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Nucleotide-Dependent Processes in the Thylakoid Lumen of Plant Chloroplasts
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Plants, algae and photosynthetic bacteria are able to harvest the sunlight and use its energy to transform water and carbon dioxide to carbohydrate molecules and oxygen, both important to sustain life on Earth. This process is called photosynthesis and is the route by which almost all energy enters the biosphere. As most simple things in life, the process of photosynthesis is easily explained but unfortunately not that easy to reproduce. If we could, we would be living in a much different world with almost unlimited energy. Light energy is harvested by chlorophyll molecules, bound to proteins in the chloroplast thylakoid membrane and drives the oxygen-evolving complex, to extract electrons from water. Electrons are then transferred to NADPH through photosystem II (PSII) to cytochrome b6f and photosystem I, the major photosynthetic protein complexes. The cytochrome b6f complex also transfers protons into the lumenal space of the thylakoid. These protons together with those from water oxidation create an electrochemical gradient across the thylakoid membrane, which fuels the ATP synthase to produce ATP. ATP, NADPH and carbon dioxide are used during the dark reactions to produce sugars in the chloroplast stroma. The thylakoid lumenal space where the water oxidation occurs has until recently been viewed as a proton sink with very few proteins. With the publication of the genome of Arabidopsis thaliana it seems to be a much more complex compartment housing a wide variety of biochemical processes.

ATP is a nucleotide and the major energy currency, but there are also other nucleotides such as AMP, ADP, GMP, GDP and GTP. Chloroplast metabolism has mostly been associated with ATP, but GTP has been shown to have a role in integration of light harversting complexes into the thylakoid. In this work, we have demonstrated the occurrence of nucleotide-dependent processes in the lumenal space of spinach by bringing evidence first for nucleotide (ATP) transport across the thylakoid membrane, second for nucleotide inter-conversion (ATP to GTP) by a nucleoside diphosphate kinase, and third the discovery that the PsbO extrinsic subunit of PSII complex can bind and hydrolyse GTP to GDP. The active PSII complex functions as a dimer but following light-induced damage, it is monomerised allowing for repair of its reaction center D1 protein. PsbO is ubiquitous in all oxygenic photosynthetic organisms and together with other extrinsic proteins stabilises the oxygen-evolving complex. We have modelled the GTP-binding site in the PsbO structure and showed that the GTPase activity of spinach PsbO induces changes in the protein structure, dissociation from the complex and stimulates the degradation of the D1 protein, possibly by inducing momerisation of damaged PSII complexes. As compared to spinach, Arabidopsis has two isoforms of PsbO, PsbO1 and PsbO2, expressed in a 4:1 ratio. A T-DNA insertion knockout mutant of PsbO1 showed a retarded growth rate, pale green leaves and a decrease in the oxygen evolution while a PsbO2 knockout mutant did not show any visual phenotype as compared to wild type. Unexpectedly, during growth under high light conditions the turnover rate of the D1 protein was impaired in the PsbO2 knockout, whereas it occurred faster in the PsbO1 knockout as compared to wild type. We concluded that the PsbO1 protein mainly functions in stabilizing the oxygen evolving complex, whereas the PsbO2 protein regulates the turnover of the D1 protein. The two PsbO proteins also differ in their GTPase-activity (PsbO2 >> PsbO1). Although their amino acid sequences are 90% identical, they differ in the GTP-binding region which could explain the difference in their GTPase activity. Based on these data, we propose that the GTPase activity of PsbO(2) leads to structural changes in interacting loops and plays a role in the initial steps of D1 turnover such as the PSII monomerisation step.

The nucleotide-dependent processes we discovered in the thylakoid lumen raise questions of transporters to facilitate these processes. As stated earlier, we provided biochemical evidence of an ATP thylakoid transporter, and most recently have identified a transporter that may be important for the export of lumenal phosphate back to the stroma. More transporters for GDP, metal ions and others solutes have still to be identified.

Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi , 2008.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1165
Keyword [en]
plant, chloroplast, thylakoid, nucleotide, PSII, PsbO
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:liu:diva-11244ISBN: 978-91-7393-965-2 (print)OAI: oai:DiVA.org:liu-11244DiVA: diva2:17657
Public defence
2008-04-04, Linden, Hus 421, Ingång 65; HU, Campus US, Linköpings universitet, Linköping, 09:30 (English)
Opponent
Supervisors
Available from: 2008-03-20 Created: 2008-03-20 Last updated: 2009-05-07
List of papers
1. Multiple evidence for nucleotide metabolism in the chloroplast thylakoid lumen
Open this publication in new window or tab >>Multiple evidence for nucleotide metabolism in the chloroplast thylakoid lumen
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2004 (English)In: Proceedings of the National Academy of Science, ISSN 0027-8424, Vol. 101, no 5, 1409-1414 p.Article in journal (Refereed) Published
Abstract [en]

The apparatus of photosynthetic energy conversion in chloroplasts is quite well characterized with respect to structure and function. Light-driven electron transport in the thylakoid membrane is coupled to synthesis of ATP, used to drive energy-dependent metabolic processes in the stroma and the outer surface of the thylakoid membrane. The role of the inner (luminal) compartment of the thylakoids has, however, remained largely unknown although recent proteomic analyses have revealed the presence of up to 80 different proteins. Further, there are no reports concerning the presence of nucleotides in the thylakoid lumen. Here, we bring three lines of experimental evidence for nucleotide-dependent processes in this chloroplast compartment. (i) The thylakoid lumen contains a protein of 17.2 kDa, catalyzing the transfer of the γ-phosphate group from ATP to GDP, proposed to correspond to the nucleoside diphosphate kinase III. (ii) The 33-kDa subunit of photosystem II, bound to the luminal side of the thylakoid membrane and associated with the water-splitting process, can bind GTP. (iii) The thylakoid membrane contains a nucleotide transport system that is suggested to be associated with a 36.5-kDa nucleotide-binding protein. Our results imply, against current dogmas, that the thylakoid lumen contains nucleotides, thereby providing unexpected aspects on this chloroplast compartment from a metabolic and regulatory perspective and expanding its functional significance beyond a pure bioenergetic function.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13003 (URN)10.1073/pnas.0308164100 (DOI)
Available from: 2008-03-20 Created: 2008-03-20 Last updated: 2010-01-26
2. Subsequent events to GTP binding by the plant PsbO protein: structural changes, GTP hydrolysis and dissociation from the photosystem II complex
Open this publication in new window or tab >>Subsequent events to GTP binding by the plant PsbO protein: structural changes, GTP hydrolysis and dissociation from the photosystem II complex
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2007 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, Vol. 1767, no 6, 500-508 p.Article in journal (Refereed) Published
Abstract [en]

Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409–1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the β-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn2+ and Ca2+ ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.

Keyword
Photosystem II, PsbO protein, GTPase, Oxygen-evolving complex, D1 protein
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13004 (URN)10.1016/j.bbabio.2006.10.009 (DOI)
Available from: 2008-03-20 Created: 2008-03-20 Last updated: 2009-05-07Bibliographically approved
3. Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction centre D1 protein
Open this publication in new window or tab >>Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction centre D1 protein
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2007 (English)In: The Plant Journal, ISSN 0960-7412, Vol. 49, no 3, 528-539 p.Article in journal (Refereed) Published
Abstract [en]

The extrinsic photosystem II (PSII) protein of 33 kDa (PsbO), which stabilizes the water-oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isoforms. Two T-DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were retarded in growth in comparison with the wild type, while differing from each other phenotypically. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less efficient than PsbO1 under photoinhibitory conditions. Prolonged high light stress led to reduced growth and fitness of the mutant lacking PsbO2 as compared with the wild type and the mutant lacking PsbO1. During a short period of treatment of detached leaves or isolated thylakoids at high light levels, inactivation of PSII electron transport in the PsbO2-deficient mutant was slowed down, and the subsequent degradation of the D1 protein was totally inhibited. The steady-state levels of in vivo phosphorylation of the PSII reaction centre proteins D1 and D2 were specifically reduced in the mutant containing only PsbO2, in comparison with the mutant containing only PsbO1 or with wild-type plants. Phosphorylation of PSII proteins in vitro proceeded similarly in thylakoid membranes from both mutants and wild-type plants. However, dephosphorylation of the D1 protein occurred much faster in the thylakoids containing only PsbO2. We conclude that the function of PsbO1 in Arabidopsis is mostly in support of PSII activity, whereas the interaction of PsbO2 with PSII regulates the turnover of the D1 protein, increasing its accessibility to the phosphatases and proteases involved in its degradation.

Keyword
Arabidopsis, photosystem II, PsbO, oxygen evolution, D1 protein degradation, high light stress
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13005 (URN)10.1111/j.1365-313X.2006.02976.x (DOI)
Available from: 2008-03-20 Created: 2008-03-20 Last updated: 2009-05-07
4. Arabidopsis PsbOs differ in their GTPase activity
Open this publication in new window or tab >>Arabidopsis PsbOs differ in their GTPase activity
2008 (English)In: Photosynthesis: Energy from the Sun / [ed] John F. Allen, Elisabeth Gantt, John H. Golbeck, Barry Osmond., Springer , 2008, 729-731 p.Chapter in book (Other academic)
Abstract [en]

Crucial for the optimal function of the oxygen-evolving complex (OEC) is the PsbO subunit of the photosystem II (PSII) complex. Previously we reported the ability of PsbO in spinach to bind and hydrolyze GTP. GTP stimulates the dissociation of PsbO from PSII following illumination and induces the degradation of the D1 protein. We have predicted four plant-specific binding motifs for GTP, which are not conserved in the sequences of the cyanobacteria or green algae PsbO proteins. We have proposed a location of the GTP-binding site inside the β-barrel exposed to the lumenal side. Arabidopsis thaliana has two PsbO isoforms encoded by two different genes: psbO1 and psbO2. Here we have measured and compared the GTPase activities of PSII membranes isolated from Arabidopsis knockouts mutants containing T-DNA insertions in one or the other of the psbO genes. The specific GTPase activity of PsbO2 is three fold higher than that of PsbO1. Furthermore, PsbO2 is more efficiently released than PsbO1 from PSII following light treatment. We conclude that PsbO2 is a better GTPase than Psb.

Place, publisher, year, edition, pages
Springer, 2008
Keyword
Photosystem II, PsbO protein, Arabidopsis thaliana, T-DNA insertionO1
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13006 (URN)10.1007/978-1-4020-6709-9_162 (DOI)978-1-4020-6707-5 (ISBN)
Available from: 2008-03-20 Created: 2008-03-20 Last updated: 2013-06-14Bibliographically approved
5. Arabidopsis ANTR1 is a thylakoid Na+-dependent phosphate transporter -functional characterization in Escherichia coli
Open this publication in new window or tab >>Arabidopsis ANTR1 is a thylakoid Na+-dependent phosphate transporter -functional characterization in Escherichia coli
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 283, no 20, 13520-13527 p.Article in journal (Refereed) Published
Abstract [en]

In this study, the putative anion transporter 1 (ANTR1) from Arabidopsis thaliana was shown to be localized to the chloroplast thylakoid membrane by Western blotting with two different peptide-specific antibodies. ANTR1 is homologous to the type I of mammalian Na+-dependent inorganic phosphate (Pi) transporters. The function of ANTR1 as a Na+-dependent Pi transporter was demonstrated by heterologous expression and uptake of radioactive Pi into Escherichia coli cells. The expression of ANTR1 conferred increased growth rates to the transformed cells and stimulated Pi uptake in a pH- and Na+-dependent manner as compared with the control cells. Among various tested effectors, Pi was the preferred substrate. Although it competed with the uptake of Pi, glutamate was not transported by ANTR1 into E. coli. In relation to its function as a Pi transporter, several physiological roles for ANTR1 in the thylakoid membrane are proposed, such as export of Pi produced during nucleotide metabolism in the thylakoid lumen back to the chloroplast stroma and balance of the trans-thylakoid H+ electrochemical gradient storage.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13007 (URN)10.1074/jbc.M709371200 (DOI)
Available from: 2008-03-20 Created: 2008-03-20 Last updated: 2009-05-13

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