Final Report Summary - COSI (Chloroplast Signals)
Summary of the project objectives
The COSI network integrated young researchers into a highly innovative interdisciplinary approach aiming at identification of regulatory networks governing chloroplast functions. The active participation of Bayer BioScience, a plant biotechnology centre within Bayer CropScience, as a full network partner ensured intersectorial industry-academia cooperation with the long term objective of improving yield and stress robustness of crops. COSI partners work on different aspects of photosynthesis in algae and higher plants, and on environmentally triggered signalling pathways. At the scientific level, COSI focuses on environmentally triggered phosphorylation events and calcium signalling in the regulation of chloroplast metabolism and the coordination of cellular and chloroplast metabolism. Regulation of chloroplast function within the metabolic network of the cell is studied in an integrated working programme, composed of five work packages (WPs). These cover:
- proteomic and genetic approaches to identify involved components;
- metabolomic and physiologic studies to analyse stress adaptation processes and
- cell biological techniques for live cell imaging of calcium signalling.
The integrated systematic training program of COSI was comprised of methodological and complementary skills workshops, accompanied by visits to partner laboratories.
Description of the work performed and main results achieved
Thylakoid protein kinases and their regulation and role in signalling of plant acclimation to environmental changes were studied by the groups of Eva-Mari Aro in Turku and Jean-David Rochaix in Geneva. The STN7 kinase was shown to be essential for providing equal distribution of excitation energy between both photosystems in Arabidopsis enabling fluent photosynthesis despite of fluctuations in light intensity (Tikkanen et al., 2010). Indeed, the growth of both the stn7 and stn7 stn8 kinase mutants is strongly compromised under fluctuating light conditions, mimicking natural growth environments. BN gel analysis indicates that Chlamydomonas Stt7 forms a core complex with the cytochrome b6f complex, and that upon activation of the kinase in state 2 large Stt7 supercomplexes are assembled, appearing to be involved in the reversible transfer of LHCII from PSII to PSI. Proteomic studies revealed striking differences in photosynthetic acclimation between diatoms and higher plants (Grouneva et al., 2011; Bailleul et al., 2010). The Leister group in Munich identified the photosynthesis altered mutant 48 (pam48), in which chloroplast-to-nucleus communication is perturbed. The PAM48 protein shares sequence similarity with human mitochondrial transcription termination factors but was localised to chloroplasts. Currently, it is analysed whether PAM48 regulates chloroplastic gene expression and could act as chloroplastic transcription termination factor (Morosetti et al., under preparation).
Calcium-dependent signalling pathways and the identification of involved components was the focus of the work in the Vothknecht group in Munich and the Teige group in Vienna (Bayer et al., 2011; Stael et al., 2012). Different proteomics approaches and phosphorylation assays identified several targets of Ca2+-dependent phosphorylation (Stael and Rocha et al., 2012). These factors involve components of photosynthesis and related metabolic pathways and putative metabolite transporters in the chloroplast envelope. Cross-talk of chloroplasts and mitochondria in diatoms is studied in the Bowler group in Paris and recent analysis indicates that this chloroplast-mitochondria interaction comprises a sustained electron flow from the chloroplast to the mitochondria, capable of generating an ATP flow in the other direction in sufficient amounts to fuel carbon assimilation in the light. Furthermore, the process also appears to be operative under physiological conditions in P. tricornutum, and photosynthesis is obligately dependent on the functioning of respiration.
We therefore conclude that marine diatoms are obligate photomixotrophs and not photoautotrophs like plants and green algae, as commonly assumed. This finding is highly novel and will require a revision of carbon flow in marine environments, and how energy is generated from light in diatoms.from genome sequencing revealed (Prihoda et al., 2012). For further studies of Ca2+-dependent signalling pathways in response to environmental signals in diatoms, the Bowler group generated reporter lines with GFP-AEQ targeted to the chloroplast or mitochondria. Similarly, to visualise environmentally triggered Ca2+-signals in different subcellular compartments in higher plants, the Vothknecht group in Munich generated a set of YFP-AEQ reporter lines (Mehlmer et al., 2012).
Functional links between stress signalling and metabolic adaptation were studied by the Jonak group in Vienna (Krasensky and Jonak, 2012), the Noctor group in Orsay, the Foyer group in Leeds, and by Bayer BioScience in Gent. A plastid-localised protein kinase, which associates with starch granules, has been studied, mutant lines for this kinase have been established and potential interaction partners have been identified. Sensitivity of its kinase activity to hydrogen peroxide points towards a redox-regulation. The photosynthetic control of gene regulation was studied in the Foyer lab in Leeds and the Noctor lab in Paris (Foyer et al., 2012), and Arabidopsis lines for specific photoreceptors in an oxidative stress genetic background were analysed at the transcriptomic, biochemical, metabolomic, and phenotypic levels in order to uncover new links between light signalling and reactive oxygen species. Glutathione (GSH) was found to co-localise with nuclear DNA at the early stages of proliferation in plant cells and GSH sequestration in the nucleus during the G1 phase of the cell cycle has a profound impact on cellular redox homeostasis, gene expression and glutathione synthesis (Diaz et al., 2010; Noctor et al., 2012). The nuclear GSH pool might act as regulator of nuclear proteins such as poly (ADP-ribose) polymerases (PARP). The influence of chemical inhibition of PARP under different abiotic stress treatments was studied by Bayer BioScience in Gent on a wide spectrum of physiological, biochemical and molecular phenotypes. PARP and Anthocyanin accumulation during stress was found to be associated with an enhanced growth of the plants under stress conditions and that these changes are further associated with photosynthetic, redox and gene expression changes (Schulz et al., 2012).
Light signals were studied by the Falciatore group in Naples (diatoms) and by the Gabrys group in Krakow (higher plants). Analysis of mutants generated in the diatome Phaeodactylum tricornutum indicates that the recently identified putative red light phytochrome (DPh) photoreceptor is involved in the cellular aggregation control, likely due to a key role in the neighbour perception and/or regulation of photo-protection mechanisms Depauw et al., under preparation). The Gabrys group in Krakow studies signalling pathways, which control blue light-induced chloroplast redistribution in the mesophyll of higher plants to identify a link between light and stress / redox signalling, and to define the exact role of calcium in the movements (Banas et al., 2012; Labuz et al., 2012). It was found that infiltration of Arabidopsis leaves with GSH resulted in an enhancement of chloroplast movement response. Using transgenic Aequorin-GFP reporter lines (generated by the Vothknecht group) it was found that chloroplasts might have an input to blue-light-mediated calcium regulation of their movement.
Expected final results and their potential impact and use
We aim at obtaining detailed knowledge of the signalling mechanisms governing chloroplast function and its coordination with the metabolic network of the entire cell. This includes involved kinases, potentially also phosphatases, and also factors involved in Ca2+-signalling and decoding of those signals. Understanding of these molecular mechanisms enabling acclimation to stress conditions is vital for sustainable agriculture in a changing environment.
The COSI network integrated young researchers into a highly innovative interdisciplinary approach aiming at identification of regulatory networks governing chloroplast functions. The active participation of Bayer BioScience, a plant biotechnology centre within Bayer CropScience, as a full network partner ensured intersectorial industry-academia cooperation with the long term objective of improving yield and stress robustness of crops. COSI partners work on different aspects of photosynthesis in algae and higher plants, and on environmentally triggered signalling pathways. At the scientific level, COSI focuses on environmentally triggered phosphorylation events and calcium signalling in the regulation of chloroplast metabolism and the coordination of cellular and chloroplast metabolism. Regulation of chloroplast function within the metabolic network of the cell is studied in an integrated working programme, composed of five work packages (WPs). These cover:
- proteomic and genetic approaches to identify involved components;
- metabolomic and physiologic studies to analyse stress adaptation processes and
- cell biological techniques for live cell imaging of calcium signalling.
The integrated systematic training program of COSI was comprised of methodological and complementary skills workshops, accompanied by visits to partner laboratories.
Description of the work performed and main results achieved
Thylakoid protein kinases and their regulation and role in signalling of plant acclimation to environmental changes were studied by the groups of Eva-Mari Aro in Turku and Jean-David Rochaix in Geneva. The STN7 kinase was shown to be essential for providing equal distribution of excitation energy between both photosystems in Arabidopsis enabling fluent photosynthesis despite of fluctuations in light intensity (Tikkanen et al., 2010). Indeed, the growth of both the stn7 and stn7 stn8 kinase mutants is strongly compromised under fluctuating light conditions, mimicking natural growth environments. BN gel analysis indicates that Chlamydomonas Stt7 forms a core complex with the cytochrome b6f complex, and that upon activation of the kinase in state 2 large Stt7 supercomplexes are assembled, appearing to be involved in the reversible transfer of LHCII from PSII to PSI. Proteomic studies revealed striking differences in photosynthetic acclimation between diatoms and higher plants (Grouneva et al., 2011; Bailleul et al., 2010). The Leister group in Munich identified the photosynthesis altered mutant 48 (pam48), in which chloroplast-to-nucleus communication is perturbed. The PAM48 protein shares sequence similarity with human mitochondrial transcription termination factors but was localised to chloroplasts. Currently, it is analysed whether PAM48 regulates chloroplastic gene expression and could act as chloroplastic transcription termination factor (Morosetti et al., under preparation).
Calcium-dependent signalling pathways and the identification of involved components was the focus of the work in the Vothknecht group in Munich and the Teige group in Vienna (Bayer et al., 2011; Stael et al., 2012). Different proteomics approaches and phosphorylation assays identified several targets of Ca2+-dependent phosphorylation (Stael and Rocha et al., 2012). These factors involve components of photosynthesis and related metabolic pathways and putative metabolite transporters in the chloroplast envelope. Cross-talk of chloroplasts and mitochondria in diatoms is studied in the Bowler group in Paris and recent analysis indicates that this chloroplast-mitochondria interaction comprises a sustained electron flow from the chloroplast to the mitochondria, capable of generating an ATP flow in the other direction in sufficient amounts to fuel carbon assimilation in the light. Furthermore, the process also appears to be operative under physiological conditions in P. tricornutum, and photosynthesis is obligately dependent on the functioning of respiration.
We therefore conclude that marine diatoms are obligate photomixotrophs and not photoautotrophs like plants and green algae, as commonly assumed. This finding is highly novel and will require a revision of carbon flow in marine environments, and how energy is generated from light in diatoms.from genome sequencing revealed (Prihoda et al., 2012). For further studies of Ca2+-dependent signalling pathways in response to environmental signals in diatoms, the Bowler group generated reporter lines with GFP-AEQ targeted to the chloroplast or mitochondria. Similarly, to visualise environmentally triggered Ca2+-signals in different subcellular compartments in higher plants, the Vothknecht group in Munich generated a set of YFP-AEQ reporter lines (Mehlmer et al., 2012).
Functional links between stress signalling and metabolic adaptation were studied by the Jonak group in Vienna (Krasensky and Jonak, 2012), the Noctor group in Orsay, the Foyer group in Leeds, and by Bayer BioScience in Gent. A plastid-localised protein kinase, which associates with starch granules, has been studied, mutant lines for this kinase have been established and potential interaction partners have been identified. Sensitivity of its kinase activity to hydrogen peroxide points towards a redox-regulation. The photosynthetic control of gene regulation was studied in the Foyer lab in Leeds and the Noctor lab in Paris (Foyer et al., 2012), and Arabidopsis lines for specific photoreceptors in an oxidative stress genetic background were analysed at the transcriptomic, biochemical, metabolomic, and phenotypic levels in order to uncover new links between light signalling and reactive oxygen species. Glutathione (GSH) was found to co-localise with nuclear DNA at the early stages of proliferation in plant cells and GSH sequestration in the nucleus during the G1 phase of the cell cycle has a profound impact on cellular redox homeostasis, gene expression and glutathione synthesis (Diaz et al., 2010; Noctor et al., 2012). The nuclear GSH pool might act as regulator of nuclear proteins such as poly (ADP-ribose) polymerases (PARP). The influence of chemical inhibition of PARP under different abiotic stress treatments was studied by Bayer BioScience in Gent on a wide spectrum of physiological, biochemical and molecular phenotypes. PARP and Anthocyanin accumulation during stress was found to be associated with an enhanced growth of the plants under stress conditions and that these changes are further associated with photosynthetic, redox and gene expression changes (Schulz et al., 2012).
Light signals were studied by the Falciatore group in Naples (diatoms) and by the Gabrys group in Krakow (higher plants). Analysis of mutants generated in the diatome Phaeodactylum tricornutum indicates that the recently identified putative red light phytochrome (DPh) photoreceptor is involved in the cellular aggregation control, likely due to a key role in the neighbour perception and/or regulation of photo-protection mechanisms Depauw et al., under preparation). The Gabrys group in Krakow studies signalling pathways, which control blue light-induced chloroplast redistribution in the mesophyll of higher plants to identify a link between light and stress / redox signalling, and to define the exact role of calcium in the movements (Banas et al., 2012; Labuz et al., 2012). It was found that infiltration of Arabidopsis leaves with GSH resulted in an enhancement of chloroplast movement response. Using transgenic Aequorin-GFP reporter lines (generated by the Vothknecht group) it was found that chloroplasts might have an input to blue-light-mediated calcium regulation of their movement.
Expected final results and their potential impact and use
We aim at obtaining detailed knowledge of the signalling mechanisms governing chloroplast function and its coordination with the metabolic network of the entire cell. This includes involved kinases, potentially also phosphatases, and also factors involved in Ca2+-signalling and decoding of those signals. Understanding of these molecular mechanisms enabling acclimation to stress conditions is vital for sustainable agriculture in a changing environment.