Thylakoid protein phosphorylation in dynamic regulation of photosystem II in higher plants

In higher plants, the photosystem (PS) II core and its several light harvesting antenna (LHCII) proteins undergo reversible phosphorylation cycles according to the light intensity. High light intensity induces strong phosphorylation of the PSII core proteins and suppresses the phosphorylation level of the LHCII proteins. Decrease in light intensity, in turn, suppresses the phosphorylation of PSII core, but strongly induces the phosphorylation of LHCII. Reversible and differential phosphorylation of the PSII-LHCII proteins is dependent on the interplay between the STN7 and STN8 kinases, and the respective phosphatases. The STN7 kinase phosphorylates the LHCII proteins and to a lesser extent also the PSII core proteins D1, D2 and CP43. The STN8 kinase, on the contrary, is rather specific for the PSII core proteins. Mechanistically, the PSII-LHCII protein phosphorylation is required for optimal mobility of the PSII-LHCII protein complexes along the thylakoid membrane. Physiologically, the phosphorylation of LHCII is a prerequisite for sufficient excitation of PSI, enabling the excitation and redox balance between PSII and PSI under low irradiance, when excitation energy transfer from the LHCII antenna to the two photosystems is efficient and thermal dissipation of excitation energy (NPQ) is minimised. The importance of PSII core protein phosphorylation is manifested under highlight when the photodamage of PSII is rapid and phosphorylation is required to facilitate the migration of damaged PSII from grana stacks to stroma lamellae for repair. The importance of thylakoid protein phosphorylation is highlighted under fluctuating intensity of light where the STN7 kinase dependent balancing of electron transfer is a prerequisite for optimal growth and development of the plant. This article is part of a Special Issue entitled: Photosystem II.     

Identification of a photosystem II phosphatase involved in light acclimation in Arabidopsis

Reversible protein phosphorylation plays a major role in the acclimation of the photosynthetic apparatus to changes in light. Two paralogous kinases phosphorylate subsets of thylakoid membrane proteins. STATE TRANSITION7 (STN7) phosphorylates LHCII, the light-harvesting antenna of photosystem II (PSII), to balance the activity of the two photosystems through state transitions. STN8, which is mainly involved in phosphorylation of PSII core subunits, influences folding of the thylakoid membranes and repair of PSII after photodamage. The rapid reversibility of these acclimatory responses requires the action of protein phosphatases. In a reverse genetic screen, we identified the chloroplast PP2C phosphatase, PHOTOSYSTEM II CORE PHOSPHATASE (PBCP), which is required for efficient dephosphorylation of PSII proteins. Its targets, identified by immunoblotting and mass spectrometry, largely coincide with those of the kinase STN8. The recombinant phosphatase is active in vitro on a synthetic substrate or on isolated thylakoids. Thylakoid folding is affected in the absence of PBCP, while its overexpression alters the kinetics of state transitions. PBCP and STN8 form an antagonistic kinase and phosphatase pair whose substrate specificity and physiological functions are distinct from those of STN7 and the counteracting phosphatase PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, but their activities may overlap to some degree.     

Photosynthesis: the processing of redox signals in chloroplasts

Recent work identifies two kinases required for phosphorylation of proteins of chloroplast thylakoid membranes. One kinase, STN7, is required for phosphorylation of light-harvesting complex II; another, STN8, is required for phosphorylation of photosystem II. How do these kinases interact, what do they do, and what are they for?     

Phosphoproteomics of Arabidopsis chloroplasts reveals involvement of the STN7 kinase in phosphorylation of nucleoid protein pTAC16

Light-regulated protein kinases STN7 and STN8 phosphorylate thylakoid membrane proteins and also affect expression of several chloroplast proteins via yet unknown mechanisms. Comparative phosphoproteomics of acetic acid protein extracts of chloroplasts from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutants yielded two previously unknown findings: (i) neither STN7 nor STN8 kinase was required for phosphorylation of Ser-48 in Lhcb1.1-1.3 proteins; and (ii) phosphorylation of Thr-451 in pTAC16 protein was STN7-dependent. pTAC16 was found distributed between thylakoids and nucleoid. Its knockout did not affect the nucleoid protein composition and the Thr-451 phosphorylated protein was excluded from the nucleoid. Thr-451 of pTAC16 is conserved in all studied plants and its phosphorylation may regulate membrane-anchoring functions of the nucleoid.     

Identification of STN7/STN8 kinase targets reveals connections between electron transport,metabolism and gene expression

The thylakoid‐associated kinases STN7 and STN8 are involved in short‐ and long‐term acclimation of photosynthetic electron transport to changing light conditions. Here we report the identification of STN7/STN8 in vivo targets that connect photosynthetic electron transport with metabolism and gene expression. Comparative phosphoproteomics with the stn7 and stn8 single and double mutants identified two proteases, one RNA‐binding protein, a ribosomal protein, the large subunit of Rubisco and a ferredoxin‐NADP reductase as targets for the thylakoid‐associated kinases. Phosphorylation of three of the above proteins can be partially complemented by STN8 in the stn7 single mutant, albeit at lower efficiency, while phosphorylation of the remaining three proteins strictly depends on STN7. The properties of the STN7‐dependent phosphorylation site are similar to those of phosphorylated light‐harvesting complex proteins entailing glycine or another small hydrophobic amino acid in the ?1 position. Our analysis uncovers the STN7/STN8 kinases as mediators between photosynthetic electron transport, its immediate downstream sinks and long‐term adaptation processes affecting metabolite accumulation and gene expression.     

Natural variation in phosphorylation of photosystem II proteins in Arabidopsis thaliana: is it caused by genetic variation in the STN kinases?

Reversible phosphorylation of photosystem II (PSII) proteins is an important regulatory mechanism that can protect plants from changes in ambient light intensity and quality. We hypothesized that there is natural variation in this process in Arabidopsis (Arabidopsis thaliana), and that this results from genetic variation in the STN7 and STN8 kinase genes. To test this, Arabidopsis accessions of diverse geographical origins were exposed to two light regimes, and the levels of phospho-D1 and phospho-light harvesting complex II (LHCII) proteins were quantified by western blotting with anti-phosphothreonine antibodies. Accessions were classified as having high, moderate or low phosphorylation relative to Col-0. This variation could not be explained by the abundance of the substrates in thylakoid membranes. In genotypes with atrazine-resistant forms of the D1 protein, low D1 and LHCII protein phosphorylation was observed, which may be due to low PSII efficiency, resulting in reduced activation of the STN kinases. In the remaining genotypes, phospho-D1 levels correlated with STN8 protein abundance in high-light conditions. In growth light, D1 and LHCII phosphorylation correlated with longitude and in the case of LHCII phosphorylation also with temperature variability. This suggests a possible role of natural variation in PSII protein phosphorylation in the adaptation of Arabidopsis to diverse environments.     

Photosystem II protein phosphorylation follows four distinctly different regulatory patterns induced by environmental cues

Differential redox regulation of thylakoid phosphoproteins was studied in winter rye plants in vivo. The redox state of chloroplasts was modulated by growing plants under different light/temperature conditions and by transient shifts to different light/temperature regimes. Phosphorylation of PSII reaction centre proteins D1 and D2, the chlorophyll a binding protein CP43, the major chlorophyll a/b binding proteins Lhcb1 and Lhcb2 (LHCII) and the minor light‐harvesting antenna protein CP29 seem to belong to four distinct regulatory groups. Phosphorylation of D1 and D2 was directly dependent on the reduction state of the plastoquinone pool. CP43 protein phosphorylation generally followed the same pattern, but often remained phosphorylated even in darkness. Phosphorylation of CP29 occurred upon strong reduction of the plastoquinone pool, and was further enhanced by low temperatures. In vitro studies further demonstrated that CP29 phosphorylation is independent of the redox state of both the cytochrome b₆/f complex and the thiol compounds. Complete phosphorylation of Lhcb1 and 2 proteins, on the contrary, required only modest reduction of the plastoquinone pool, and was subject to inhibition upon increase in the thiol redox state of the stroma. Furthermore, the reversible phosphorylation of Lhcb1 and 2 proteins appeared to be an extremely dynamic process, being rapidly modulated by short‐term fluctuations in chloroplast redox conditions.     

The STN8 kinase‐PBCP phosphatase system is responsible for high‐light‐induced reversible phosphorylation of the PSII inner antenna subunit CP29 in rice

Reversible phosphorylation of thylakoid light‐harvesting proteins is a mechanism to compensate for unbalanced excitation of photosystem I (PSI) versus photosystem II (PSII) under limiting light. In monocots, an additional phosphorylation event on the PSII antenna CP29 occurs upon exposure to excess light, enhancing resistance to light stress. Different from the case of the major LHCII antenna complex, the STN7 kinase and its related PPH1 phosphatase were proven not to be involved in CP29 phosphorylation, indicating that a different set of enzymes act in the high‐light (HL) response. Here, we analyze a rice stn8 mutant in which both PSII core proteins and CP29 phosphorylation are suppressed in HL, implying that STN8 is the kinase catalyzing this reaction. In order to identify the phosphatase involved, we produced a recombinant enzyme encoded by the rice ortholog of AtPBCP, antagonist of AtSTN8, which catalyzes the dephosphorylation of PSII core proteins. The recombinant protein was active in dephosphorylating P‐CP29. Based on these data, we propose that the activities of the OsSTN8 kinase and the antagonistic OsPBCP phosphatase, in addition to being involved in the repair of photo‐damaged PSII, are also responsible for the HL‐dependent reversible phosphorylation of the inner antenna CP29.     

Impaired photosystem I oxidation induces STN7-dependent phosphorylation of the light-harvesting complex I protein Lhca4 in Arabidopsis thaliana

Reduction of the plastoquinone (PQ) pool is known to activate phosphorylation of thylakoid proteins. In the Arabidopsis thaliana mutants psad1-1 and psae1-3, oxidation of photosystem I (PSI) is impaired, and the PQ pool is correspondingly over-reduced. We show here that, under these conditions, the antenna protein Lhca4 of PSI becomes a target for phosphorylation. Phosphorylation of the mature Lhca4 protein at Thr16 is suppressed in stn7 psad1 and stn7 psae1 double mutants. Thus, under extreme redox conditions, hyperactivation of thylakoid protein kinases and/or reorganization of thylakoid protein complex distribution increase the susceptibility of PSI to phosphorylation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Anna Ihnatowicz and Paolo Pesaresi contributed equally to the article.     

Radiation inactivation analysis of thylakoid protein kinase systems in light and in darkness

Chloroplast thylakoid contains several membrane-bound protein kinases that phosphorylate thylakoid polypeptides for the regulation of photosynthesis. Thylakoid protein phosphorylation is activated when the plastoquinone pool is reduced either by light-dependent electron flow through photosystem 2 (PS2) or by adding exogenous reductants such as durohydroquinone in the dark. The major phosphorylated proteins on thylakoid are components of light-harvesting complex 2 (LHC2) and a PS2 associated 9 kDa phosphoprotein. Radiation inactivation technique was employed to determine the functional masses of various kinases for protein phosphorylation in thylakoids. Under the photosynthetically active radiation (PAR), the apparent functional masses of thylakoid protein kinase systems (TPKXs) for catalyzing phosphorylation of LHC2 27 and 25 kDa polypeptides were 540±50 and 454±35 kDa as well as it was 448±23 kDa for PS2 9 kDa protein phosphorylation. Furthermore, the functional sizes of dark-regulated TPKXs for 25 and 9 kDa proteins were 318±25 and 160±8 kDa. The 9 kDa protein phosphorylation was independent of LHC2 polypeptides phosphorylation with regard to its TPKX functional mass. Target size analysis of protein phosphorylation mentioned above indicates that thylakoid contains a group of distinct protein kinase systems. A working model is accordingly proposed to interpret the interaction between these protein kinase systems.     

A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes

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.     

State transitions at the crossroad of thylakoid signalling pathways

In order to maintain optimal photosynthetic activity under a changing light environment, plants and algae need to balance the absorbed light excitation energy between photosystem I and photosystem II through processes called state transitions. Variable light conditions lead to changes in the redox state of the plastoquinone pool which are sensed by a protein kinase closely associated with the cytochrome b ₆ f complex. Preferential excitation of photosystem II leads to the activation of the kinase which phosphorylates the light-harvesting system (LHCII), a process which is subsequently followed by the release of LHCII from photosystem II and its migration to photosystem I. The process is reversible as dephosphorylation of LHCII on preferential excitation of photosystem I is followed by the return of LHCII to photosystem II. State transitions involve a considerable remodelling of the thylakoid membranes, and in the case of Chlamydomonas, they allow the cells to switch between linear and cyclic electron flow. In this alga, a major function of state transitions is to adjust the ATP level to cellular demands. Recent studies have identified the thylakoid protein kinase Stt7/STN7 as a key component of the signalling pathways of state transitions and long-term acclimation of the photosynthetic apparatus. In this article, we present a review on recent developments in the area of state transitions.     

Analysis of the Chloroplast Protein Kinase Stt7 during State Transitions

State transitions allow for the balancing of the light excitation energy between photosystem I and photosystem II and for optimal photosynthetic activity when photosynthetic organisms are subjected to changing light conditions. This process is regulated by the redox state of the plastoquinone pool through the Stt7/STN7 protein kinase required for phosphorylation of the light-harvesting complex LHCII and for the reversible displacement of the mobile LHCII between the photosystems. We show that Stt7 is associated with photosynthetic complexes including LHCII, photosystem I, and the cytochrome b₆f complex. Our data reveal that Stt7 acts in catalytic amounts. We also provide evidence that Stt7 contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys that are critical for its activity and state transitions. On the basis of these data, we propose that the activity of Stt7 is regulated through its transmembrane domain and that a disulfide bond between the two lumen Cys is essential for its activity. The high-light–induced reduction of this bond may occur through a transthylakoid thiol–reducing pathway driven by the ferredoxin-thioredoxin system which is also required for cytochrome b₆f assembly and heme biogenesis.     

A Survey of Chloroplast Protein Kinases and Phosphatases in Arabidopsis thaliana

Protein phosphorylation is a major mode of regulation of metabolism, gene expression and cell architecture. In chloroplasts, reversible phosphorylation of proteins is known to regulate a number of prominent processes, for instance photosynthesis, gene expression and starch metabolism. The complements of the involved chloroplast protein kinases (cpPKs) and phosphatases (cpPPs) are largely unknown, except 6 proteins (4 cpPKs and 2 cpPPs) which have been experimentally identified so far. We employed combinations of programs predicting N-terminal chloroplast transit peptides (cTPs) to identify 45 tentative cpPKs and 21 tentative cpPPs. However, test sets of 9 tentative cpPKs and 13 tentative cpPPs contain only 2 and 7 genuine cpPKs and cpPPs, respectively, based on experimental subcellular localization of their N-termini fused to the reporter protein RFP. Taken together, the set of enzymes known to be involved in the reversible phosphorylation of chloroplast proteins in A. thaliana comprises altogether now 6 cpPKs and 9 cpPPs, the function of which needs to be determined in future by functional genomics approaches. This includes the calcium-regulated PK CIPK13 which we found to be located in the chloroplast, indicating that calcium-dependent signal transduction pathways also operate in this organelle.Key Words: Arabidopsis thaliana, chloroplast, chloroplast transit peptide, protein kinase, protein phosphatase, protein phosphorylation, proteomics.     

Inorganic-pyrophosphate-dependent phosphorylation of spinach thylakoid proteins.

It has been shown for the first time that several photosystem-II thylakoid proteins and the main chlorophyll-a/b light-harvesting complex can be phosphorylated with inorganic pyrophosphate as phosphate donor. With pyrophosphate, as with ATP, the protein-kinase reaction is dependent on light or a strong reducing agent. The reaction which can be demonstrated in well-washed spinach thylakoids is dependent on electron transport and is controlled by the redox state of the plastoquinone pool. It is suggested that the pyrophosphate-dependent thylakoid protein phosphorylation is mediated by the same kinase which is responsible for the ATP-dependent protein phosphorylation. This pyrophosphate-dependent kinase activity may be derived from an evolutionary precursor from which ATP-dependent protein phosphorylation also developed.     

Coregulation of light-harvesting complex II phosphorylation and lhcb mRNA accumulation in winter rye

Winter rye plants grown under contrasting environmental conditions or just transiently shifted to varying light and temperature conditions, were studied to elucidate the chloroplast signal involved in regulation of photosynthesis genes in the nucleus. Photosystem II excitation pressure, reflecting the plastoquinone redox state, and the phosphorylation level of thylakoid light-harvesting proteins (LHCII and CP29) were monitored together with changes occurring in the accumulation of lhcb, rbcS and psbA mRNAs. Short-term shifts of plants to changed conditions, from 1 h to 2 d, were postulated to reveal signals crucial for the initiation of the acclimation process. Comparison of these results with those obtained from plants acclimated during several weeks' growth at contrasting temperature and in different light regimes, allow us to make the following conclusions: (1) LHCII protein phosphoylation is a sensitive tool to monitor redox changes in chloroplasts; (2) LHCII protein phosphorylation and lhcb mRNA accumulation occur under similar conditions and are possibly coregulated via an activation state of the same kinase (the LHCII kinase); (3) Maximal accumulation of lhcb mRNA during the diurnal light phase seems to require an active LHCII kinase whereas inactivation of the kinase is accompanied by dampening of the circadian oscillation in the amount of lhcb mRNA; (4) Excitation pressure of photosystem II (reduction state of the plastoquinone pool) is not directly involved in the regulation of lhcb mRNA accumulation. Instead (5) the redox status of the electron acceptors of photosystem I in the stromal compartment seems to be highly regulated and crucial for the regulation of lhcb gene expression in the nucleus.     

Short-term down-regulation of zeaxanthin epoxidation in Arabidopsis thaliana in response to photo-oxidative stress conditions

The epoxidation of zeaxanthin (Zx) to violaxanthin after exposure to different light stress conditions has been studied in Arabidopsis (Arabidopsis thaliana). Formation of Zx was induced by illumination of intact leaves for up to 8 h at different light intensities and temperatures. The kinetics of epoxidation was found to be gradually retarded with increasing light stress during pre-illumination, indicating a gradual down-regulation of the Zx epoxidase activity. Retardation of the epoxidation rates by a factor of up to 10 was inducible either by increasing the light intensity or by extending the illumination time or by decreasing the temperature during pre-illumination. The retardation of the epoxidation kinetics was correlated with a decrease of the PSII quantum efficiency after the pre-illumination treatment. Experiments with the stn7/stn8 mutant of Arabidopsis indicated that the thylakoid protein kinases STN7 and STN8, which are required for the phosphorylation of PSII proteins, are not involved in the short-term down-regulation of Zx epoxidation. However, the retardation of Zx epoxidation was maintained in thylakoids isolated from pre-illuminated leaves, indicating that a direct modification of the Zx epoxidase is most likely involved in the light-induced down-regulation.