Phosphorylation Of CP29 Research Articles

Combinatory actions of CP29 phosphorylation by STN7 and stability regulate leaf age-dependent disassembly of photosynthetic complexes

A predominant physiological change that occurs during leaf senescence is a decrease in photosynthetic efficiency. An optimal organization of photosynthesis complexes in plant leaves is critical for efficient photosynthesis. However, molecular mechanisms for regulating photosynthesis complexes during leaf senescence remain largely unknown. Here we tracked photosynthesis complexes alterations during leaf senescence in Arabidopsis thaliana. Grana stack is significantly thickened and photosynthesis complexes were disassembled in senescing leaves. Defects in STN7 and CP29 led to an altered chloroplast ultrastructure and a malformation of photosynthesis complex organization in stroma lamella. Both CP29 phosphorylation by STN7 and CP29 fragmentation are highly associated with the photosynthesis complex disassembly. In turn, CP29 functions as a molecular glue to facilitate protein complex formation leading phosphorylation cascade and to maintain photosynthetic efficiency during leaf senescence. These data suggest a novel molecular mechanism to modulate leaf senescence via CP29 phosphorylation and fragmentation, serving as an efficient strategy to control photosynthesis complexes.

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.

High light-dependent phosphorylation of photosystem II inner antenna CP29 in monocots is STN7 independent and enhances nonphotochemical quenching.

Phosphorylation of the photosystem II antenna protein CP29 has been reported to be induced by excess light and further enhanced by low temperature, increasing resistance to these stressing factors. Moreover, high light-induced CP29 phosphorylation was specifically found in monocots, both C3 and C4, which include the large majority of food crops. Recently, knockout collections have become available in rice (Oryza sativa), a model organism for monocots. In this work, we have used reverse genetics coupled to biochemical and physiological analysis to elucidate the molecular basis of high light-induced phosphorylation of CP29 and the mechanisms by which it exerts a photoprotective effect. We found that kinases and phosphatases involved in CP29 phosphorylation are distinct from those reported to act in State 1-State 2 transitions. In addition, we elucidated the photoprotective role of CP29 phosphorylation in reducing singlet oxygen production and enhancing excess energy dissipation. We thus established, in monocots, a mechanistic connection between phosphorylation of CP29 and nonphotochemical quenching, two processes so far considered independent from one another.

The significance of CP29 reversible phosphorylation in thylakoids of higher plants under environmental stresses

Reversible phosphorylation of proteins is a key event in many fundamental cellular processes. Under stressful conditions, many thylakoid membrane proteins in photosynthetic apparatus of higher plants undergo rapid phosphorylation and dephosphorylation in response to environmental changes. CP29 is the most frequently phosphorylated protein among three minor antennae complexes in higher plants. CP29 phosphorylation in dicotyledons has been known for several decades and is well characterized. However, CP29 phosphorylation in monocotyledons is less studied and appears to have a different phosphorylation pattern. In this review, we discuss recent advancements in CP29 phosphorylation and dephosphorylation studies and its physiological significance under environmental stresses in higher plants, especially in the monocotyledonous crops. Physiologically, the phosphorylation of CP29 is likely to be a prerequisite for state transitions and the disassembly of photosystem II supercomplexes, but not involved in non-photochemical quenching (NPQ). CP29 is phosphorylated in monocots exposed to environmental cues, with its subsequent lateral migration from grana stacks to stroma lamellae. However, neither CP29 phosphorylation nor its lateral migration occurs in dicotyledonous plants after drought, cold, or salt stress. Since the molecular mechanisms of differential CP29 phosphorylation under stresses are not fully understood, this review provides insights for future studies regarding the physiological function of CP29 reversible phosphorylation.

High Light Induced Disassembly of Photosystem II Supercomplexes in Arabidopsis Requires STN7-Dependent Phosphorylation of CP29

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 plants lacking the STN7 protein kinase. Blue native gel electrophoresis followed by immunological and mass spectrometric analyses of the membrane protein complexes revealed that the high light treatment of the wild type caused redistribution of CP29 from 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 PSII supercomplexes in plants exposed to high light involves 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, as previously reported.

Phosphorylation of Photosynthetic Antenna Protein CP29 and Photosystem II Structure Changes in Monocotyledonous Plants under Environmental Stresses

Kinetic studies of protein dephosphorylation in thylakoid membranes showed that the minor light-harvesting antenna protein CP29 could be phosphorylated in barley (C3) and maize (C4) seedlings, but not in spinach under water [Liu, W. J., et al. (2009) Biochim. Biophys. Acta 1787, 1238-1245], salt, or cold stress [Pursiheimo, S., et al. (2003) Plant Cell Environ. 26, 1995-2003], suggesting that phosphorylation of CP29 is a general phenomenon in monocots, but not in dicots under environmental stresses. Abscisic acid (ABA), reactive oxygen species (ROS), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), NO, and the scavenger of H(2)O(2) had weak effects on CP29 phosphorylation. However, three protein kinase inhibitors, U0126, W7, and K252a (for mitogen-activated protein kinase, Ca(2+)-dependent protein kinase, and Ser/Thr protein kinases, respectively), decrease the level of CP29 phosphorylation in barley apparently under environmental stresses. Therefore, these three protein kinases are involved in CP29 phosphorylation. We also found that most CP29 phosphorylation was accompanied by its lateral migration from granum membranes to stroma-exposed thylakoid regions, and the instability of PSII supercomplexes and LHCII trimers under environmental stresses.

Dephosphorylation of photosystem II proteins and phosphorylation of CP29 in barley photosynthetic membranes as a response to water stress

Kinetic studies of protein dephosphorylation in barley thylakoid membranes revealed accelerated dephosphorylation of photosystem II (PSII) proteins, and meanwhile rapidly induced phosphorylation of a light-harvesting complex (LHCII) b4, CP29 under water stress. Inhibition of dephosphorylation aggravates stress damages and hampers photosystem recovery after rewatering. This increased dephosphorylation is catalyzed by both intrinsic and extrinsic membrane protein phosphatase. Water stress did not cause any thylakoid destacking, and the lateral migration from granum membranes to stroma-exposed lamellae was only found to CP29, but not other PSII proteins. Activation of plastid proteases and release of TLP40, an inhibitor of the membrane phosphatases, were also enhanced during water stress. Phosphorylation of CP29 may facilitate disassociation of LHCII from PSII complex, disassembly of the LHCII trimer and its subsequent degradation, while general dephosphorylation of PSII proteins may be involved in repair cycle of PSII proteins and stress-response-signaling.

Light‐harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions

The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light-harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI-PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox-dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.

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

ABSTRACTDifferential 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.

Light-harvesting complex II kinase and chloroplast redox signaling

Winter rye plants were grown under contrasting environmental conditions, or just transiently shifted to varying light/temperature regimes, to elucidate the acclimative processes of the light-harvesting complex II. Photosystem II (PSII) excitation pressure, reflecting the plastoquinone redox state, and the steady-state phosphorylation level of the thylakoid light-harvesting proteins (LHCII and CP29), together with accumulation of lhcb mRNA were monitored in vivo. Regulatory mechanisms of the phosphorylation of LHCII and CP29 were additionally explored in vitro. Obtained results allow us to make the following conclusions. (i) LHCII protein phosphorylation is a sensitive tool to monitor redox changes in chloroplasts. (ii) LHCII protein phosphorylation and lhcb mRNA accumulation are co-regulated, possibly via an activation state of the LHCII kinase. (iii) Excitation pressure of photosystem II (reduction state of the plastoquinone pool) is not directly involved in the regulation of lhcb mRNA accumulation. Instead, (iv) the redox status of the electron acceptors of photosystem I in the stromal compartment seems to be highly regulated and crucial for regulation of both the LHCII protein phosphorylation and lhcb gene expression in the nucleus. (v) Phosphorylation of CP29 is induced under plastoquinol-reducing conditions, and does not involve thiol redox-regulation

A study of the relation between CP29 phosphorylation, zeaxanthin content and fluorescence quenching parameters in Zea mays leaves

The hypothesis that phosphorylation of the minor photosystem II antenna complex CP29 (CP34 formation) in Zea mays (cv. Dekalb DK300), under conditions of illumination and low temperature stress, may constitute a protective mechanism against photoinhibition, has been investigated. It is demonstrated that illumination at low temperature induces a marked increase in reversible non‐photochemical quenching yield of chlorophyll fluorescence, together with CP34 formation. These two parameters, however, are not related as CP34 dephosphorylates to CP29 in the dark, with a half‐time of about 10 min, while the enhanced non‐photochemical quenching yield is stable for many hours. The enhanced non‐photochemical quenching yield seems to correlate with zeaxanthin formation. The influence of CP34 formation on photoinhibition was also directly investigated. No measurable effect on this parameter could be observed after treatment with high light. It is concluded that CP34 is probably not directly involved in photoprotective processes.

Cold-Resistant and Cold-Sensitive Maize Lines Differ in the Phosphorylation of the Photosystem II Subunit, CP29.

The effects of low temperature on the relative contributions of the reaction center and the antenna activities to photosystem II (PSII) electron transport were estimated by chlorophyll fluorescence. The inhibition of PSII photochemistry resulted from photo-damage to the reaction center and/or a reduced probability of excitation energy trapping by the reaction center. Although chill treatment did not modify the proportion of the dimeric to monomeric PSII, it destabilized its main light-harvesting complex. Full protection of the reaction center was achieved only in the presence of the phosphorylated PSII subunit, CP29. In a nonphosphorylating genotype the chill treatment led to photoinhibitory damage. The phosphorylation of CP29 modified neither its binding to the PSII core nor its pigment content. Phosphorylated CP29 was isolated by flat-bed isoelectric focusing. Its spectral characteristics indicated a depletion of the chlorophyll spectral forms with the highest excitation transfer efficiency to the reaction center. It is suggested that phosphorylated CP29 performs its regulatory function by an yet undescribed mechanism based on a shift of the equilibrium for the excitation energy toward the antenna.

Conformational changes induced by phosphorylation in the CP29 subunit of photosystem II.

Light energy absorbed by the chloroplast membranes of higher plants is dissipated by nonradiative de-excitation in order to protect against photodamage. In photosystem II, which is the photosynthetic component most sensitive to photoinhibition, three pigment binding subunits, called CP24, CP26, and CP29, have been proposed to act in the regulation of the chlorophyll excited states concentration. In heavy stress conditions, CP29 becomes phosphorylated in its stroma-exposed portion, and this process is reversed by returning to normal conditions. In this study, we have used the pigments bound to the intramembrane hydrophobic portion of the protein as intrinsic probes to detect conformational changes induced by phosphorylation. We isolated the phosphorylated and unphosphorylated forms of the protein and showed that, although they have the same pigment complement, spectral differences can be consistently detected by absorption, linear dichroism, and circular dichroism spectroscopy. Alkaline phosphatase treatment of the phosphoprotein restores both the electrophoretic mobility and the spectral properties of the unphosphorylated CP29. The results of this study show that phosphorylation of CP29 can modulate the spectral properties of this photosystem II subunit and provide a possible mechanism for the regulation of excitation energy supply to the reaction center.