The Arabidopsis thaliana ADP Glucose Pyrophosphorylase Large Subunit Gene, adg2 (Accession No.: AB022891). Plant Physiol. 119:1652.
- Sun, J., Okita, T.W. and Edwards, G.E. 1999 Feedback Inhibition of Photosynthesis in Rice Measured by O2 Dependent Transients. Photosynth. Res. 59:187-200.
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Progress 01/01/98 to 12/31/98
Outputs
Funds from this project were used to cover travel expenses for the two P.I.s to attend the annual NC-142 meeting at Spokane, WA. At the meeting, an overview of ongoing research at WSU in photosynthetic carbon partitioning was presented. Specifically, using nonevasive techniques to measure the photosynthetic potential of the flag leaf of flowering rice plants, it was demonstrated that carbon dioxide assimilation was saturated at near atmospheric levels of CO2. This response by rice plants, which is unusual for a C3 plant, suggests that under current levels of CO2, moderate temperature and light, and high humidity, photosynthesis in rice is limited not by photorespiration but by the capacity for triose phosphate (triose-P) utilization. Results from further studies showed that transient inhibition of photosynthesis under low O2 occurs in rice with increasing light and increasing levels of CO2 (conditions which favor increased ability to generate triose-P during
photosynthesis) and with decreasing temperature where the capacity of RuBP carboxylase tends to be greater than the capacity for carbohydrate synthesis. The degree of transient inhibition is correlated with low O2 enhancement of photosynthesis under steady-state. The limitation on triose-P utilization may be associated with limited capacity for carbohydrate synthesis in the leaves. In support of this hypothesis, significant correlation was evident between CO2 assimilation and starch synthesis and between cumulative leaf area and starch synthesis in Arabidopsis plants. In collaboration with Dr. Douglas Randall, University of Missouri, transgenic rice plants containing the tomato fructokinase gene under either the CaMV 35S or rice glutelin promoters were obtained. These transgenic plants were analyzed for their growth and seed yield potential under different environmental conditions. Results from studies to evaluate the structure-function relationships of ADPglucose pyrophosphorylase
(AGP) using a biochemical-genetic approach were presented as well. Extensive sequence alignment analysis indicates the presence of 3 conserved cysteine residues in both the large and small subunit. Mutagenesis of the proximal cysteine residue in either the large or small subunit results in a significant reduction in subunit production, indicating that this residue is essential for polypeptide folding. Mutations in the other two residues in both the large and small subunit are under study. A series of allosteric mutants ranging from up-regulatory to down-regulatory in phenotype were transformed into Arabidopsis under the control of the Rubisco small subunit promoter. Surprisingly, plants containing the up-regulatory AGP to wildtype AGP that were germinated and grown on sucrose media for two weeks before being transferred to soil grew significantly faster and produced larger quantities of seed than wildtype plants. The basis for this increased performance in plant growth and development
is currently being explored.
Impacts
(N/A)
Publications
- Laughlin, M.J., Payne, J.W. and Okita, T.W. 1998. The generation of substrate mutants by random mutagenesis of potato ADP-glucose phyrophosphorylase. Phytochemistry 47:621-629.
- Choi, S.B., Zhang, Y., Ito, H., Stephens, K., Winder, T., Edwards, G.E. and Okita, T.W. 1998. Increasing rice productivity by manipulation of starch biosynthesis during seed development. In "Feeding a World Population of More Than Eight Billion People: A Challenge to Science" (Waterford, J.C., Armstrong, D.G., Fowden, L. and Riley, R., eds.), Oxford University Press, New York, pp. 137-149.
- Laughlin, M.J., Chantler, S.E. and Okita, T.W. 1998. N- and C-terminal peptide sequences are essential for enzyme assembly, allosteric, and/or catalytic properties of ADP-glucose pyrophosphorylase. Plant J. 14:159-168.
- Greene, T.W., Kavakli, I.H., Kahn, M.L. and Okita, T.W. 1998. Generation of up-regulated allosteric variants of potato ADP-glucose phyrophosphorylase by reversion genetics. Proc. Natl. Acad. Sci. USA 95:10322-10327.
- Winder, T.L., Sun, J., Okita, T.W. and Edwards, G.E. 1998. Evidence for the occurrence of feedback inhibition of photosynthesis in rice. Plant Cell Physiol. 39:813-820.
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Progress 01/01/97 to 12/31/97
Outputs
During the last year we continued studies on rice source-sink relationships. We evaluated whether the rate of photosynthesis in rice is limited by feedback conditions (i.e., triose-P utilization limited) under varying environmental conditions. This was examined by response to change in O2. An increase in the rate of photosynthesis in rice on transfer from atmospheric to low levels of O2 indicates that the plant can make use of additional photosynthate (triose-P production limited); whereas a restriction or loss of the response to O2 (loss of O2 sensitivity) indicates a restriction in the capacity to utilize triose-P. Rice shows strong limitations on its capacity to utilize triose-P over a range of environmental conditions. Only under low light or sub-atmospheric levels of CO2 does the rate photosynthesis become fully limited by the rate of production of triose-P. Both transient and steady-state responses to low O2 indicates restrictions on utilization of triose-P.
Both feedback responses are consistent with a limitation on utilization of triose-P for synthesis of carbohydrates, and decreased availability of inorganic phosphate resulting in decreased production of RuBP. Measurements of assimilatory charge as an indicator of the RuBP pool indicates that the rate of CO2 assimilation is strongly correlated with the RuBP pool size in rice.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/96 to 12/30/96
Outputs
Studies on expression of ADPglucose pyrophosphorylase small subunit gene during potato development continue. Various portions of 5' & 3'flanking sequences of small subunit gene were fused to GUS reporter gene & transferred into potato by Agrobacterium-mediated transformation techniques. Chimeric gene was expressed at high levels in tubers & many other starch containing cells throughout plant. Deletion analysis of 5' flanking region revealed observed spatial patterns of expression was due to different regions of promoter working in combination to confer cell- & organ-specific patterns of expression. Addition of 3' flanking sequence resulted in enhancement, suppression or alteration of transgene expression depending on tissue examined. Studies were initiated to study structure-function relationships of potato ADPglucose pyrophosphorylase using biochemical-genetic approach. Mature cDNA sequences for large & small subunit cDNAs were inserted into 2 separate bacterial
expression vectors & transformed into E coli. Functional enzyme activity wasn't detected unless both large & small cDNAs were co-expressed together. Recombinant plant enzyme was able to complement function of bacterial ADPglucose pyrophosphorylase as evident by capacity of glgC- mutants to accumulate glycogen. This resulted in development of powerful mutagenesis system which generated hundreds of enzyme mutants. Two of these mutants were found to be defective only in allosteric regulation & not in catalytic function.
Impacts
(N/A)
Publications
- Greene, Chantler, Kahn, Barry, Preiss, & Okita. 1996. Mutagenesis of the Potato ADPglucose Pyrophosphorylase and Characterization of an Allosteric Mutant Defective in 3-Phosphoglycerate Activation. Proc. Natl. Acad. Sci. USA 93:1509-1513.
- Greene, T.W., Woodbury, R.L. and Okita, T.W. 1996. Aspartic Acid 413 is Important for the Normal Allosteric Functioning of ADP-Glucose Pyrophosphorylase. Plant Physiol. 112:1315-1320.
- Giroux, M., Shaw, J., Barry, G., Cobb, B.G., Greene, T.W., Okita, T.W. and Hannah, L.C. 1996. A Single Gene Mutation that Increases Maize Seed Weight. Proc. Natl. Acad. Sci. USA 93:5824-5829.
- Nakata, P.A. and Okita, T.W. 1996. Cis-Elements Important for the Expression of the ADP-Glucose Pyrophosphorylase Small-Subunit are Located Both Upstream and Downstream from Its Structural Gene. Mol. Gen. Genet. 250:581-592.
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Progress 01/01/95 to 12/30/95
Outputs
Studies on the expression of the ADPglucose pyrophosphorylase large and small subunit genes during potato development were continued. Temporal analysis revealed that the coordinate expression of the large and small subunits, which collectively constitute the heterotetrameric holoenzyme, is primarily under transcriptional control during tuber development. Unlike the coordinate steady state mRNA and subunit levels evident in tubers, noncoordinate expression of the subunit mRNAs was observed in leaf tissue where steady state levels of the small subunit transcripts were present at several-fold excess over large subunit transcript levels. Despite the vast differences in steady state transcript levels, immunoblot analysis revealed that the large and small subunit levels were present at nearly equimolar amounts indicating that a posttranscriptional event coordinates subunit polypeptide levels. This posttranscriptional control was also evident in leaves subjected to a
photoperiod regime and during sucronalling cascade involved in induced oleoresin formation. The employment of different regulatory processes in controlling the expression of ADPglucose pyrophosphorylase in tubers and leaves is consistent with the distinct pathways of carbon partitioning and with the type and function of starch syntheses that occurs within each tissue.
Impacts
(N/A)
Publications
- Nakata, P.A. & Okita, T.W. 1994. Studies to Enhance Starch Biosynthesis by Manipulation of ADPglucose Pyrophosphorylase Genes. Molecular & Cellular Biology of the Potato, Second Edition, CAB Intl, Wallingford, UK, pp. 31-44.
- Nakata, P.A. and Okita, T.W. 1995. Differential Regulation of ADP-Glucose Pyrophosphorylase in the Sink and Source Tissues of Potato. Plant Physiol. 108:361-368.
- Ballicora, M.A., et al. 1995. ADP-glucose Pyrophosphorylase from Potato Tuber. Significance of the N-terminal of the Small Subunit for Heat Stability and Phosphate Inhibition. Plant Physiol. 109:245-251.
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Progress 01/01/94 to 12/30/94
Outputs
Studies on the structure, organization and expression of the gene that encodes the ADPglucose pyrophosphorylase (AGP) small subunit were completed. DNA sequence analysis revealed that the small subunit gene from potato is structurally similar to the rice endosperm sAGP in containing multiple introns at identical positions within the coding sequence. Unlike rice and other plants which contain multiple sAGP genes that are expressed in specific tissues, potato contains only a single sAGP gene which is expressed in multiple tissues. This conclusion was substantiated by the observed spatial and tissue expression patterns in transgenic potato of a B-glucuronidase reporter gene under the control of the sAGP promoter. Although the same sAGP gene is expressed in both leaves and tubers, the sAGP gene is controlled by different mechanisms in these tissues. In tubers, the sAGP gene is controlled mainly at the transcriptional level, whereas in leaves, the gene is controlled
post-transcriptionally. This post-transcriptional control is also readily evident when leaves are incubated in sucrose solutions. Despite the dramatic increases of large and small subunit transcripts, protein and enzyme activity levels either decreased (small subunit) or remained largely unchanged (large subunit).
Impacts
(N/A)
Publications
- NAKATA, PA, ANDERSON, JM & OKITA, TW. 1994. Structure and expression of the potato ADP-glucose pyrophosphorylase small subunit. J Biol Chem 269:30798-30807.
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