Progress 09/01/07 to 08/31/11
Outputs
OUTPUTS: Project researchers conducted fundamental research to characterize metabolic processes involved in the acclimation of microalgae to low CO2, especially how the protein LCIB functions in the inducible carbon dioxide (CO2)-concentrating mechanism (CCM) of the model microalga Chlamydomonas reinhardtii. Researchers determined that LCIB is located in the aqueous space (stroma) outside the chloroplast photosynthetic membranes, but that it sometimes clusters around the pyrenoid (suborganeller structure in algae that contains the ribulose-bisphosphate carboxylase/oxygenase). Researchers demonstrated that LCIB interacts with a related protein, LCIC, to form a complex that can be stably isolated from the cells. In published work, researchers demonstrated the role of candidate CO2 transport proteins, HLA3 and LCIA, in the Chlamydomonas CCM, as well as their interaction with LCIB. Specific depression of HLA3 abundance, when combined either with LCIB mutations and/or specific depression of LCIA abundance, resulted in defective growth and photosynthesis under alkaline pH conditions. Researchers concluded that HLA3 is involved in the energy-dependent transport into the microalgal cells of bicarbonate, a form of CO2 that predominates in alkaline conditions, and that LCIA functions in transport of CO2 or bicarbonate into the chloroplast. Researchers identified 11 mutations (suppressor mutations) that suppress the lethal phenotype of LCIB mutants, allowing the double mutants to survive. In published work, researchers demonstrated that two of these are in the gene CAH3, which codes for a thylakoid lumen carbonic anhydrase, and researchers concluded that LCIB functions downstream of CAH3 to prevent loss of CO2 in the CCM. The specific gene defects in the other 9 suppressor mutations have not yet been determined, but researchers have published the characterization of 4 of the mutations, su1, su4, su5 and su8, genetically and physiologically. Researchers investigated global changes in Chlamydomonas gene expression in response to exposure to low-CO2 (less than 0.04% CO2) or very-low-CO2 (less than 0.02% CO2) relative to high-CO2 (5% CO2) using next-generation sequencing of gene transcripts from a wild-type strain and a strain, cia5, that is totally defective in induction of the CCM. Over 3,000 genes changed expression in this study, and a manuscript describing these changes is under review. Researchers demonstrated that over-expression of LCIB in 5% CO2 conditions, where it normally is not expressed, increases photosynthetic CO2 assimilation and biomass accumulation, especially when combined with over-expression of LCIA. This increased photosynthetic CO2 assimilation appears to result from increased CCM-like activity, and a manuscript is in preparation. Results of the project experiments reported here either have been or will be disseminated to the scientific and public community through further peer-reviewed publications in nationally and internationally renowned journals. PARTICIPANTS: Dr. Martin H. Spalding (P.I.); Dr. Peng Liu (Co-P.I.; responsible for statistical analyses); Dr. Deqiang Duanmu (former graduate student); Mr. Bo Chen (graduate student; Ms. Han Gao (graduate student); Mr. Wei Fang (graduate student), Ms. Soujanya Akella (graduate student). The project researchers have been involved in the research based training, education and mentoring of the graduate students listed above, as well as several undergraduates participating in the project for hourly wages and/or academic credit and one postdoctoral associate (Dr. Yingjun Wang) not directly supported by salary from this project. TARGET AUDIENCES: Target audiences for this research include the scientific research community, especially those involved in research on plant productivity, as well as those involved in education at the undergraduate and graduate level for whom the outputs of this project should provide opportunities for update or revision of educational materials and curricula to reflect the new knowledge disseminated. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The activities of the project researchers have contributed to major changes in the understanding of the functional components of the microalgal CCM. Prior to this research, little was known about the critical roles of the proteins LCIB, LCIA or HLA3 in the microalgal CCM. A full understanding of the functional components of the microalgal CCM is an important step in understanding the role of the CCM in controlling microalgal productivity, especially with regard to production of biofuels and other renewable bioproducts from microalgae. Many of the observations made by researchers represent novel and critically important advances in the understanding of the CCM and its role in normal microalgal growth and photosynthesis, as well as understanding how the CCM might be modified to improve photosynthesis and biomass yield of microalgae under industrial conditions. In addition, this fundamental knowledge is key to the process of transferring a microalgal-type CCM into higher plants to improve their productivity. However, although our long-term goals involve transfer of a functional CCM into economically important plants, the introduction of Ci transport genes and other CCM components into a model plant was not included in the scope of this project.
Publications
- Duanmu, D, MH Spalding 2011 Insertional suppressors of Chlamydomonas reinhardtii that restore growth of air-dier lcib mutants in low CO2. Photosynthesis Research 109:123-132.
- Duanmu, D, AR Miller, KM Horken, DP Weeks, MH Spalding, 2009 Knockdown of a limiting-CO2-inducible gene HLA3 decreases bicarbonate transport and photosynthetic Ci-affinity in Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences USA 106:5990-5995.
- Duanmu, D, Y Wang, MH Spalding, 2009 Thylakoid lumen carbonic anhydrase (CAH3) mutation suppresses air-dier phenotype of LCIB mutant in Chlamydomonas reinhardtii. Plant Physiology 149:929-937.
- Spalding, MH 2008 Microalgal carbon-dioxide-concentrating mechanisms: Chlamydomonas inorganic carbon transporters. Journal of Experimental Botany. 59:1463-1473.
- Merchant, S, SE Prochnik, O Vallon, EH Harris, SJ Karpowicz, GB Witman, A Terry, A Salamov, LK Fritz-Laylin, L Marechal-Drouard, WF Marshall, L-H Qu, DR Nelson, AA Sanderfoot, MH Spalding, VV Kapitonov, Q Ren, P Ferris, E Lindquist, H Shapiro, J Grimwood, J Schmutz, IV Grigoriev, Chlamydomonas Annotation Group, DS Rokhsar, AR Grossman 2007 The Chlamydomonas genome reveals evolutionary insights into key animal and plant functions. Science 318:245-250.
- Grossman, AR, M Croft, VN Gladyshev, S Merchant, M Posewitz, S Prochnik, MH Spalding 2007 Novel metabolism in Chlamydomonas through the lens of genomics. Current Opinion in Plant Biology 10:190-198.
- Wang, Y, D Duanmu, D, MH Spalding 2011 Carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture. Photosynthesis Research 109:115-122.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Project researchers have conducted fundamental research to characterize unique metabolic processes involved in the acclimation of microalgae to low CO2 availability, especially how the protein LCIB functions in the inducible carbon dioxide (CO2)-concentrating mechanism (CCM) of the model microalga Chlamydomonas reinhardtii. Researchers have determined that LCIB is located in the aqueous space (stroma) outside the photosynthetic membranes of the chloroplast, but that it is found clustered around the pyrenoid (suborganeller structure in algae that contains the ribulose-bisphosphate carboxylase/oxygenase). Researchers also demonstrated that LCIB interacts with a related protein, LCIC, to form a complex that can be stably isolated from the cells. Characteristics of this complex are under active investigation. Researchers identified eleven new mutations (suppressor mutations) that suppress the lethal phenotype of LCIB mutants, allowing the double mutants to survive. In published work, researchers demonstrated that two of these are in the gene CAH3, which codes for a known thylakoid lumen carbonic anhydrase, and researchers concluded that LCIB functions downstream of CAH3 to prevent loss of CO2 in the CCM metabolic pathway, which represents novel findings regarding the function of LCIB and the CCM pathway in microalgae. The specific gene defects in the other 9 suppressor mutations have not yet been determined, but researchers have characterized 4 of the mutations, su1, su4, su5 and su8, genetically and physiologically and submitted this work for publication. Researchers also are actively mapping two of these mutations to identify the responsible genes. Researchers also investigated the global changes in gene expression that occur in Chlamydomonas in response to exposure to low-CO2 (less than 0.04% CO2) or very-low-CO2 (less than 0.01% CO2) relative to control, high-CO2 (5% CO2) using next-generation sequencing of gene transcripts from the three conditions. These experiments were conducted in a wild-type strain and in a strain, cia5, that is totally defective in induction of the CCM. Over 3,000 genes were observed to change expression under some condition in this study, and researchers are currently preparing a manuscript for publication describing the changes observed. Results of the project experiments reported here will be disseminated to the scientific and public community through further peer-reviewed publications in nationally and internationally renowned journals within the next year. PARTICIPANTS: Dr. Martin H. Spalding (P.I.); Dr. Peng Liu (Co-P.I.; responsible for statistical analyses); Mr. Deqiang Duanmu (former graduate student; graduated in August 2009); Mr. Bo Chen (graduate student; Ms. Han Gao (graduate student); Mr. Wei Fang (graduate student). The project researchers have been involved in the research based training, education and mentoring of the graduate students listed above, as well as multiple undergraduates participating in the project for hourly wages and/or academic credit and one postdoctoral associate (Dr. Yingjun Wang) not directly supported by salary from this project. TARGET AUDIENCES: Target audiences for this research include the scientific research community, especially those involved in research on plant productivity, as well as those involved in education at the undergraduate and graduate level for whom the outputs of this project should provide opportunities for update or revision of educational materials and curricula to reflect the new knowledge disseminated. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The activities of the project researchers have contributed to major changes in the understanding of the functional components of the microalgal CCM. Prior to this research, little was known about the critical roles of the proteins LCIB or HLA3 in the microalgal CCM. A full understanding of the functional components of the microalgal CCM is an important step in understanding the role of the CCM in controlling microalgal productivity, especially with regard to production of biofuels and other renewable bioproducts from microalgae. In addition, this fundamental knowledge is key to the process of transferring a microalgal-type CCM into higher plants to improve their productivity. However, although our long-term goals involve transfer of a functional CCM into economically, as well as -important plants, the introduction of Ci transport genes and other CCM components into a model plant is still beyond the scope of the current project.
Publications
- No publications reported this period
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Project researchers have conducted fundamental research to characterize unique metabolic processes involved in the acclimation of microalgae to low CO2 availability, especially how the protein LCIB functions in the inducible carbon dioxide (CO2)-concentrating mechanism (CCM) of the model microalga Chlamydomonas reinhardtii. Researchers have determined that LCIB is located in the aqueous space (stroma) outside the photosynthetic membranes of the chloroplast. Researchers identified eleven new mutations (suppressor mutations) that suppress the lethal phenotype of LCIB mutants, allowing the double mutants to survive. Two of these suppressor mutations, su6 and su7, are in the gene CAH3, which codes for a known enzyme, carbonic anhydrase, located in the aqueous space (lumen) between the photosynthetic membranes of the chloroplast. Based on the localization and the CAH3 suppressor mutation results, researchers concluded that LCIB functions downstream of CAH3 in the CCM metabolic pathway, which represents novel findings regarding the function of LCIB and the CCM pathway in microalgae. The specific gene defects in the other 9 suppressor mutations have not yet been determined, but researchers have characterized 4 of the mutations, su1, su4, su5 and su8, genetically and physiologically. Genetic analysis identified 2 allelic, dominant suppressors (su4 and su5), and two recessive suppressors (su1 and su8). Consistent with the suppression phenotype, the relative affinities of photosynthetic O2 evolution and the internal inorganic carbon (Ci) accumulation in all four suppressors were substantially increased relative to lcib mutants. Although the growth and relative Ci affinity of su1 and su8 were similar to those of wild-type cells, the relative Ci affinity of su4/su5 was only intermediate between wild type and lcib mutants. These results reveal complex LCIB-dependent and independent active Ci uptake/accumulation systems and provide insight into the roles of LCIB in limiting Ci acclimation. Researchers also investigated the potential roles of additional candidate CO2 transport proteins, HLA3 and LCIA, in the Chlamydomonas CCM. Specific depression of HLA3 abundance combined either with an LCIB mutation and/or specific depression of LCIA abundance resulted in defective growth and photosynthesis specifically under alkaline pH conditions. Based on these results, researchers concluded that HLA3 is directly or indirectly involved in the energy-dependent transport into the microalgal cells of bicarbonate, a form of CO2 that predominates in alkaline conditions, and that LCIA functions in transport of CO2 or bicarbonate into the chloroplast. Researchers also used a modern, high throughput system (microarray analysis) for evaluation of global changes in gene expression that result when Chlamydomonas is exposed to low CO2 environments and identified several new genes that increase expression during acclimation to low CO2 concentrations. Results of the project experiments reported here will be disseminated to the scientific and public community through further peer-reviewed publications in nationally and internationally renowned journals within the next year. PARTICIPANTS: Dr. Martin H. Spalding (P.I.); Dr. Peng Liu (Co-P.I., responsible for statistical analysis); Mr. Deqiang Duanmu (graduate student); Mr. Bo Chen (graduate student; Ms. Han Gao (graduate student); Mr. Wei Fang (graduate student). The project researchers have been involved in the research based training, education and mentoring of the graduate students listed above, as well as multiple undergraduates participating in the project for hourly wages and/or academic credit and one postdoctoral associate (Dr. Yingjun Wang) not directly supported by salary from this project. TARGET AUDIENCES: Target audiences for this research include the scientific research community, especially those involved in research on plant productivity, as well as those involved in education at the undergraduate and graduate level for whom the outputs of this project should provide opportunities for update or revision of educational materials and curricula to reflect the new knowledge disseminated. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The activities of the project researchers have contributed to major changes in the understanding of the functional components of the microalgal CCM. Prior to this research, little was known about the critical roles of the proteins LCIB or HLA3 in the microalgal CCM. A full understanding of the functional components of the microalgal CCM is an important step in understanding the role of the CCM in controlling microalgal productivity, especially with regard to production of biofuels and other renewable bioproducts from microalgae. In addition, this fundamental knowledge is key to the process of transferring a microalgal-type CCM into higher plants to improve their productivity. However, although our long-term goals involve transfer of a functional CCM into economically-important plants, the introduction of Ci transport genes and other CCM components into a model plant is still beyond the scope of the current project.
Publications
- Duanmu, D, AR Miller, KM Horken, DP Weeks, MH Spalding, 2009 Knockdown of a limiting-CO2-inducible gene HLA3 decreases bicarbonate transport and photosynthetic Ci-affinity in Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences USA 106:5990-5995.
- Duanmu, D, Y Wang, MH Spalding, 2009 Thylakoid lumen carbonic anhydrase (CAH3) mutation suppresses air-dier phenotype of LCIB mutant in Chlamydomonas reinhardtii. Plant Physiology 149:929-937.
- Vallon, O, MH Spalding 2009 Amino acid metabolism. In (D Stern, ed, E Harris, Series ed) The Chlamydomonas Sourcebook 2nd Edition, Volume 2: Organellar and Metabolic Processes. Elsevier Publishers, Amsterdam, The Netherlands. (invited chapter).
- Spalding, MH 2009 CO2-concentrating mechanism and carbon assimilation. In (D Stern, ed, E Harris, Series ed) The Chlamydomonas Sourcebook 2nd Edition, Volume 2: Organellar and Metabolic Processes. Elsevier Publishers, Amsterdam, The Netherlands. (invited chapter).
- Spalding, MH 2008 Microalgal carbon-dioxide-concentrating mechanisms: Chlamydomonas inorganic carbon transporters. Journal of Experimental Botany. 59:1463-1473 (refereed, invited review)
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Project researchers have conducted fundamental research to characterize unique metabolic processes involved in the acclimation of microalgae to low CO2 availability, especially exploring how the previously identified protein LCIB functions in the inducible carbon dioxide (CO2)-concentrating mechanism (CCM) of the model microalga Chlamydomonas reinhardtii. Researchers identified six new mutations (suppressor mutations) that suppress the lethal phenotype of mutants defective in LCIB, allowing the double mutants to survive. Two of these suppressor mutations are in the gene CAH3, which codes for a known enzyme, carbonic anhydrase, located in the aqueous space (lumen) between the photosynthetic membranes of the chloroplast. The nature of the other four suppressor mutations have not yet been determined. LCIB also has been determined to be located in the aqueous space (stroma) outside the photosynthetic membranes of the chloroplast. Based on the results reported here, researchers concluded that LCIB functions downstream of CAH3 in the metabolic pathway of the CCM, which represents novel findings informative about the function of the CCM pathway in microalgae. Researchers also have investigated the potential role of additional candidate CO2 transport proteins, HLA3 and LCIA, in the CCM of Chlamydomonas, as well as their interaction with the protein LCIB. Specific depression of HLA3 abundance, when combined either with mutations in LCIB and/or specific depression of LCIA abundance resulted in defective growth and photosynthesis specifically under alkaline pH conditions. Based on these results, researchers concluded that HLA3 is directly or indirectly involved in the energy-dependent transport into the microalgal cells of bicarbonate, a form of CO2 that predominates in alkaline conditions, and that LCIA functions in transport of CO2 or bicarbonate into the chloroplast. Project researchers also used a modern, high throughput system (microarray analysis) for evaluation of global changes in gene expression that result when Chlamydomonas is exposed to low CO2 environments and identified several new genes that increase expression during acclimation to low CO2 concentrations. Results of the project experiments reported here will be disseminated to the scientific and public community through peer-reviewed publications in nationally and internationally renowned journals within the next year, but in the first year this project researchers have published four publications of relevance to this project although not directly funded by this project. PARTICIPANTS: Dr. Martin H. Spalding (P.I.); Dr. Peng Liu (Co-P.I.); Mr. Deqiang Duanmu (graduate student); Mr. Bo Chen (graduate student; Ms. Han Gao (graduate student); Mr. Wei Fang (graduate student). The project researchers have been involved in the research based training, education and mentoring of the graduate students listed above, as well as multiple undergraduates participating in the project for hourly wages and/or academic credit and one postdoctoral associate (Dr. Yingjun Wang) not directly supported by salary from this project. TARGET AUDIENCES: Target audiences for this research include the scientific research community, especially those involved in research on plant productivity, as well as those involved in education at the undergraduate and graduate level for whom the outputs of this project should provide opportunities for update or revision of educational materials and curricula to reflect the new knowledge disseminated. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The activities of the project researchers have contributed to major changes in the understanding of the functional components of the microalgal CCM. A full understanding of the functional components of the microalgal CCM is an important step in the process of transferring a microalgal-type CCM into higher plants to improve their productivity. However, although our long-term goals involve transfer of a functional CCM into economically-important plants, the introduction of Ci transport genes and other CCM components into a model plant is still beyond the scope of the current project.
Publications
- Grossman, AR, M Croft, VN Gladyshev, S Merchant, M Posewitz, S Prochnik, MH Spalding. 2007. Novel metabolism in Chlamydomonas through the lens of genomics. Current Opinion in Plant Biology 10:190-198.
- Wang, Y, MH Spalding. 2007. CO2 concentrating mechanisms in photosynthetic microorganisms. Functional Plant Science and Biotechnology 1:120-128.
- Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L, Marshall WF, Qu LH, Nelson DR, Sanderfoot AA, Spalding MH, Kapitonov VV, Ren Q, Ferris P, Lindquist E, Shapiro H, Lucas SM, Grimwood J, Schmutz J, Cardol P, Cerutti H, Chanfreau G, Chen CL, Cognat V, Croft MT, Dent R, Dutcher S, Fernandez E, Fukuzawa H, Gonzalez-Ballester D, Gonzalez-Halphen D, Hallmann A, Hanikenne M, Hippler M, Inwood W, Jabbari K, Kalanon M, Kuras R, Lefebvre PA, Lemaire SD, Lobanov AV, Lohr M, Manuell A, Meier I, Mets L, Mittag M, Mittelmeier T, Moroney JV, Moseley J, Napoli C, Nedelcu AM, Niyogi K, Novoselov SV, Paulsen IT, Pazour G, Purton S, Ral JP, Riano-Pachon DM, Riekhof W, Rymarquis L, Schroda M, Stern D, Umen J, Willows R, Wilson N, Zimmer SL, Allmer J, Balk J, Bisova K, Chen CJ, Elias M, Gendler K, Hauser C, Lamb MR, Ledford H, Long JC, Minagawa J, Page MD, Pan J, Pootakham W, Roje S, Rose A, Stahlberg E, Terauchi AM, Yang P, Ball S, Bowler C, Dieckmann CL, Gladyshev VN, Green P, Jorgensen R, Mayfield S, Mueller-Roeber B, Rajamani S, Sayre RT, Brokstein P, Dubchak I, Goodstein D, Hornick L, Huang YW, Jhaveri J, Luo Y, Martinez D, Ngau WC, Otillar B, Poliakov A, Porter A, Szajkowski L, Werner G, Zhou K, Grigoriev IV, Rokhsar DS, Grossman AR. 2007. The Chlamydomonas genome reveals evolutionary insights into key animal and plant functions. Science 318:245-250.
- Spalding, MH. 2008. Microalgal carbon-dioxide-concentrating mechanisms: Chlamydomonas inorganic carbon transporters. Journal of Experimental Botany. 59:1463-1473
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