Phosphorylation Of Light-harvesting Complex Research Articles

Formation of light-harvesting complex II aggregates from LHCII–PSI–LHCI complexes in rice plants under high light

During low light- (LL) induced state transitions in dark-adapted rice (Oryza sativa) leaves, light-harvesting complex (LHC) II become phosphorylated and associate with PSI complexes to form LHCII-PSI-LHCI supercomplexes. When the leaves are subsequently transferred to high light (HL) conditions, phosphorylated LHCII complexes are no longer phosphorylated. Under the HL-induced transition in LHC phosphorylation status, we observed a new green band in the stacking gel of native green-PAGE, which was determined to be LHCII aggregates by immunoblotting and 77K chlorophyll fluorescence analysis. Knockout mutants of protein phosphatase 1 (PPH1) which dephosphorylates LHCII failed to form these LHCII aggregates. In addition, the ability to develop non-photochemical quenching in the PPH1 mutant under HL was less than for wild-type plants. As determined by immunoblotting analysis, LHCII proteins present in LHCII-PSI-LHCI supercomplexes included the Lhcb1 and Lhcb2 proteins. In this study, we provide evidence suggesting that LHCII in the LHCII-PSI-LHCI supercomplexes are dephosphorylated and subsequently form aggregates to dissipate excess light energy under HL conditions. We propose that this LHCII aggregation, involving LHCII L-trimers, is a newly observed photoprotective light-quenching process operating in the early stage of acclimation to HL in rice plants.

Modeling the Role of LHCII-LHCII, PSII-LHCII, and PSI-LHCII Interactions in State Transitions

The light-dependent reactions of photosynthesis take place in the plant chloroplast thylakoid membrane, a complex three-dimensional structure divided into the stacked grana and unstacked stromal lamellae domains. Plants regulate the macro-organization of photosynthetic complexes within the thylakoid membrane to adapt to changing environmental conditions and avoid oxidative stress. One such mechanism is the state transition that regulates photosynthetic light harvesting and electron transfer. State transitions are driven by changes in the phosphorylation of light harvesting complex II (LHCII), which cause a decrease in grana diameter and stacking, a decrease in energetic connectivity between photosystem II (PSII) reaction centers, and an increase in the relative LHCII antenna size of photosystem I (PSI) compared to PSII. Phosphorylation is believed to drive these changes by weakening the intramembrane lateral PSII-LHCII and LHCII-LHCII interactions and the intermembrane stacking interactions between these complexes, while simultaneously increasing the affinity of LHCII for PSI. We investigated the relative roles and contributions of these three types of interaction to state transitions using a lattice-based model of the thylakoid membrane based on existing structural data, developing a novel algorithm to simulate protein complex dynamics. Monte Carlo simulations revealed that state transitions are unlikely to lead to a large-scale migration of LHCII from the grana to the stromal lamellae. Instead, the increased light harvesting capacity of PSI is largely due to the more efficient recruitment of LHCII already residing in the stromal lamellae into PSI-LHCII supercomplexes upon its phosphorylation. Likewise, the increased light harvesting capacity of PSII upon dephosphorylation was found to be driven by a more efficient recruitment of LHCII already residing in the grana into functional PSII-LHCII clusters, primarily driven by lateral interactions.

The High Light Response in Arabidopsis Requires the Calcium Sensor Protein CAS, a Target of STN7- and STN8-Mediated Phosphorylation.

Reversible phosphorylation of thylakoid proteins contributes to photoacclimation responses in photosynthetic organisms, enabling the fine-tuning of light harvesting under changing light conditions and promoting the onset of photoprotective processes. However, the precise functional role of many of the described phosphorylation events on thylakoid proteins remains elusive. The calcium sensor receptor protein (CAS) has previously been indicated as one of the targets of the state transition kinase 8 (STN8). Here we show that in Arabidopsis thaliana, CAS is also phosphorylated by the state transition kinase 7 (STN7), as well as by another, so-far unknown, Ca2+-dependent kinase. Phosphoproteomics analysis and in vitro phosphorylation assays on CAS variants identified the phylogenetically conserved residues Thr-376, Ser-378, and Thr-380 as the major phosphorylation sites of the STN kinases. Spectroscopic analyses of chlorophyll fluorescence emission at 77K further showed that, while the cas mutant is not affected in state transition, it displays a persistent strong excitation of PSI under high light exposure, similar to the phenotype previously observed in other mutants defective in photoacclimation mechanisms. Together with the observation of a strong concomitant phosphorylation of light harvesting complex II (LHCII) and photosynthetic core proteins under high irradiance in the cas mutant this suggests a role for CAS in the STN7/STN8/TAP38 network of phosphorylation-mediated photoacclimation processes in Arabidopsis.

Chlororespiration induces non-photochemical quenching of chlorophyll fluorescence during darkness in lichen chlorobionts.

Lichens and their algal partners are desiccation‐tolerant organisms and as such survive after the complete loss of water. This trait is the consequence of several physiological, biochemical and structural features, including specific mechanisms dissipating excess light to avoid photooxidative stress. The maximum quantum yield of photosystem II (PSII; Fv/Fm) is widely used as a sensitive indicator of photosynthetic performance and is calculated after complete relaxation in darkness of the fluorescence quenching associated with active light energy dissipation mechanisms. Unexpectedly, we observed that lichens and isolated chlorobionts (chlorophyte symbionts in lichen) maintained in darkness for several hours showed a strong decrease in the ratio Fv/Fm, which was reversible after re‐illumination. We analyzed this dark‐induced Fv/Fm decay in the chlorobiont Asterochloris erici through steady‐state and fast‐induction kinetics of chlorophyll a fluorescence and simultaneous P700 oxidation measurements. We found that the gradual decay of Fv/Fm in darkness was caused by reversible dark‐induced inactivation of some PSII reaction centers that was accompanied by a decrease in the flux of electrons to PSI. Darkness induced the plastoquinone‐reductase activity associated with chlororespiration and the phosphorylation of light harvesting complex (LHC). We propose that upon phosphorylation the LHC detaches from PSII, resulting in a decrease of exciton‐trapping by PSII reaction centers and, consequently, an increased dissipation of light energy. This mechanism probably serves an ecophysiological function in lichens to prevent the damage at dawn or under strong fluctuating light conditions when lichens are in a hydrated state.

A stromal region of cytochrome b6f subunit IV is involved in the activation of the Stt7 kinase in Chlamydomonas

The cytochrome (cyt) b6f complex and Stt7 kinase regulate the antenna sizes of photosystems I and II through state transitions, which are mediated by a reversible phosphorylation of light harvesting complexes II, depending on the redox state of the plastoquinone pool. When the pool is reduced, the cyt b6f activates the Stt7 kinase through a mechanism that is still poorly understood. After random mutagenesis of the chloroplast petD gene, coding for subunit IV of the cyt b6f complex, and complementation of a ΔpetD host strain by chloroplast transformation, we screened for impaired state transitions in vivo by chlorophyll fluorescence imaging. We show that residues Asn122, Tyr124, and Arg125 in the stromal loop linking helices F and G of cyt b6f subunit IV are crucial for state transitions. In vitro reconstitution experiments with purified cyt b6f and recombinant Stt7 kinase domain show that cyt b6f enhances Stt7 autophosphorylation and that the Arg125 residue is directly involved in this process. The peripheral stromal structure of the cyt b6f complex had, until now, no reported function. Evidence is now provided of a direct interaction with Stt7 on the stromal side of the membrane.

A thioredoxin-like/β-propeller protein maintains the efficiency of light harvesting in Arabidopsis.

The light-harvesting complexes of plants have evolved the ability to switch between efficient light harvesting and quenching forms to optimize photosynthesis in response to the environment. Several distinct mechanisms, collectively termed "nonphotochemical quenching" (NPQ), provide flexibility in this response. Here we report the isolation and characterization of a mutant, suppressor of quenching 1 (soq1), that has high NPQ even in the absence of photosystem II subunit S (PsbS), a protein that is necessary for the rapidly reversible component of NPQ. The formation of NPQ in soq1 was light intensity-dependent, and it exhibited slow relaxation kinetics and other characteristics that distinguish it from known NPQ components. Treatment with chemical inhibitors or an uncoupler, as well as crosses to mutants known to affect other NPQ components, showed that the NPQ in soq1 does not require a transthylakoid pH gradient, zeaxanthin formation, or the phosphorylation of light-harvesting complexes, and it appears to be unrelated to the photosystem II damage-and-repair cycle. Measurements of pigments and chlorophyll fluorescence lifetimes indicated that the additional NPQ in soq1 is the result of a decrease in chlorophyll excited-state lifetime and not pigment bleaching. The SOQ1 gene was isolated by map-based cloning, and it encodes a previously uncharacterized thylakoid membrane protein with thioredoxin-like and β-propeller domains located in the lumen and a haloacid-dehalogenase domain exposed to the chloroplast stroma. We propose that the role of SOQ1 is to prevent formation of a slowly reversible form of antenna quenching, thereby maintaining the efficiency of light harvesting.

Protein tyrosine phosphorylation in the transition to light state 2 of chloroplast thylakoids.

Redox dependent protein phosphorylation in chloroplast thylakoids regulates distribution of excitation energy between the two photosystems of photosynthesis, PS I and PS II. Several thylakoid phosphoproteins are known to be phosphorylated on N-terminal threonine residues exposed to the chloroplast stroma. Phosphorylation of light harvesting complex II (LHC II) on Thr-6 is thought to account for redistribution of light energy from PS II to PS I during the transition to light state 2. Here, we present evidence that a protein tyrosine kinase activity is required for the transition to light state 2. With an immunological approach using antibodies directed specifically towards either phospho-tyrosine or phospho-threonine, we observed that LHC II became phosphorylated on both tyrosine and threonine residues. The specific protein tyrosine kinase inhibitor genistein, at concentrations causing no direct effect on threonine kinase activity, was found to prevent tyrosine phosphorylation of LHC II, the transition to light state 2, and associated threonine phosphorylation of LHC II. Possible reasons for an involvement of tyrosine phosphorylation in light state transitions are proposed and discussed.

ATP control on state transitions in vivo in Chlamydomonas reinhardtii

We have attempted to depress the ATP content in dark adapted cells of the unicellular green algae, C. reinhardtii, by inhibiting ATP synthesis coupled to mitochondrial electron flow. Whether we used uncouplers, ATP synthase inhibitors, inhibitors of mitochondrial electron transport or a mutant altered in mitochondrial cytochrome b, we observed a fluorescence quenching at room temperature associated with an increased reduction of the intersystem electron carriers of the photosynthetic apparatus. This fluorescence quenching reflected a genuine transition to state II, since: (i) it was associated with an increase in light-harvesting complex phosphorylation and (ii) it did not occur in mutants lacking the b 6 f complex which are blocked in State I. The complete reversion to state I occurred only into experimental conditions which allowed both ATP synthesis and reoxidation of intersystem electron carriers. We conclude that the intracellular demand for ATP controls state transitions in vivo. The interplay between intracellular ATP concentrations and the redox state of the kinase activator at the thylakoid membrane level is discussed.

Changes in phosphorylation of thylakoid membrane proteins in light-harvesting complex mutants from Chlamydomonas reinhardtii

Phosphorylation of thylakoid membrane proteins was assessed in vivo in the BF3 and BF4 mutants from Chlamydomonas reinhardtii which are deficient in light-harvesting complex (LHC). Thylakoid membranes from the BF3 mutant, which was lacking in chlorophyll b, nevertheless contained significant amounts of LHC apoproteins. The phosphorylation pattern of thylakoid membrane proteins in the mutant was similar to that in the wild type. Incubation of BF3 cells in reducing conditions induced an increase in LHC phosphorylation and a fluorescence quenching at room temperature, thereby indicating that kinase-controlled state transitions still occurred in this mutant. In contrast to the BF3 mutant, the BF4 mutant still contained chlorophyll b but lacked most of the LHC apoproteins. It contained no LHC phosphoproteins and did not undergo state transitions as shown by the absence of fluorescence quenching in reducing conditions in vivo. Surprisingly, phosphorylation of the PS II subunits D2 and 6 (CP IV apoprotein) was totally abolished in this mutant. However, the two PS II subunits of low molecular weight, L5 and L6, were still slightly phosphorylated.

State Transitions in the Green Alga Scenedesmus Obliquus Probed by Time-Resolved Chlorophyll Fluorescence Spectroscopy and Global Data Analysis

Decay-associated fluorescence spectra of the green alga Scenedesmus obliquus have been measured by single-photon timing with picosecond resolution in various states of light adaptation. The data have been analyzed by applying a global data analysis procedure. The amplitudes of the decay-associated spectra allow a determination of the relative antenna sizes of the photosystems. We arrive at the following conclusions: (a) The fluorescence kinetics of algal cells with open PS II centers (F(0) level) have to be described by a sum of three exponential components. These decay components are attributed to photosystem (PS) I (tau approximately 85 ps, lambda(max) (em) approximately 695-700 nm), open PS II alpha-centers (tau approximately 300 ps, lambda(max) (em) = 685 nm), and open PS II beta-centers (tau approximately 600 ps, lambda(max) (em) = 685 nm). A fourth component of very low amplitude (tau approximately 2.2-2.3 ns, lambda(max) (em) = 685 nm) derives from dead chlorophyll. (b) At the F(max) level of fluorescence there are also three decay components. They originate from PS I with properties identical to those at the F(0) level, from closed PS II alpha-centers (tau approximately 2.2 ns, lambda(max) (em) = 685 nm) and from closed PS beta-centers (tau approximately 1.2 ns, lambda(max) (em) = 685 nm). (c) The major effect of light-induced state transitions on the fluorescence kinetics involves a change in the relative antenna size of alpha- and beta-units brought about by the reversible migration of light-harvesting complexes between alpha-centers and beta-centers. (d) A transition to state II does not measurably increase the direct absorption cross-section (antenna size) of PS I. Our data can be rationalized in terms of a model of the antenna organization that relates the effects of state transitions and light-harvesting complex phosphorylation with the concepts of PS II alpha,beta-heterogeneity. We discuss why our results are in disagreement with those of a recent lifetime study of Chlorella by M. Hodges and I. Moya (1986, Biochim. Biophys. Acta., 849:193-202).