D1 Protein Phosphorylation Research Articles

Terrestrial Plants Evolve Highly Assembled Photosystem Complexes in Adaptation to Light Shifts.

It has been known that PSI and PSII supercomplexes are involved in the linear and cyclic electron transfer, dynamics of light capture, and the repair cycle of PSII under environmental stresses. However, evolutions of photosystem (PS) complexes from evolutionarily divergent species are largely unknown. Here, we improved the blue native polyacrylamide gel electrophoresis (BN-PAGE) separation method and successfully separated PS complexes from all terrestrial plants. It is well known that reversible D1 protein phosphorylation is an important protective mechanism against oxidative damages to chloroplasts through the PSII photoinhibition-repair cycle. The results indicate that antibody-detectable phosphorylation of D1 protein is the latest event in the evolution of PS protein phosphorylation and occurs exclusively in seed plants. Compared to angiosperms, other terrestrial plant species presented much lower contents of PS supercomplexes. The amount of light-harvesting complexes II (LHCII) trimers was higher than that of LHCII monomers in angiosperms, whereas it was opposite in gymnosperms, pteridophytes, and bryophytes. LHCII assembly may be one of the evolutionary characteristics of vascular plants. In vivo chloroplast fluorescence measurements indicated that lower plants (bryophytes especially) showed slower changes in state transition and nonphotochemical quenching (NPQ) in response to light shifts. Therefore, the evolution of PS supercomplexes may be correlated with their acclimations to environments.

Progesterone increases photochemical efficiency of photosystem II in wheat under heat stress by facilitating D1 protein phosphorylation

Experiments were conducted to investigate the effects of exogenous progesterone on photochemical efficiency of PSII and turnover of D1 protein under heat stress during the grain-filling stage. Heat stress resulted in increases of hydrogen peroxide production, malondialdehyde content, and relative electrolytic leakage in wheat leaves, but these responses were alleviated by foliar application of progesterone. Meanwhile, activities of superoxide dismutase, catalase, and peroxidase were significantly improved in progesterone-pretreated leaves. Along with the alleviation of oxidative stress, higher abundances of STN8 and phosphorylated D1 protein and lower total D1 protein content in the PSII reactive center were observed in progesterone-pretreated leaves relative to controls. Consequently, progesterone raised the potential photochemical efficiency, actual photochemical efficiency, and electron transfer rate. These results indicate that foliar application of progesterone can effectively alleviate heat-induced PSII damage by enhancing antioxidant capability and regulating phosphorylation of D1 protein in wheat leaves.

Effect of hydrogen sulfide on D1 protein in wheat under drought stress

Drought usually induces plant growth inhibition and oxidative damage. This report showed the protections of hydrogen sulfide (H2S) against drought-induced damage in wheat, which was manifested in the better growth, higher relative water content (RWC), lower thiobarbituric acid-reactive substances (TBARS), and hydrogen peroxide (H2O2) as well as the increased activities of superoxide dismutase (SOD) and catalase (CAT). Drought often causes photosystem II (PS II) damage, which was observed in the decreased potential photochemical efficiency (F v/F m), actual photochemical efficiency (ΦPS II), photochemical quenching (qP), electron transfer rate (ETR), and increased non-photochemical quenching (qN), however, these effects could be alleviated by NaHS (H2S donor). D1 protein in PS II reaction center is the most sensitive target of PS II damage. Compared to water treatment, a higher level of transcription but less D1 protein and phosphorylated D1 protein was detected in wheat leaves when exposed to NaHS under drought stress, which may result from the higher expression of STN8 (catalyze D1 protein phosphorylation) and D1 protein degradation-related gene (Deg1, Deg5, Deg8, FtsH2, and FtsH5). These results suggested that H2S alleviated drought-induced PS II damage owing to fast D1 protein turnover rather than D1 protein content.

The short-term response of Arabidopsis thaliana (C3) and Zea mays (C4) chloroplasts to red and far red light.

Light quality has various effects on photochemistry and protein phosphorylation in Zea mays and Arabidopsis thaliana thylakoids due to different degrees of light penetration across leaves and redox status in chloroplasts. The effect of the spectral quality of light (red, R and far red, FR) on the function of thylakoid proteins in Zea mays and Arabidopsis thaliana was investigated. It was concluded that red light stimulates PSII activity in A. thaliana thylakoids and in maize bundle sheath (BS) thylakoids, but not in mesophyll (M) thylakoids. The light quality did not change PSI activity in M thylakoids of maize. FR used after a white light period increased PSI activity significantly in maize BS and only slightly in A. thaliana thylakoids. As shown by blue native (BN)-PAGE followed by SDS-PAGE, proteins were differently phosphorylated in the thylakoids, indicating their different functions. FR light increased dephosphorylation of LHCII proteins in A. thaliana thylakoids, whereas in maize, dephosphorylation did not occur at all. The rate of phosphorylation was higher in maize BS than in M thylakoids. D1 protein phosphorylation increased in maize and decreased in A. thaliana upon irradiation with both R and growth light (white light, W). Light variations did not change the level of proteins in thylakoids. Our data strongly suggest that response to light quality is a species-dependent phenomenon. We concluded that the maize chloroplasts were differently stimulated, probably due to different degrees of light penetration across the leaf and thereby the redox status in the chloroplasts. These acclimation changes induced by light quality are important in the regulation of chloroplast membrane flexibility and thus its function.

Protecting effect of phosphorylation on oxidative damage of D1 protein by down-regulating the production of superoxide anion in photosystem II membranes under high light

The physiological significance of photosystem II (PSII) core protein phosphorylation has been suggested to facilitate the migration of oxidative damaged D1 and D2 proteins, but meanwhile the phosphorylation seems to be associated with the suppression of reactive oxygen species (ROS) production, and it also relates to the degradation of PSII reaction center proteins. To more clearly elucidate the possible protecting effect of the phosphorylation on oxidative damage of D1 protein, the degradation of oxidized D1 protein and the production of superoxide anion in the non-phosphorylated and phosphorylated PSII membranes were comparatively detected using the Western blotting and electron spin resonance spin-trapping technique, respectively. Obviously, all of three ROS components, including superoxide anion, hydrogen peroxide and hydroxyl radical are responsible for the degradation of oxidized D1 protein, and the protection of the D1 protein degradation by phosphorylation is accompanied by the inhibition of superoxide anion production. Furthermore, the inhibiting effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a competitor to Q(B), on superoxide anion production and its protecting effect on D1 protein degradation are even more obvious than those of phosphorylation. Both DCMU effects are independent of whether PSII membranes are phosphorylated or not, which reasonably implies that the herbicide DCMU and D1 protein phosphorylation probably share the same target site in D1 protein of PSII. So, altogether it can be concluded that the phosphorylation of D1 protein reduces the oxidative damage of D1 protein by decreasing the production of superoxide anion in PSII membranes under high light.

Abstract A13: Chemoprevention agent SHetA2 induces G1 arrest through modulation of a biological system driven by Cyclin D1

Abstract A13 Flexible heteroarotinoid (Flex-Het) compounds induce apoptosis through direct targeting of mitochondria and Bcl-2 proteins, inhibit angiogenesis through regulation of cytokine expression and induce differentiation through increased E-Cadherin expression. In addition, the lead Flex-Het, SHetA2, induces G1 cell cycle arrest through induction of Cyclin D1 protein phosphorylation, ubiquitination and degradation. Overexpression of Cyclin D1 abrogated the G1 arrest. SHetA2 counteraction of carcinogen-induced Cyclin D1 expression was associated with SHetA2 chemoprevention of transformation in an organotypic model. Cyclin D1 is a tightly controlled protein that drives G1 cell cycle progression. Overexpression of Cyclin D1 is frequently observed in ovarian cancer and is believed to be an early event in ovarian tumorigenesis. The hypothesis of this study was that SHetA2 induces G1 arrest through modulation of a biological system driven by Cyclin D1. Cyclin D1 degradation was associated with several expected down-stream events in 2 ovarian cancer cell lines. Co-IP assays demonstrated that Cyclin D1 loss was associated with relocation of p21 from the Cyclin D1 complex to the Cyclin E2 complex. Consistent with the stimulatory effect of p21 on the CyclinD1-Cdk4/6 complex and the inhibitory effect on the CyclinE2-Cdk2 kinase activity, Western blot analysis demonstrated that phosphorylation of Rb on serine 780, known to be induced by CyclinD1-Cdk4/6-p21, was decreased. This was followed by decreased Rb phosphorylation on serine 612, which is known to be induced by CyclinE2-Cdk2. SHetA2 inhibition of Cyclin A protein expression, confirmed that the repression of Rb phosphorylation prevented the release of E2F from Rb, thus preventing expression of S Phase genes. An rtPCR array demonstrated that 7 genes involved in cell cycle regulation were significantly altered above background in both ovarian cancer cell lines. Ingenuity analysis demonstrated that several of these genes are involved in an interacting biological system driving the cell cycle. A Cdk4 inhibitor, p16, and the down-stream gene Bax were up-regulated, while several Cyclin D1 and E2F interacting genes were also regulated. Study of upstream events driving Cyclin D1 degradation demonstrated that the GSK3β kinase, known to induce Cyclin D1 threonine 286 phosphorylation, was phosphorylated/inhibited by SHetA2 and not involved in the cell cycle arrest. This phosphorylation event could be due to the SHetA2 induced phosphorylation of Akt serine 473, which has been shown to lead to increased GSK3β phosphorylation. Experiments with libraries of chemical and siRNA kinase inhibitors demonstrated that inhibition of kinases, such as EGF-R, mTOR, PKC, DNA-PK, contributed to SHetA2 growth inhibition, depending on their expression and activity patterns in the specific cell lines evaluated. In conclusion, SHetA2 inhibition of Cyclin D1 induced a number of changes in down-stream and associated targets consistent with G1 arrest. Experiments are ongoing to identify the specific kinase responsible for SHetA2 induction of Cyclin D1 phosphorylation. Knowledge of SHetA2 modulation of the biological system regulating cell cycle progression provides important information, such as response biomarkers, that can be used in translational research associated with planned chemoprevention trials. Supported by CA106713 Citation Information: Cancer Prev Res 2008;1(7 Suppl):A13.

Photoinhibition and D1 protein degradation in mesophyll and agranal bundle sheath thylakoids of maize.

Susceptibility of photosystem II complex (PSII) to photoinhibition and degradation of D1 protein has been described in the chloroplasts of C3 plants but so far, the PSII turnover has not been characterised in any C4 plant, which contains two types of chloroplasts differing biochemically and structurally. In maize (Zea mays L. Oleńka), chloroplasts located in mesophyll (M) develop grana, while bundle sheath (BS) chloroplasts are agranal. In this paper, we report the D1 protein phosphorylation, damage and proteolysis in mesophyll as well as in agranal bundle sheath thylakoids of maize plants. Photoinhibitory treatment (1800 μmol photons m-2 s-1) of isolated thylakoids led to donor side inhibition of PSII electron transport and then to damage of reaction centre in both M and BS thylakoids. Rate of D1 degradation rate was faster in BS than in M thylakoids, and the addition of ATP to incubation medium delayed D1 degradation in both types of thylakoids. Furthermore, we demonstrated that the proteases belonging to FtsH and Deg families were present but their amounts significantly differed in M and BS thylakoids. Protease inhibitor studies revealed that serine- and metallo-proteases were involved in degradation of D1 protein. Apparent existence of D1 degradation cycle and the presence of proteolytic enzymes responsible for this process in BS thylakoids confirm that PSII plays an important role in agranal membranes, and when damaged, D1 can be rapidly degraded to enable PSII repair and restoration in these membranes.

Modulation of PsbS and flexible vs sustained energy dissipation by light environment in different species

Contrasting acclimation strategies of photosynthesis and photoprotection were identified for annual mesophytes (spinach, pumpkin, and Arabidopsis) vs the tropical evergreen Monstera deliciosa. The annual species utilized full sunlight for photosynthesis to a much greater extent than the evergreen species. Conversely, the evergreen species exhibited a greater capacity for photoprotective thermal energy dissipation as well as a greater expression of the PsbS protein in full sun than the annual species. In all species, the majority of thermal energy dissipation [assessed as non‐photochemical fluorescence quenching (NPQ)] was the flexible, ΔpH‐dependent form of NPQ over the entire range of growth light environments. However, in response to a transfer of shade‐grown plants to high light, the evergreen species exhibited a high level of sustained thermal dissipation (qI), but the annual species did not. This sustained energy dissipation in the evergreen species was not ΔpH‐dependent nor did the low level of PsbS in shade leaves increase upon transfer to high light for several days. Sustained ΔpH‐independent NPQ was correlated (a) initially, with sustained D1 protein phosphorylation and xanthophyll cycle arrest and (b) subsequently, with an accumulation over several days of PsbS‐related one‐helix proteins and newly synthesized zeaxanthin and lutein.

D1 protein phosphorylation/dephosphorylation alone has no effect on the electron transport activity of photosystem II in soybean leaves

In order to explore the relationship between the phosphorylation of D1 protein and the function of PSII reaction center, the changes in chlorophyll fluorescence parameters, PSII electron transport activity and D1 protein amount were observed after dephosphorylation of phosphorylated D1 proteins caused by FSBA (5′- p-fluorosulfonylbenzoyl adenosine, an inhibitor of protein kinase) treatment. Also, phosphorylated and non-phosphorylated D1 proteins (D1* and D1, respectively) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting. The following results were obtained. (1) D1* could be up to 74% of the total D1 proteins in dark-adapted soybean leaves and could be completely dephosphorylated by FSBA (1 mM) treatment for 2 h. (2) Dephosphorylation of D1* resulted in neither substantial net loss of D1 proteins nor the significant changes in value of chlorophyll fluorescence parameters. (3) The electron transport activity of PS II (H 2O→1,4-BQ) was not changed significantly after D1* was completely dephosphorylated. Based on these results, it is concluded that D1 protein phosphorylation/dephosphorylation alone has no significant effect on the function of PS II reaction center in soybean leaves.

Influence of protein phosphorylation on the electron-transport properties of Photosystem II.

Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q(A) (-) to Q(B)(Q(B) (-)) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin(-)) and the EPR signal from the Q(A) (-) Fe(2+) complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S(2) state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S(2) multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S(2) multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b (559) was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed.

Phosphorylation/dephosphorylation of D1 protein alone has no effect on the electron transport activity of photosystem II in soybean leaves

D1 protein, as a core component of the photosystem II (PSII) reaction center complex, can be reversibly phosphorylated/dephosphorylated in higher plants. The phosphorylated D1 protein (D1*) and non-phosphorylated D1 protein (D1), having slightly different mobilities on denaturing polyacrylamide gels, can be resolved by using SDS-PAGE and Western Blotting [Callahan et al, J Biol Chem 1990, 265: 15357-15360]. The relationship between the phosphorylation of D1 protein and the electron transport activity of PSII was investigated by changing the phosphorylation level of D1 proteins with an inhibitor of protein kinase, 5?-p-fluorosulfonylbenzoyl adenosine (FSBA). The following results were obtained. (1) D1* could be up to 74% of total D1 proteins in fully dark-adapted soybean leaves and could be completely dephosphorylated by FSBA (1 mM) treatment for 2 hours. (2) Dephosphorylation of D1* resulted in neither substantial net loss of D1 proteins nor the significant changes of chlorophyll a fluorescence parameters. (3) The electron transport activity of PS II (H2O ® 1,4-BQ) had no significant change after D1* was completely dephosphorylated. Based on these results, it is concluded that phosphorylation/dephosphorylation of D1 protein alone has no significant effect on the function of PS II reaction centers in soybean leaves.

Protein phosphorylation and magnesium status regulate the degradation of the D1 reaction centre protein of Photosystem II

Illumination of higher plant leaves (pumpkin, wheat and pea) at a photon flux density of 1200 μmol m −2 s −1 induced strong phosphorylation of the D1 reaction centre protein of photosystem II. Phosphorylation of D1 protein was not observed in a moss Ceratodon purpureus. To study the effect of protein phosphorylation on D1 degradation, thylakoids isolated from pumpkin leaves and Ceratodon were phosphorylated in vitro in the presence of ATP before being subjected to photoinhibitory illumination. The rate of photoinhibition of Photosystem II oxygen evolution was not influenced by phosphorylation conditions in pumpkin or Ceratodon thylakoids. D1 protein in its phosphorylated form in pumpkin thylakoids was degraded significantly more slowly whereas in Ceratodon the degradation of D1 protein occurred rapidly with or without the presence of ATP in the photoinhibitory medium. Depletion of Mg 2+ ions also blocked the degradation of unphosphorylated D1 protein in pumpkin thylakoid membranes. We conclude that degradation of the D1 protein in higher plants is regulated by phosphorylation on the substrate level and its optimal rate requires the presence of Mg 2+.

Light‐dependent phosphorylation of D1 reaction centre protein of photosystem II: hypothesis for the functional role in vivo

Reversible phosphorylation of the D1 reaction centre protein of photosystem II (PSII) occurs in thylakoid membranes of higher plants. The significance of D1 protein phosphorylation in the function of PSII is not yet clear. This paper summarizes the data implying that phosphorylation of D1 protein in higher plants is involved in the regulation of the repair cycle of photoinhibited PSII centres. Photoinhibition of PSII, D1 protein phosphorylation and degradation have been studied in vivo in higher plant leaves acclimated to different growth irradiances. It is shown that photoinhibitory illumination induces maximal phosphorylation of the D1 protein. Under these conditions D1 turnover is also saturated. We postulate that phosphorylation retards the degradation of damaged D1 protein under conditions where rapid replacement by a new D1 copy is not possible. This would protect PSII from total disassembly and degradation of all PSII subunits. We conclude that the phosphorylation of D1 protein and the regulation of D1 protein degradation may have evolved together. Furthermore, these characteristics seem to be related to the highly organized structure of higher‐plant type thylakoid membranes, since the capability to phosphorylate D1 protein is restricted to seed plants.

Regulation of thylakoid protein phosphorylation in intact chloroplasts by the activity of kinases and phosphatases

Regulation of thylakoid protein phosphorylation from saturating up to photoinhibitory light conditions was studied in intact chloroplasts with high rates of CO 2 fixation and functioning protein biosynthesis. Comparing steady-state level phosphorylation with the phosphorylation in the presence of the phosphatase inhibitor NaF, evidence was found that phosphorylation of light harvesting chlorophyll-protein complex II (LHC II) and Photosystem II (PS II) polypeptides was catalysed by at least two different kinase / phosphatase systems. Whereas the steady-state level of LHC II phosphorylation declined with increasing light intensity, the phosphorylation level of PS II polypeptides remained stable even at photoinhibitory conditions. Decrease of steady-state level of LHC II phosphorylation at higher light intensities was partially due to an inhibition of the phosphorylation reaction. Its inactivation was observed before any significant loss of PS II electron transfer activity occurred. Quenching analysis of chlorophyll fluorescence revealed that the high light inhibition of the LHC II phosphorylation reaction was caused by an increased membrane energetisation, and not by an oxidation of the plastoquinone pool. In contrast, the kinase activity responsible for the phosphorylation of the PS II polypeptides seemed to be exclusively under the redox control of the plastoquinone pool and was not influenced by membrane energetisation. Evidence was found that also the phosphatase activities specific for LHC II and PS II proteins were different. As indicated by the high turnover of phosphate groups bound to LHC II, the LHC II specific phosphatase showed a high activity. Its activity was stimulated at higher light intensities and determined to a main extent the steady state level of LHC II phosphorylation. The phosphatase specific for the PS II phosphoproteins showed almost no activity in the light as indicated by the absence of a phosphate group turnover in the light. Dephosphorylation of PS II could only be observed in the dark and in contrast to LHC II dephosphorylation could not be inhibited by NaF. Even the increased turnover of the D1 protein at higher light intensities, which was followed by the light-dependent incorporation of [ 14C]leucine into the protein, did not accelerate the D1 protein phosphorylation. The steady-state level of PS II protein phosphorylation was therefore in principle determined by its kinase activity.