Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes

Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO(2) availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes.     

Biogenesis of the chloroplast-encoded D1 protein: regulation of translation elongation, insertion, and assembly into photosystem II

Regulation of translation elongation, membrane insertion, and assembly of the chloroplast-encoded D1 protein of photosystem II (PSII) was studied using a chloroplast translation system in organello. Translation elongation of D1 protein was found to be regulated by (1) a redox component that can be activated not only by photosynthetic electron transfer but also by reduction with DTT; (2) the trans-thylakoid proton gradient, which is absolutely required for elongation of D1 nascent chains on the thylakoid membrane; and (3) the thiol reactants N-ethylmaleimide (NEM) and iodosobenzoic acid (IBZ), which inhibit translation elongation with concomitant accumulation of distinct D1 pausing intermediates. These results demonstrate that D1 translation elongation and membrane insertion are tightly coupled and highly regulated processes in that proper insertion is a prerequisite for translation elongation of D1. Cotranslational and post-translational assembly steps of D1 into PSII reaction center and core complexes occurred independently of photosynthetic electron transfer or trans-thylakoid proton gradient but were strongly affected by the thiol reactants DTT, NEM, and IBZ. These compounds reduced the stability of the early PSII assembly intermediates, hampered the C-terminal processing of the precursor of D1, and prevented the post-translational reassociation of CP43, indicating a strong dependence of the D1 assembly steps on proper redox conditions and the formation of disulfide bonds.     

Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes

Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO₂ availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes.     

Up-regulation of a photosystem II core protein phosphatase inhibitor and sustained D1 phosphorylation in zeaxanthin-retaining, photoinhibited needles of overwintering Douglas fir

Overwintering needles of the evergreen conifer Douglas fir exhibited an association between arrest of the xanthophyll cycle in the dissipating state (as zeaxanthin + antheraxanthin; Z + A) with a strongly elevated predawn phosphorylation state of the D1 protein of the photosystem II (PSII) core. Furthermore, the high predawn phosphorylation state of PSII core proteins was associated with strongly increased levels of TLP40, the cyclophilin-like inhibitor of PSII core protein phosphatase, in winter versus summer. In turn, decreases in predawn PSII efficiency, F_v/F_m, in winter were positively correlated with pronounced decreases in the non-phosphorylated form of D1. In contrast to PSII core proteins, the light-harvesting complex of photosystem II (LHCII) did not exhibit any nocturnally sustained phosphorylation. The total level of the D1 protein was found to be the same in summer and winter in Douglas fir when proteins were extracted in a single step from whole needles. In contrast, total D1 protein levels were lower in thylakoid preparations of overwintering needles versus needles collected in summer, indicating that D1 was lost during thylakoid preparation from overwintering Douglas fir needles. In contrast to total D1, the ratio of phosphorylated to non-phosphorylated D1 as well as the levels of the PsbS protein were similar in thylakoid versus whole needle preparations. The level of the PsbS protein, that is required for pH-dependent thermal dissipation, exhibited an increase in winter, whereas LHCII levels remained unchanged.     

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.     

Differential changes in the steady state levels of thylakoid membrane proteins during senescence in Cucumis sativus cotyledons

Chloroplast structure and function is known to alter during foliar senescence. Besides, the alterations in the structural organisation of thylakoid membranes changes in the steady state levels of thylakoid membrane proteins occur due to leaf ageing. We monitored temporal changes in some of the specific proteins of thylakoid membrane protein complexes by western blotting in the Cucumis sativus cotyledons as a function of the cotyledon age. We observed that the levels of D1 and D2 proteins of photosystem II started declining at the early stages of senescence of Cucumis cotyledons and continued to decline with the progress of cotyledon age. Similarly the level of Cyt f of Cyt b6/f complex declined rapidly with progress of senescence in these cotyledons. The reaction centre proteins of photosystem I were relatively found to be more stable than that of photosystem II reaction centre proteins reflecting possibly the disorganisation of photosystem II prior to photosystem I. The 33 kDa extrinsic protein (MSP) of oxygen evolving complex, the LHCII apoprotein and the beta-subunit of ATPsynthase showed the declined levels with the progress of cotyledon age. However, the extents of loss of these proteins were not as high as the reaction centre proteins of photosystem II and the Cyt f. These results provide that during senescence, proteins of thylakoid membranes degrade in a specific temporal sequence and thereby affect the temporal photochemical functions in Cucumis sativus cotyledons.     

Thylakoid protein phosphorylation,state 1-state 2 transitions,and photosystem stoichiometry adjustment: redox control at multiple levels of gene expression

In photosynthesis in chloroplasts, control of thylakoid protein phosphorylation by redox state of inter-photosystem electron carriers makes distribution of absorbed excitation energy between the two photosystems self-regulating. During operation of this regulatory mechanism, reduction of plastoquinone activates a thylakoid protein kinase which phosphorylates the light-harvesting complex LHC II, causing a change in protein recognition that results in redistribution of energy to photosystem I at the expense of photosystem II, thus tending to oxidise the reduced plastoquinone pool. These events correspond to the transition from light-state 1 to light-state 2. The reverse transition (to light-state 1) is initiated by transient oxidation of plastoquinone, inactivation of the LHC II kinase, and return of dephosphorylated LHC II from photosystem I to photosystem II, supplying excitation energy to photosystem II and thereby reducing plastoquinone. State 1-state 2 transitions therefore operate by means of redox control of reversible, post-translational modification of pre-existing proteins. A balance in the rates of light utilization by photosystem I and photosystem II can also be achieved, on longer time-scales and between wider limits, by adjustment of the relative quantities, or stoichiometry, of photosystem I and photosystem II. Recent evidence suggests that adjustment of photosystem stoichiometry is also a response to perturbation of the redox state of inter-photosystem electron carriers, and involves specific redox control of de novo protein synthesis, assembly, and breakdown. It is therefore suggested that the same redox sensor initiates these different adaptations by control of gene expression at different levels, according to the time-scale and amplitude of the response. This integrated feedback control may serve to maintain redox homeostasis, and, as a result, quantum yield. Evidence for the components required by such systems is discussed.     

Deletion Mutagenesis of the Cytochrome b559 Protein Inactivates the Reaction Center of Photosystem II

In green plant-like photosynthesis, oxygen evolution is catalyzed by a thylakoid membrane-bound protein complex, photosystem II. Cytochrome b559, a protein component of the reaction center of this complex, is absent in a genetically engineered mutant of the cyanobacterium, Synechocystis 6803 [Pakrasi, H.B., Williams, J.G.K., and Arntzen, C.J. (1988). EMBO J. 7, 325-332]. In this mutant, the genes psbE and psbF, encoding cytochrome b559, were deleted by targeted mutagenesis. Two other protein components, D1 and D2 of the photosystem II reaction center, are also absent in this mutant. However, two chlorophyll-binding proteins, CP47 and CP43, as well as a manganese-stabilizing extrinsic protein component of photosystem II are stably assembled in the thylakoids of this mutant. Thus, this deletion mutation destabilizes the reaction center of photosystem II only. The mutant also lacks a fluorescence maximum peak at 695 nm (at 77 K) even though the CP47 protein, considered to be the origin of this fluorescence peak, is present in this mutant. We propose that the fluorescence at 695 nm originates from an interaction between the reaction center of photosystem II and CP47. The deletion mutant shows the absence of variable fluorescence at room temperature, indicating that its photosystem II complex is photochemically inactive. Also, photoreduction of QA, the primary acceptor quinone in photosystem II, could not be detected in the mutant. We conclude that cytochrome b559 plays at least an essential structural role in the reaction center of photosystem II.     

光系统II蛋白磷酸化及其生理意义

蛋白磷酸化修饰在几乎所有的生命活动中都起重要的调节作用.该文结合作者研究组的研究工作,概述了光系统II(PS II)蛋白磷酸化的调节及其生理功能.PS II复合体中的核心组分D1、D2、CP43和PsbH蛋白以及外周捕光天线(LHC II)蛋白都可以发生磷酸化.PS II蛋白磷酸化受质醌(PQ)的氧化还原状态、细胞色素b6f (Cyt b6f ) 和硫氧还蛋白以及光调节.PS II蛋白磷酸化可以调节激发能在两种光系统(PS I和PS II)之间的分配,减轻光胁迫对PS II的压力,保护核心蛋白免于光破坏,稳定PS II复合体的结构.     

Dithiol oxidant and disulfide reductant dynamically regulate the phosphorylation of light-harvesting complex II proteins in thylakoid membranes

Light-induced phosphorylation of light-harvesting chlorophyll a/b complex II (LHCII) proteins in plant thylakoid membranes requires an activation of the LHCII kinase via binding of plastoquinol to cytochrome b(6)f complex. However, a gradual down-regulation of LHCII protein phosphorylation occurs in higher plant leaves in vivo with increasing light intensity. This inhibition is likely to be mediated by increasing concentration of thiol reductants in the chloroplast. Here, we have determined the components involved in thiol redox regulation of the LHCII kinase by studying the restoration of LHCII protein phosphorylation in thylakoid membranes isolated from high-light-illuminated leaves of pumpkin (Cucurbita pepo), spinach (Spinacia oleracea), and Arabidopsis. We demonstrate an experimental separation of two dynamic activities associated with isolated thylakoid membranes and involved in thiol regulation of the LHCII kinase. First, a thioredoxin-like compound, responsible for inhibition of the LHCII kinase, became tightly associated and/or activated within thylakoid membranes upon illumination of leaves at high light intensities. This reducing activity was completely missing from membranes isolated from leaves with active LHCII protein phosphorylation, such as dark-treated and low-light-illuminated leaves. Second, hydrogen peroxide was shown to serve as an oxidant that restored the catalytic activity of the LHCII kinase in thylakoids isolated from leaves with inhibited LHCII kinase. We propose a dynamic mechanism by which counteracting oxidizing and reducing activities exert a stimulatory and inhibitory effect, respectively, on the phosphorylation of LHCII proteins in vivo via a novel membrane-bound thiol component, which itself is controlled by the thiol redox potential in chloroplast stroma.     

Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

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.     

Phosphorylation of Photosystem II Components, CP43 Apoprotein, D1, D2, and 10 to 11 Kilodalton Protein in Chloroplast Thylakoids of Higher Plants

Phosphorylated thylakoid proteins of spinach (Spinacia oleracea L.) and pea (Pisum sativum L.) were solubilized, fractionated by sucrose density gradient centrifugation, and analyzed by gel electrophoresis and crossed immunoelectrophoresis to identify the phosphoproteins. It was found that in addition to intense phosphorylation of light-harvesting chlorophyll complex II, four photosystem II components, CP43 apoprotein, D1, D2, and a 10 to 11 kilodalton protein, are substantially phosphorylated in the light. Furthermore, the CP43 apoprotein, D1 and D2 can be resolved into two electrophoretic subspecies, only one of which is phosphorylated. This indicates that only a fraction of the PSII polypeptides is phosphorylated. Finally, analysis of detergent procedures suggests that the 10 to 11 kilodalton phosphoprotein is a peripheral component of the O₂-evolving PSII reaction center complex.     

Redox signalling and the structural basis of regulation of photosynthesis by protein phosphorylation

In photosynthesis in chloroplasts and cyanobacteria, redox control of thylakoid protein phosphorylation regulates distribution of absorbed excitation energy between the two photosystems. When electron transfer through chloroplast photosystem II (PSII) proceeds at a rate higher than that through photosystem I (PSI), chemical reduction of a redox sensor activates a thylakoid protein kinase that catalyses phosphorylation of light-harvesting complex II (LHCII). Phosphorylation of LHCII increases its affinity for PSI and thus redistributes light-harvesting chlorophyll to PSI at the expense of PSII. This short-term redox signalling pathway acts by means of reversible, post-translational modification of pre-existing proteins. A long-term equalisation of the rates of light utilisation by PSI and PSII also occurs: by means of adjustment of the stoichiometry of PSI and PSII. It is likely that the same redox sensor controls both state transitions and photosystem stoichiometry. A specific mechanism for integration of these short- and long-term adaptations is proposed. Recent evidence shows that phosphorylation of LHCII causes a change in its 3-D structure, which implies that the mechanism of state transitions in chloroplasts involves control of recognition of PSI and PSII by LHCII. The distribution of LHCII between PSII and PSI is therefore determined by the higher relative affinity of phospho-LHCII for PSI, with lateral movement of the two forms of the LHCII being simply a result of their diffusion within the membrane plane. Phosphorylation-induced dissociation of LHCII trimers may induce lateral movement of monomeric phospho-LHCII, which binds preferentially to PSI. After dephosphorylation, monomeric, unphosphorylated LHCII may trimerize at the periphery of PSII.     

Chlorophyll triplet states associated with photosystem II of thylakoids

The analysis of FDMR thylakoid spectra, determined at multiple emission wavelengths, by a global decomposition technique, has revealed the presence of three previously undescribed triplet populations at emission wavelengths characteristic of Photosystem II chlorophyll/protein complexes. Their zero-field splitting parameters have been determined in order to compare them with the well-studied PSII recombination triplet state. None of these triplets have the zero-field splitting parameters characteristic of the recombination triplet and are therefore probably not generated directly in the reaction center. On the basis of their microwave-induced emission spectra, it is suggested that two are probably generated in the core complex(es) while the third may be generated in the external antenna. These triplets are formed under nonreducing redox conditions, when the recombination triplet is undetectable. It is suggested that they may be involved in the photoinhibitory damage of Photosystem II. The triplet-minus-singlet spectrum associated with the recombination triplet state has been determined for thylakoids after reduction of the secondary acceptors. Its main peak is at 685 nm, slightly red shifted with respect to earlier reports, with a weak signal, of opposite sign at approximately 675 nm. The 685 nm peak indicates that at cryogenic temperatures, the triplet is located on the long-wavelength chlorophyll state present in the reaction center complex of Photosystem II (D1.D2.Cytb(559) complex). From the absence of a clear structure in the 680 nm absorption region, this long-wavelength absorbing state does not appear to be strongly coupled to P(680), though it must be associated with one of the "inner core" pigments recently identified in the photosystem II crystallographic structure [Zouni et al. (2001) Nature 408, 739-743].     

The D1 protein of the photosystem II reaction-centre complex accumulates in the absence of D2: analysis of a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking cytochrome d559

The reaction center core of photosystem II, a multiprotein membrane bound complex, is composed of a heterodimer of two proteins, D1 and D2. A random mutagenesis technique was used to isolate a photosystem II deficient mutant, CP6t16, of the unicellular cyanobacterium, Synechocystis sp. PCC 6803. Nucleotide sequence analysis showed that the primary lesion in CP6t16 is an ochre mutation introducing a translational stop codon in the psbE gene, encoding the alpha-subunit of cytochrome b559, an integral component of the PSII complex. Analysis of the protein composition of CP6t16 thylakoid membranes isolated in the presence of serine protease inhibitors revealed that, in the absence of cytochrome b559, the D2 protein is also absent. However, the D1 protein is stably incorporated in these membranes, suggesting that the synthesis and integration of D1 are independent of those of D2 and cytochrome b559.     

A small zinc finger thylakoid protein plays a role in maintenance of photosystem II in Arabidopsis thaliana

This work identifies LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), a Zn finger protein that shows disulfide isomerase activity, interacts with the photosystem II (PSII) core complex, and may act in repair of photodamaged PSII complexes. Two mutants of an unannotated small Zn finger containing a thylakoid membrane protein of Arabidopsis thaliana (At1g75690; LQY1) were found to have a lower quantum yield of PSII photochemistry and reduced PSII electron transport rate following high-light treatment. The mutants dissipate more excess excitation energy via nonphotochemical pathways than wild type, and they also display elevated accumulation of reactive oxygen species under high light. After high-light treatment, the mutants have less PSII-light-harvesting complex II supercomplex than wild-type plants. Analysis of thylakoid membrane protein complexes showed that wild-type LQY1 protein comigrates with the PSII core monomer and the CP43-less PSII monomer (a marker for ongoing PSII repair and reassembly). PSII repair and reassembly involve the breakage and formation of disulfide bonds among PSII proteins. Interestingly, the recombinant LQY1 protein demonstrates a protein disulfide isomerase activity. LQY1 is more abundant in stroma-exposed thylakoids, where key steps of PSII repair and reassembly take place. The absence of the LQY1 protein accelerates turnover and synthesis of PSII reaction center protein D1. These results suggest that the LQY1 protein may be involved in maintaining PSII activity under high light by regulating repair and reassembly of PSII complexes.     

Structural organization of proteins on the oxidizing side of photosystem II. Two molecules of the 33-kDa manganese-stabilizing proteins per reaction center.

The 33-kDa manganese-stabilizing protein stabilizes the manganese cluster in the oxygen-evolving complex. There has been, however, a considerable amount of controversy concerning the stoichiometry of this photosystem II (PS II) component. In this paper, we have verified the extinction coefficient of the manganese-stabilizing protein by amino acid analysis, determined the manganese content of oxygen-evolving photosystem II membranes and reaction center complex using inductively coupled plasma spectrometry, and determined immunologically the amount of the manganese-stabilizing protein associated with photosystem II. Oxygen-evolving photosystem II membranes and reaction center complex preparations contained 258 +/- 11 and 67 +/- 3 chlorophyll, respectively, per tetranuclear manganese cluster. Immunoquantification of the manganese-stabilizing protein using mouse polyclonal antibodies on "Western blots" demonstrated the presence of 2.1 +/- 0.2 and 2.0 +/- 0.3 molecules of the manganese-stabilizing protein/tetranuclear manganese cluster in oxygen-evolving PS II membranes and highly purified PS II reaction center complex, respectively. Since the manganese-stabilizing protein co-migrated with the D2 protein in our electrophoretic system, accurate immunoquantification required the inclusion of CaCl2-washed PS II membrane proteins or reaction center complex proteins in the manganese-stabilizing protein standards to compensate for the possible masking effect of the D2 protein on the binding of the manganese-stabilizing protein to Immobilon-P membranes. Failure to include these additional protein components in the manganese-stabilizing protein standards leads to a marked underestimation of the amount of the manganese-stabilizing protein associated with these photosystem II preparations.     

Phosphorylation-dephosphorylation of light-harvesting complex II as a response to variation in irradiance is thiol sensitive and thylakoid sufficient: modulation of the sensitivity of the phenomenon by a peripheral component

Downregulation of phosphorylation of chlorophyll a/b-binding proteins (LHCII) of the photosystem II at high irradiance could only be demonstrated with leaf discs but not in isolated thylakoids. The present view suggests this phenomenon to be regulated by stromal thioredoxin. Here, we show that high-light inactivation of LHCII phosphorylation can be reproduced in isolated thylakoids and have explained the apparent absence of inactivation in vitro to be due to the derepressed activity of a peripheral kinase. We investigated this phenomenon with Arachis hypogea thylakoids prepared with (Th:A) or without (Th:B) tricine, where tricine is known for removing peripheral proteins from thylakoids. While LHCII remained phosphorylated at high irradiance in Th:B, the response of Th:A mimicked Arachis leaflets where LHCII was transiently phosphorylated with irradiance. LHCII phosphorylation in Th:A was sensitive to thiol reducing conditions, but in Th:B, the phenomenon became insensitive to thiol reduction following illumination. Washing Th:B with tricine made them resemble Th:A, and conversely, Th:A reconstituted with the Tricine extract resembled Th:B with respect to both irradiance response and thiol sensitivity. In vitro phosphorylation reactions indicated a thiol insensitive kinase activity to be present in the Tricine extract that was capable of phosphorylating histone H1 as well as purified LHCII. This peripherally associated kinase activity explained the sustenance of LHCII phosphorylation as well as its thiol insensitivity at high irradiance in Th:B thylakoids. Contrary to the current view, our results clearly show that irradiance dependent phosphorylation and dephosphorylation of LHCII is a thylakoid sufficient phenomenon, although it remained open to regulation by thiol redox state modulation.