Final Report Summary - CALIPSO (Calcium- and light signals in photosynthetic organisms)
Summary of the project objectives
CALIPSO (http://itn-calipso.univie.ac.at/) aimed at identifying environmentally triggered regulatory calcium signals and protein phosphorylation events that control photosynthesis and metabolism. CALIPSO partners work with a wide range of different organisms covering the full phylogenetic spectrum from algae to higher plants including economically important crops. In four scientific work packages, they combine a wide spectrum of different technologies to uncover how photosynthetic organisms acclimate to changing environmental conditions or stress. The long-term aim of the projects was to identify the components of an integrative model of acclimation in photosynthetic organisms and translation into crops and biotechnologically relevant alga. State-of-the-art technology from various disciplines of life science ranging from molecular biology and genetics (transgenic algae and plants, gene expression analysis), cell biology (protein localization, Ca2+ live cell imaging), biochemistry (kinase/phosphatase assays, proteomics, protein purification), and high throughput biology (GC-MS based metabolomics and Mass-western analysis) were combined with bioinformatics (motif and domain identification, network analysis) to trace the evolution of key proteins and mechanisms to finally understand conserved functions across different species. In CALIPSO, these four highly interconnected scientific work packages were complemented by training and management work packages. Most employees were active within more than one of the four scientific WPs.
Description of the work performed and main results achieved
Signal perception and processing by protein phosphorylation
Functional links between stress signalling, reversible protein phosphorylation, and metabolic adaptation were studied by the Jonak and the Teige groups in Vienna, the group of Michel Goldschmidt-Clermont in Geneva, and by Bayer Crop Science in Gent. A plastid-localised protein kinase, which associates with starch granules, has been studied in the Jonak group. Mutant lines for this kinase have been established and potential interaction partners have been identified. Within the framework of CALIPSO, the localization of a novel protein kinase to the chloroplast has been verified. Interestingly, the presence of this kinase in chloroplasts appeared to be regulated by different environmental cues. Genetic analyses indicate that this novel chloroplast-localized protein kinase is involved in regulating plant vitality under unfavourable environmental conditions.
Furthermore, three chloroplast proteins have been identified as direct phosphorylation targets of this kinase and might link protein phosphorylation events to Ca2+ signalling in the chloroplast. Thylakoid protein phosphorylation and its regulation and role in signalling of acclimation to environmental changes were studied in the group of Michel Goldschmidt-Clermont in Geneva in Chlamydomonas using chloroplast transformation. Mutants of the major phosphorylation sites in the PSII subunits PsbC, PsbD and PsbH were obtained, and non-phosphorylatable mutants of single subunits did not show any significant phenotype, an indication that the different sites may have redundant roles. Chlamydomonas lines with mutations in multiple subunits have now been obtained, and their characterization should help clarify this issue.
The laboratory of Halina Gabrys at the Jagiellonian University of Krakow (PL) studied the expression of phototropins and their dephosphorylation in wild type Arabidopsis plants and in mutants showing differences in chloroplast responses to blue light pulses. The results provide evidence that phototropins cooperate rather than compete in eliciting chloroplast movements. To link phototropin signalling leading to chloroplast redistribution with phototropin phosphorylation status, chloroplast responses to light pulses were examined in mutants of selected subunits of PP2A phosphatase. The phosphatase was shown to affect expression of both phototropins but it does not seem to participate in the chloroplast movement signaling pathway (Sztatelman et al. 2016).
Signal perception and processing by calcium
This work was carried on in the labs of Marc Knight (Durham University, UK) and of Ute Vothknecht in Bonn (DE) using higher plants and in the group of Eva-Mari Aro in Turku (FI) in cyanobacteria. Our specific research interest was how calcium affects the chloroplast functioning as well as the role of chloroplast calcium increases in controlling plant physiology. The Calcium sensing protein CaS was investigated in the Vothknecht lab in Bonn. It was found that CaS revealed a strong differential regulation by diurnal variation of protein content as well as phosphorylation. CaS content is highest near the end of the day and lowest at the end of night. One of the major stresses for the chloroplast is heat, as it impairs photosynthesis. A chloroplast-specific calcium response was identified in response to heat in Durham University (UK), and no corresponding increase in calcium was seen in the cytosol. The properties of this chloroplast calcium response have been investigated and the problem of how calcium signal is decoded was approached by making a mathematical model of the immune response in Arabidopsis. This model is able to describe the flow of information in the biological pathway and predict the plant response to any calcium kinetics, giving insights how calcium signalling is decoded in plants.
The study of Ca2+ signalling in cyanobacteria, the photosynthetic ancestors of the plant chloroplast, in the lab of Eva-Mari Aro revealed that Ca2+ is used to communicate changes in the metabolic states in cyanobacteria, modifying gene expression and adjusting the carbon:nitrogen balance of the cell (Walter et al. 2016). A novel cyanobacterial Ca2+-binding protein was discovered, and was shown to be important for cell differentiation and for harvesting of atmospheric nitrogen (Walter et al. submitted). This work has demonstrated the importance of Ca2+ signalling at the earliest stages of photosynthetic evolution.
Crosstalk between Ca2+ signalling and phosphorylation
Novel Ca2+-binding proteins with localization in the chloroplast stroma were further characterized in the Teige group in Vienna. These two proteins are functionally redundant and their complete knockout or strong overexpression resulted in severe phenotypes strongly affecting photosynthesis and chloroplast development.
It was further found that thylakoid protein phosphorylation and the chloroplast ultrastructure were strongly altered in mutants of those Ca2+-binding proteins. Similar observations were made by the Vothknecht lab in Bonn, where it was found that also the CaS protein can be phosphorylated at Serine 373 by a unknown calcium-dependent stromal kinase but also by a thylakoid-localized kinase in a non-calcium dependent manner. Analysis of cas mutant plants further revealed a difference in thylakoid stacking during the daytime when compared to the wildtype, while no differences could be observed in the night or under far red light conditions.
The group of Halina Gabryś in Krakow (PL) studied Calcium fluxes induced by blue light in Arabidopsis mesophyll cells by transmission electron microscopy followed by energy-dispersive X-ray microanalysis (Łabuz et al. 2016). This study revealed that calcium re-localization after blue light treatment requires the presence of phototropin2, as it is absent in the phot2 mutant which lacks chloroplast avoidance in strong light and in the phot1phot2 mutant. In general, phototropin2 was shown to be involved in the control of Ca2+ homeostasis in mesophyll cells.
Signal integration and translation into crops and biotech
Exploiting the genetic diversity of photosynthetic traits in a relevant mapping population of bread wheat has the focus of Bayer Crop Science in Gent (BE). The establishment of phenotyping protocols to assess photosynthetic characteristics, including growth rate, leaf photosynthesis and metabolite fingerprinting, helped us to better understand the genetic basis of improved photosynthetic efficiency in wheat. Data obtained from field and growth chamber grown plants strengthened that approach. Those insights and knowledge will provide valuable input in our wheat breeding activities in the future. In general, it is stated that under water deficit photosynthesis is downregulated, but little is known on possible drought-specific reorganization of the photosynthetic apparatus. By combining structural and functional data, the Teige group in Vienna found that drought-induced re-configurations of light reactions, allows the adjustment of photosynthetic activity under drought upon fluctuations of light intensity, in wheat and Arabidopsis. Interestingly, wheat and Arabidopsis appear to use different strategies to adjust the photosynthetic apparatus to water deficit: in wheat such re-configuration includes a change of steady-state PSII-LHCII phosphorylation, while in Arabidopsis this re-configuration is independent of phosphorylation.
Dynamic acclimation responses of algae to environmental fluctuations or nutrient limitations are particularly relevant for biotech applications. In this context, the Weckwerth group at the University of Vienna (AT) investigated the proteome and phosphoproteome acclimation of Chlamydomonas to nitrogen depletion and recovery as well as to a rapamycin treatment. The aim of those studies was to investigate the dynamic adaptation of the AMPK-TOR signalling during nutrient stress in micro-algae, which was also done in collaboration with Ebergsberger group in Frankfurt. The main focus of the Ebersberger group at the Goethe University in Frankfurt (DE) was the tracing of functional protein networks from model to non-model organisms. The toolbox of available bioinformatics methods for this purpose was extended by the development and implementation of protTrace (https://github.com/BIONF/protTrace) to estimate for individual proteins the evolutionary distances beyond which the sequence similiarity between orthologs becomes too low to infer common ancestry. The concept of evolutionary traceability, and the applicability of protTrace was initially investigated on the example of the yeast gene set (Jain et al. submitted), the tracing of functional protein interaction networks was demonstrated on the AMPK-TOR pathway (Roustan et al. 2016, Jain et al. 2018), and the tracing of the plant heat stress response pathway across photosynthesizing organisms is about to be completed.
The group of Angela Falciatore at UPMC (Paris, FR) provided novel information on the light perception and acclimation mechanisms in diatoms. By discovering the first Phytochrome photoreceptor mediating far-red light signalling in marine algae, our findings open completely novel perspectives on the evolution and functional diversification of aquatic light sensors. The possibile role of diatom Phytochrome as depth detector or as sensor of neighbour cells, by perceiving far-red lights produced by biotic sources (e.g. cholorophyll fluorescence of photosynthetic organisms) is currently investigated. Moreover, UPMC uncovered the key function of members of the light-harvesting complex stress-related family, LHCXs, in the regulation of photoprotective mechanisms. We found that multiple abiotic stress signals and chloroplast-mediated signals converge to regulate the LHCX content of cells. The expansion of the LHCX gene family found in diatoms reflects functional diversification of its members and provides a way to fine-tune light harvesting and photoprotection under different environmental conditions.
Socio economic impact of our work
In CALIPSO, we aimed 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.
CALIPSO (http://itn-calipso.univie.ac.at/) aimed at identifying environmentally triggered regulatory calcium signals and protein phosphorylation events that control photosynthesis and metabolism. CALIPSO partners work with a wide range of different organisms covering the full phylogenetic spectrum from algae to higher plants including economically important crops. In four scientific work packages, they combine a wide spectrum of different technologies to uncover how photosynthetic organisms acclimate to changing environmental conditions or stress. The long-term aim of the projects was to identify the components of an integrative model of acclimation in photosynthetic organisms and translation into crops and biotechnologically relevant alga. State-of-the-art technology from various disciplines of life science ranging from molecular biology and genetics (transgenic algae and plants, gene expression analysis), cell biology (protein localization, Ca2+ live cell imaging), biochemistry (kinase/phosphatase assays, proteomics, protein purification), and high throughput biology (GC-MS based metabolomics and Mass-western analysis) were combined with bioinformatics (motif and domain identification, network analysis) to trace the evolution of key proteins and mechanisms to finally understand conserved functions across different species. In CALIPSO, these four highly interconnected scientific work packages were complemented by training and management work packages. Most employees were active within more than one of the four scientific WPs.
Description of the work performed and main results achieved
Signal perception and processing by protein phosphorylation
Functional links between stress signalling, reversible protein phosphorylation, and metabolic adaptation were studied by the Jonak and the Teige groups in Vienna, the group of Michel Goldschmidt-Clermont in Geneva, and by Bayer Crop Science in Gent. A plastid-localised protein kinase, which associates with starch granules, has been studied in the Jonak group. Mutant lines for this kinase have been established and potential interaction partners have been identified. Within the framework of CALIPSO, the localization of a novel protein kinase to the chloroplast has been verified. Interestingly, the presence of this kinase in chloroplasts appeared to be regulated by different environmental cues. Genetic analyses indicate that this novel chloroplast-localized protein kinase is involved in regulating plant vitality under unfavourable environmental conditions.
Furthermore, three chloroplast proteins have been identified as direct phosphorylation targets of this kinase and might link protein phosphorylation events to Ca2+ signalling in the chloroplast. Thylakoid protein phosphorylation and its regulation and role in signalling of acclimation to environmental changes were studied in the group of Michel Goldschmidt-Clermont in Geneva in Chlamydomonas using chloroplast transformation. Mutants of the major phosphorylation sites in the PSII subunits PsbC, PsbD and PsbH were obtained, and non-phosphorylatable mutants of single subunits did not show any significant phenotype, an indication that the different sites may have redundant roles. Chlamydomonas lines with mutations in multiple subunits have now been obtained, and their characterization should help clarify this issue.
The laboratory of Halina Gabrys at the Jagiellonian University of Krakow (PL) studied the expression of phototropins and their dephosphorylation in wild type Arabidopsis plants and in mutants showing differences in chloroplast responses to blue light pulses. The results provide evidence that phototropins cooperate rather than compete in eliciting chloroplast movements. To link phototropin signalling leading to chloroplast redistribution with phototropin phosphorylation status, chloroplast responses to light pulses were examined in mutants of selected subunits of PP2A phosphatase. The phosphatase was shown to affect expression of both phototropins but it does not seem to participate in the chloroplast movement signaling pathway (Sztatelman et al. 2016).
Signal perception and processing by calcium
This work was carried on in the labs of Marc Knight (Durham University, UK) and of Ute Vothknecht in Bonn (DE) using higher plants and in the group of Eva-Mari Aro in Turku (FI) in cyanobacteria. Our specific research interest was how calcium affects the chloroplast functioning as well as the role of chloroplast calcium increases in controlling plant physiology. The Calcium sensing protein CaS was investigated in the Vothknecht lab in Bonn. It was found that CaS revealed a strong differential regulation by diurnal variation of protein content as well as phosphorylation. CaS content is highest near the end of the day and lowest at the end of night. One of the major stresses for the chloroplast is heat, as it impairs photosynthesis. A chloroplast-specific calcium response was identified in response to heat in Durham University (UK), and no corresponding increase in calcium was seen in the cytosol. The properties of this chloroplast calcium response have been investigated and the problem of how calcium signal is decoded was approached by making a mathematical model of the immune response in Arabidopsis. This model is able to describe the flow of information in the biological pathway and predict the plant response to any calcium kinetics, giving insights how calcium signalling is decoded in plants.
The study of Ca2+ signalling in cyanobacteria, the photosynthetic ancestors of the plant chloroplast, in the lab of Eva-Mari Aro revealed that Ca2+ is used to communicate changes in the metabolic states in cyanobacteria, modifying gene expression and adjusting the carbon:nitrogen balance of the cell (Walter et al. 2016). A novel cyanobacterial Ca2+-binding protein was discovered, and was shown to be important for cell differentiation and for harvesting of atmospheric nitrogen (Walter et al. submitted). This work has demonstrated the importance of Ca2+ signalling at the earliest stages of photosynthetic evolution.
Crosstalk between Ca2+ signalling and phosphorylation
Novel Ca2+-binding proteins with localization in the chloroplast stroma were further characterized in the Teige group in Vienna. These two proteins are functionally redundant and their complete knockout or strong overexpression resulted in severe phenotypes strongly affecting photosynthesis and chloroplast development.
It was further found that thylakoid protein phosphorylation and the chloroplast ultrastructure were strongly altered in mutants of those Ca2+-binding proteins. Similar observations were made by the Vothknecht lab in Bonn, where it was found that also the CaS protein can be phosphorylated at Serine 373 by a unknown calcium-dependent stromal kinase but also by a thylakoid-localized kinase in a non-calcium dependent manner. Analysis of cas mutant plants further revealed a difference in thylakoid stacking during the daytime when compared to the wildtype, while no differences could be observed in the night or under far red light conditions.
The group of Halina Gabryś in Krakow (PL) studied Calcium fluxes induced by blue light in Arabidopsis mesophyll cells by transmission electron microscopy followed by energy-dispersive X-ray microanalysis (Łabuz et al. 2016). This study revealed that calcium re-localization after blue light treatment requires the presence of phototropin2, as it is absent in the phot2 mutant which lacks chloroplast avoidance in strong light and in the phot1phot2 mutant. In general, phototropin2 was shown to be involved in the control of Ca2+ homeostasis in mesophyll cells.
Signal integration and translation into crops and biotech
Exploiting the genetic diversity of photosynthetic traits in a relevant mapping population of bread wheat has the focus of Bayer Crop Science in Gent (BE). The establishment of phenotyping protocols to assess photosynthetic characteristics, including growth rate, leaf photosynthesis and metabolite fingerprinting, helped us to better understand the genetic basis of improved photosynthetic efficiency in wheat. Data obtained from field and growth chamber grown plants strengthened that approach. Those insights and knowledge will provide valuable input in our wheat breeding activities in the future. In general, it is stated that under water deficit photosynthesis is downregulated, but little is known on possible drought-specific reorganization of the photosynthetic apparatus. By combining structural and functional data, the Teige group in Vienna found that drought-induced re-configurations of light reactions, allows the adjustment of photosynthetic activity under drought upon fluctuations of light intensity, in wheat and Arabidopsis. Interestingly, wheat and Arabidopsis appear to use different strategies to adjust the photosynthetic apparatus to water deficit: in wheat such re-configuration includes a change of steady-state PSII-LHCII phosphorylation, while in Arabidopsis this re-configuration is independent of phosphorylation.
Dynamic acclimation responses of algae to environmental fluctuations or nutrient limitations are particularly relevant for biotech applications. In this context, the Weckwerth group at the University of Vienna (AT) investigated the proteome and phosphoproteome acclimation of Chlamydomonas to nitrogen depletion and recovery as well as to a rapamycin treatment. The aim of those studies was to investigate the dynamic adaptation of the AMPK-TOR signalling during nutrient stress in micro-algae, which was also done in collaboration with Ebergsberger group in Frankfurt. The main focus of the Ebersberger group at the Goethe University in Frankfurt (DE) was the tracing of functional protein networks from model to non-model organisms. The toolbox of available bioinformatics methods for this purpose was extended by the development and implementation of protTrace (https://github.com/BIONF/protTrace) to estimate for individual proteins the evolutionary distances beyond which the sequence similiarity between orthologs becomes too low to infer common ancestry. The concept of evolutionary traceability, and the applicability of protTrace was initially investigated on the example of the yeast gene set (Jain et al. submitted), the tracing of functional protein interaction networks was demonstrated on the AMPK-TOR pathway (Roustan et al. 2016, Jain et al. 2018), and the tracing of the plant heat stress response pathway across photosynthesizing organisms is about to be completed.
The group of Angela Falciatore at UPMC (Paris, FR) provided novel information on the light perception and acclimation mechanisms in diatoms. By discovering the first Phytochrome photoreceptor mediating far-red light signalling in marine algae, our findings open completely novel perspectives on the evolution and functional diversification of aquatic light sensors. The possibile role of diatom Phytochrome as depth detector or as sensor of neighbour cells, by perceiving far-red lights produced by biotic sources (e.g. cholorophyll fluorescence of photosynthetic organisms) is currently investigated. Moreover, UPMC uncovered the key function of members of the light-harvesting complex stress-related family, LHCXs, in the regulation of photoprotective mechanisms. We found that multiple abiotic stress signals and chloroplast-mediated signals converge to regulate the LHCX content of cells. The expansion of the LHCX gene family found in diatoms reflects functional diversification of its members and provides a way to fine-tune light harvesting and photoprotection under different environmental conditions.
Socio economic impact of our work
In CALIPSO, we aimed 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.