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Regulatory Functions of Protein Phosphorylation in Plant Photosynthetic Membranes
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Oxygenic photosynthesis is the process in plants, algae and cyanobacteria which converts light energy from the sun into carbohydrates and at the same time produces oxygen from water. Both carbohydrates and oxygen are essential to sustain life on earth. Sunlight is thus a necessity for life, but it can also cause severe problems for photosynthetic organisms, which have evolved several remarkable acclimation systems to cope with light fluctuations in the environment. In higher plants the light driven reactions of photosynthesis proceed in the chloroplast thylakoid membranes highly organized into stacked regions of grana and interconnecting stroma  lamellae. The grana structure is thought to provide functional benefits in the processes of acclimation of the photosynthetic apparatus, particularly in the quality control of photosystem II (PSII) were photodamaged PSII is repaired in a stepwise manner. These processes in the thylakoid membranes were suggested to be regulated by reversible phosphorylation of several proteins in PSII and in its light harvesting antennae complexes (LHCII). Two thylakoid protein kinases, called STN8 and STN7, have been previously identified as responsible for the phosphorylation of PSII and LHCII, respectively. However, molecular mechanisms and the exact functions of these protein phosphorylation events remained largely unknown.

In this thesis research I have demonstrated that the PSII protein phosphorylation is needed for the maintenance of the thylakoid structure in Arabidopsis thaliana chloroplasts. A big part of the work on characterization of proteins and their phosphorylation has been done using novel mass spectrometry techniques, and we further developed a label-free method for quantitative studies of protein phosphorylation. The phosphorylation of PSII proteins was found to be diurnal regulated and required for maintenance of the cation-dependent functional stacking of the thylakoid membranes. This phosphorylation was further shown to be important for the regulated turnover of the D1 protein of PSII.

Phosphorylation of the plant specific TSP9 protein was found to be dependent on STN7 kinase, and plants deficient in TSP9 showed reduced ability to perform the photosynthetic state transitions and to execute thermal dissipation of excess light energy under high light conditions. I also accomplished characterization of the protein phosphorylation in thylakoids from Arabidopsis plants subjected to high light treatment and discovered STN7-dependent phosphorylation of the antenna protein CP29 required for the adaptive disassembly of PSII supercomplexes in conditions of high light stress.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2010. , 42 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1212
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-62303ISBN: 978-91-7393-301-8 (print)OAI: oai:DiVA.org:liu-62303DiVA: diva2:372590
Public defence
2010-12-17, Linden, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2010-11-26 Created: 2010-11-26 Last updated: 2010-11-26Bibliographically approved
List of papers
1. Phosphorylation of Photosystem II Controls Functional Macroscopic Folding of Photosynthetic Membranes in Arabidopsis
Open this publication in new window or tab >>Phosphorylation of Photosystem II Controls Functional Macroscopic Folding of Photosynthetic Membranes in Arabidopsis
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2009 (English)In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 21, no 12, 3950-3964 p.Article in journal (Refereed) Published
Abstract [en]

Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstructed lateral migration of the photosystem II reaction center protein D1 and of processing protease FtsH between the stacked and unstacked membrane domains, suppressing turnover of damaged D1 in the leaves exposed to highlight. These findings show that the high level of photosystem II phosphorylation in plants is required for adjustment of macroscopic folding of large photosynthetic membranes modulating lateral mobility of membrane proteins and sustained photosynthetic activity.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-54057 (URN)10.1105/tpc.109.069435 (DOI)000274196000019 ()
Available from: 2010-02-22 Created: 2010-02-22 Last updated: 2017-12-12
2. A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes
Open this publication in new window or tab >>A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes
2010 (English)In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 6Article in journal (Refereed) Published
Abstract [en]

Phosphorylation of photosystem II (PSII) proteins affects macroscopic structure of thylakoid photosynthetic membranes in chloroplasts of the model plant Arabidopsis. In this study, light-scattering spectroscopy revealed that stacking of thylakoids isolated from wild type Arabidopsis and the mutant lacking STN7 protein kinase was highly influenced by cation (Mg++) concentrations. The stacking of thylakoids from the stn8 and stn7stn8 mutants, deficient in STN8 kinase and consequently in light-dependent phosphorylation of PSII, was increased even in the absence of Mg++. Additional PSII protein phosphorylation in wild type plants exposed to high light enhanced Mg++-dependence of thylakoid stacking. Protein phosphorylation in the plant leaves was analyzed during day, night and prolonged darkness using three independent techniques: immunoblotting with anti-phosphothreonine antibodies; Diamond ProQ phosphoprotein staining; and quantitative mass spectrometry of peptides released from the thylakoid membranes by trypsin. All assays revealed dark/night-induced increase in phosphorylation of the 43 kDa chlorophyll-binding protein CP43, which compensated for decrease in phosphorylation of the other PSII proteins in wild type and stn7, but not in the stn8 and stn7stn8 mutants. Quantitative mass spectrometry determined that every PSII in wild type and stn7 contained on average 2.5 +/- 0.1 or 1.4 +/- 0.1 phosphoryl groups during day or night, correspondingly, while less than every second PSII had a phosphoryl group in stn8 and stn7stn8. It is postulated that functional cation-dependent stacking of plant thylakoid membranes requires at least one phosphoryl group per PSII, and increased phosphorylation of PSII in plants exposed to high light enhances stacking dynamics of the photosynthetic membranes.

Place, publisher, year, edition, pages
Public Library of Science (PLoS), 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-57385 (URN)10.1371/journal.pone.0010963 (DOI)000278380500009 ()
Note
Original Publication: Rikard Fristedt, Pontus Granath and Alexander Vener, A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes, 2010, PLOS ONE, (5), 6. http://dx.doi.org/10.1371/journal.pone.0010963 Copyright: Public Library of Science (PLoS) http://www.plos.org/ Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2010-11-26
3. Intrinsically Unstructured Phosphoprotein TSP9 Regulates Light Harvesting in Arabidopsis thaliana
Open this publication in new window or tab >>Intrinsically Unstructured Phosphoprotein TSP9 Regulates Light Harvesting in Arabidopsis thaliana
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2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 2, 499-509 p.Article in journal (Refereed) Published
Abstract [en]

Thylakoid-soluble phosphoprotein of 9 kDa, TSP9, is an intrinsically unstructured plant-specific protein [Song, J., et al. (2006) Biochemistry 45, 15633-15643] with unknown function but established associations with light-harvesting proteins and peripheries of both photosystems [Hansson, M., et al. (2007) J. Biol. Chem. 282, 16214-16222]. To investigate the function of this protein, we used a combination of reverse genetics and biochemical and fluorescence measurement methods in Arabidopsis thaliana. Differential gene expression analysis of plants with a T-DNA insertion in the TSP9 gene using an array of 24000 Arabidopsis genes revealed disappearance of high light-dependent induction of a specific set of mostly signaling and unknown proteins. TSP9-deficient plants had reduced levels of in vivo phosphorylation of light-harvesting complex II polypeptides. Recombinant TSP9 was phosphorylated in light by thylakoid membranes isolated from the wild-type and mutant plants lacking STN8 protein kinase but not by the thylakoids deficient in STN7 kinase, essential for photosynthetic state transitions. TSP9-lacking mutant and RNAi plants with downregulation of TSP9 showed reduced ability to perform state transitions. The nonphotochemical quenching of chlorophyll fluorescence at high light intensities was also less efficient in the mutant compared to wild-type plants. Blue native electrophoresis of thylakoid membrane protein complexes revealed that TSP9 deficiency increased relative stability of photosystem II dimers and supercomplexes. It is concluded that TSP9 regulates plant light harvesting acting as a membrane-binding protein facilitating dissociation of light-harvesting proteins from photosystem II.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16524 (URN)10.1021/bi8016334 (DOI)
Available from: 2009-01-30 Created: 2009-01-30 Last updated: 2017-12-14Bibliographically approved
4. High light induced disassembly of photosystem II supercomplexes in Arabidopsis requires STN7-dependent phosphorylation of CP29
Open this publication in new window or tab >>High light induced disassembly of photosystem II supercomplexes in Arabidopsis requires STN7-dependent phosphorylation of CP29
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9Article in journal (Refereed) Published
Abstract [en]

Photosynthetic oxidation of water and production of oxygen by photosystem II (PSII) in thylakoid membranes of plant chloroplasts is highly affected by changes in light intensities. To minimize damage imposed by excessive sunlight and sustain the photosynthetic activity PSII, organized in supercomplexes with its light harvesting antenna, undergoes conformational changes, disassembly and repair via not clearly understood mechanisms. We characterized the phosphoproteome of the thylakoid membranes from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutant plants exposed to high light. The high light treatment of the wild type and stn8 caused specific increase in phosphorylation of Lhcb4.1 and Lhcb4.2 isoforms of the PSII linker protein CP29 at five different threonine residues. Phosphorylation of CP29 at four of these residues was not found in stn7 and stn7stn8 lacking the STN7 protein kinase. Blue native gel electrophoresis followed by immunological and mass spectrometric analyses of the protein complexes revealed that the high light treatment of the wild type caused migration of CP29 from the PSII supercomplexes to PSII dimers and monomers. A similar high-light-induced disassembly of the PSII supercomplexes occurred in stn8, but not in stn7 and stn7stn8. Transfer of the high-light-treated wild type plants to normal light relocated CP29 back to PSII supercomplexes. We postulate that disassembly of the PSII supercomplexes in plants exposed to high light operate via the STN7-kinase-dependent phosphorylation of the linker protein CP29. Disruption of this adaptive mechanism can explain dramatically retarded growth of the stn7 and stn7stn8 mutants under fluctuating normal/high light conditions.

Place, publisher, year, edition, pages
PloS, 2011
Keyword
Photosynthesis, chloroplast, thylakoid, protein phosphorylation, photosystem II supercomplexes, CP29
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-62302 (URN)10.1371/journal.pone.0024565 (DOI)000294802500061 ()
Note
Funding agencies|Swedish Research Council||Swedish Research Council for Environment, Agriculture and Spatial Planning||Available from: 2010-11-26 Created: 2010-11-26 Last updated: 2017-12-12Bibliographically approved

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