Involvement of protein phosphorylation in the sensitivity of photosystem II to strong illumination

Four types of differently phosphorylated hylakoids isolated from field grown spinach ( Spinacia oleracea L.) were tested for the sensitivity of photosystem II (PSII) to photoinactivation. Phosphorylation of light-harvesting II complexes (LHCII) protected PSII electron transfer from photoinhibitory damage, while the phosphorylation of the PSII core polypeptides slightly accelerated the decline of electron transfer during high irradiance treatment. Dephosphorylation of the CP43 apoprotein and PsbH protein by an alkaline phosphatase resulted in an extreme sensitivity of the thylakoids to strong illumination. The PSII photoinactivation of thylakoids with the impaired oxygen-evolving complex was found to be independent of phosphorylation.
The thylakoids of the thermophilic cyanobacterium Synechococcus elongates were used in order to compare the plants with an organism where LHCII complexes are missing and the PSII core proteins are not phosphorylated.     

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.     

Light affects the accessibility of the thylakoid light harvesting complex II (LHCII) phosphorylation site to the membrane protein kinase(s)

Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30 micromol m(-2) s(-1)) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 micromol m(-2) s(-1)) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5 degrees C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.     

Specific loss of LHCII phosphorylation in the Lemna mutant 1073 lacking the cytochrome b6/f complex

The thylakoid protein kinase(s) activity of Lemna perpusilla strain 6746 (wild type, WT) and the cytochrome (cyt) b₆/f-less mutant 1073 was compared. Isolated thylakoids of both WT and mutant phosphorylated the polypeptides of 9–15, 29, 32–34 and 40–45 kDa. This kinase(s) activity was light-dependent and could be elicited by addition of duroquinol in the dark. Thylakoids from both WT and mutant phosphorylated histone III-S at comparable rates. However, the redox-controlled phosphorylation of the LHCII polypeptide which could be demonstrated in vitro and in vivo in the WT thylakoids could not be detected under any experimental condition in the cyt b₆/f-less thylakoids. Halogenated quinone analogues known to inhibit reduction of the cyt b₆/f complex inhibited both the electron flow and duroquinol-activated LHCII phosphorylation, but had no effect on the duroquinol-dependent phosphorylation of the other thylakoid polypeptides. These results indicate that the Lemna thylakoids contain at least two redox-activated protein kinase(s). A quinone-binding site is involved in the activation of the LHCII kinase system which is rendered inactive in the absence of the cyt b₆/f complex.     

Environmental and metaboliccontrol of LHCII protein phosphorylation: revealing the mechanismsfor dual regulation of the LHCII kinase

Light‐harvesting complex II (LHCII) protein phosphorylation inplant chloroplasts is under complex regulation. Combination of the invivo monitoring of LHCII protein phosphorylation (by immunoblotting)with the in vitro[γ‐³²P]ATPphosphorylation assays revealed that the basic activation/deactivationmodel of the LHCII kinase, regulated by reversible occupation/releaseof plastoquinol at the plastoquinol oxidation (Q_o) siteof the cytochrome b₆f (cyt b₆f) complex, isconsistent with, but not sufficient to explain the data obtainedwith isolated chloroplasts, leaf discs or intact leaves. Not onlythe light conditions but also the metabolic state of the entireplant, particularly the sugar metabolism, exerted a control overLHCII protein phosphorylation. Feeding of leaves with glucose (alsowith glutathione) activated the LHCII kinase in darkness. On the otherhand, independently of the basic activation/deactivationmechanism of the kinase, a strong inhibition of LHCII protein phosphorylationoccurred in vivo at increasing irradiances and even at lowlight conditions, depending on the metabolic state of the plant.Both the experiments with intact chloroplasts and the reconstitutionexperiments with isolated thylakoids to mimic LHCII kinase inhibition,disclosed that the kinase in its activated state (plastoquinol at theQ_o site of cyt b₆f complex) is protected againstinhibition by thiol reductants. However, directly upon deactivationof the kinase (release of plastoquinol from the Q_o site) itbecomes a target for inhibition by thiol reductants. Thus the twointerdependent regulatory systems of the LHCII kinase, the constantlyoccurring activation and deactivation on the one hand and the inhibitionby thiol reductants on the other, are strongly dependent on theconcentration of reducing equivalents in the chloroplast stroma.A scheme demonstrating the interconversion of activated, deactivated andinhibited states of the LHCII kinase in the chloroplast environmentof intact leaves is presented.     

Differential kinase systems are involved in the rapidly turning over phosphorylation of prominent nuclear proteins

The activity of endogenous nuclear protein kinases has been probed in an vitro assay system of isolated nuclei from Chironomus salivary gland cells. The phosphorylation of a set of seven prominent rapidly phosphorylated non-histone proteins and of histones H3, H2A and H4 was analyzed using ATP or GTP as phosphoryl donor and heparin as protein kinase effector. The core histones H2A and H3 both incorporate 32P from [gamma-32P]ATP as well as from [gamma-32P]GTP but their phosphorylation is differentially affected by heparin. The phosphorylation of H2A is blocked by heparin while that of H3 is even stimulated in the presence of heparin when ATP is used as phosphate donor. H4 is unable to incorporate phosphate groups from GTP but its ATP-based phosphorylation is heparin sensitive. Of the non-histone protein kinase substrates, we could only detect two, the 44-kDa and 115-kDa proteins, which are heparin sensitive with either ATP or GTP and, thus, strictly meet the criteria for casein kinase type II-specific phosphorylation. The investigated histones and non-histone proteins can be grouped into three broad categories on the basis of their phosphorylation properties. (A) Proteins very likely affected by casein kinase NII. (B) Proteins phosphorylated by strictly ATP-specific protein kinases. (C) Proteins phosphorylated by ATP as well as GTP utilizing protein kinase(s) other than casein NII. Category B proteins can be subdivided into proteins phosphorylated in a heparin-resistant (B1) and heparin-sensitive (B2) manner. The phosphorylation of category C proteins may be heparin sensitive with ATP only (C1), heparin sensitive with GTP only (C2), heparin insensitive with both ATP and GTP (C3) or stimulated by heparin (C4).     

Phosphorylation of thylakoid proteins during chloroplast biogenesis in greening etiolated and light-grown wheat leaves

Phosphorylation of polypeptides in isolated thylakoids was examined during chloroplast biogenesis in greening etiolated wheat leaves and 4 day-old wheat leaves grown under a diurnal light regime. At early stages of plastid development standard thylakoid preparations were heavily contaminated with nuclear proteins, which distorted the polypeptide phosphorylation profiles. Removal of contamination from membranes by sucrose density centrifugation demonstrated that the major membrane phosphoprotein in etioplasts was at 35 kDa. During etioplast greening a number of phosphoproteins appeared, of which the 25–27 kDa apoproteins of the light-harvesting chlorophylla/b protein complex associated with photosystem II (LHCII) became the most dominant. At the early stages of thylakoid development found at the base of the 4-day-old light grown leaf the LHCII apoproteins were evident as phosphoproteins; however the major phosphoprotein was polypeptide atca. 9kDA. Phosphorylation of both the LHCII apoproteins and the 9 kDa polypeptide in these thylakoids was not light-dependent. In the older thylakoids isolated from the leaf tip the LHCII apoproteins were the major phosphoproteins and their phosphorylation had become light-regulated; however phosphorylation of the 9 kDa polypeptide remained insensitive to light.     

Differentiation and development of bundle sheath and mesophyll thylakoids in maize. Thylakoid polypeptide composition, phosphorylation, and organization of photosystem II

Photosynthetic electron flow, polypeptide pattern, presence of chlorophyll-protein complexes, and phosphorylation of thylakoid polypeptides have been investigated in differentiated mesophyll (M) and bundle sheath (B) thylakoids of the C4 plant Zea mays. The polypeptide pattern of M thylakoids and their photosynthetic electron flow are comparable to those of other green plants. B thylakoids exhibit only photosystem I (PSI) activity, contain only traces of the PSII light harvesting (LHCII) polypeptide, do not bind [3H] diuron, and lack polypeptides of the water-oxidation complex of PSII and the herbicide binding 32-kDa polypeptide, as detected by specific antibodies. However, B thylakoids possess a partially active PSII reaction center, as demonstrated by light-dependent reduction of silicomolybdate with 1,5-diphenylcarbazide (DPC) as an electron donor, and the presence of the PSII reaction center polypeptides of 44-47 kDa. Only one chlorophyll a-protein complex, corresponding to the PSI reaction center-core antenna, was detectable in B thylakoids, as opposed to chlorophyll a and chlorophyll a,b-protein complexes present in M thylakoids. The light-dependent, membrane-bound kinase activity present in M thylakoids could not be detected in B thylakoids which, nevertheless, contain a protein kinase able to phosphorylate casein. A total of 19 differences between the electrophoretic pattern of B and M thylakoid polypeptides were observed. The mRNA coding for the LHCII polypeptide is primarily, if not exclusively, localized in M cells. The development of PSII complex precedes that of PSI during the differentiation of B and M chloroplasts in expanding leaves of light-grown plants and during the greening of dark-grown etiolated seedlings. The differentiation of the maize leaf into cells programmed to form B or M chloroplasts does not require light. In light-grown plants, the differentiation of B and M thylakoids occurred progressively from the base of the leaf and was completed at 4-5 cm from the leaf base.     

Trans-thylakoid ∆pH dependent oscillation of FPSI/FPSII under continuous irradiance in isolated thylakoids

Energy distribution between photosystems (PSI & PSII) under prolonged and continuous white light irradiance was assessed by monitoring the progress of their fluorescence emission (F_PSI/F_PSII) at 77 K. Our observations indicate F_PSI/F_PSII to oscillate with the progress of irradiance treatments at all intensities tested (100, 200, 500, and 800 μE m^?2 S^?1). The amplitude of the oscillation increased with the progress, whereas the periodicity of the oscillation increased with the intensity of the incident irradiance. Spectral analysis indicated fluctuation of F_PSI to be the major determinant of the observed oscillation. The first rise and fall of F_PSI/F_PSII overlapped with phosphorylation and dephosphorylation of LHCII, but oscillation of F_PSI/F_PSII continued for several cycles without any further phosphorylation of LHCII. Moreover, in presence of DCMU where linear electron flow (LEF) is suppressed and LHCII phosphorylation is completely abolished, the oscillation of F_PSI/F_PSII was not abolished. These data indicated that LHCII phosphorylation was not essential for the observed oscillation of energy distribution between the photosystems. In contrast, in the presence of inhibitors of cyclic electron flow (CEF) like Antimycin A (AA) and rotenone, the oscillation of F_PSI/F_PSII was either abolished or severely dampened. Additionally, the oscillation was also abolished in presence of uncouplers like NH₄Cl and nigericin that cancels the trans-thylakoid ?pH. Thus, trans-thylakoid ?pH, generated through CEF, appear to be an important determinant of oscillation of F_PSI/F_PSII in isolated thylakoids. The phenomenon of oscillation could be associated with a CEF mediated chromatic adaptation of PSI in presence of excess irradiance.     

Light-modulated exposure of the light-harvesting complex II (LHCII) to protein kinase(s) and state transition in Chlamydomonas reinhardtii xanthophyll mutants

Reversible phosphorylation of chl a/b protein complex II (LHCII), the mobile light-harvesting antenna, regulates its association and energy transfer/dissipation to photosystem (PS) II or I (state transition). Excitation of LHCII induces conformational changes affecting the exposure of the phosphorylation site at the N-terminal domain to protein kinase(s) [Zer, H., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282; Zer, H., et al. (2003) Biochemistry 42, 728-738]. Thus, it was of interest to examine whether the pigment composition of LHCII affects the light-induced modulation of LHCII phosphorylation and state transition. To this end, we have used thylakoids of wild-type Chlamydomonas reinhardtii and xanthophyll deficient mutants npq1, lor1, npq2, npq1 lor1, and npq2 lor1. Phosphorylated protein bands P11, P13, and P17 are considered components of the mobile C. reinhardtii LHCII complex. The protein composition of these bands has been analyzed by mass spectrometry using Qtof-2 with a nanospray attachment. P11 and P13 contain C. reinhardtii light-harvesting chlorophyll a/b binding protein LhcII type I. P17 contains C. reinhardtii LhcII types III and IV. Illumination of isolated thylakoids inhibits the redox-controlled phosphorylation of polypeptide bands P13 and P17 and to a lower extent that of P11. The light-induced inhibition of LHCII phosphorylation and the state transition process are not influenced by extensive differences in the xanthophyll composition of the mutants. Thus, LHCII can be visualized as possessing two functionally distinct, independent domains: (i) the pigment binding transmembrane domain regulating the extent of energy transfer/dissipation and (ii) the surface-exposed phosphorylation site regulating the association of LHCII with PSII or PSI.     

Light modulation of serine and threonine phosphorylation in histone III by thylakoids

Thylakoids from dark-adapted leaves phosphorylated histone III faster in the light than in the dark, such process being the resultant of a light activation of a threonine protein phosphorylation and a light inactivation of a serine protein phosphorylation. Phosphorylation of histone III by thylakoids from dark-adapted leaves showed an optimal pH of 7.1 in the light and of 8.5 in the dark. Storage of the thylakoids at −20°C for 3 weeks completely abolished threonine phosphorylation, but had minor effect on serine protein phosphorylation which was also inhibited by illumination during the protein kinase assay. Preillumination of the spinach leaves inactivated the serine protein phosphorylation catalyzed by thylakoids. These results are consistent with the presence of two distinguishable thylakoid-bound protein kinase activities in terms of their response to light, optimal pH and specificity for amino acid residue in the protein substrate.     

Interaction between light harvesting chlorophyll-a/b protein (LHCII) kinase and cytochrome b6/f complex. In vitro control of kinase activity

We have previously reported that the cytochrome b6/f complex may be involved in the redox activation of light harvesting chlorophyll-a/b protein complex of photosystem II (LHCII) kinase in higher plants (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210). The aim of this work was to establish whether a relation between the cytochrome b6/f and LHCII kinase activation can be demonstrated in vitro. Preparations enriched in cytochrome b6/f obtained from spinach thylakoids by detergent extraction and precipitation with ammonium sulfate followed by different procedures of purification, contained various amounts of LHCII kinase activity. Analysis of the cytochrome b6/f content and kinase activity of fractions obtained by histone-Sepharose and immunoaffinity columns, immunoprecipitation and sucrose density centrifugation, indicate functional association of kinase and cytochrome b6/f. Phosphorylation of LHCII by fractions containing both cytochrome b6/f and kinase was enhanced by addition of plastoquinol-1. LHCII phosphorylation and kinase activation could be obtained in fractions prepared by use of beta-D-octyl glucoside but not when 3-[(cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate was used as the solubilizing detergent. Kinase activity could be inhibited by halogenated quinone analogues (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2,3-diiodo-5-t-butyl-p-benzoquinone) known to inhibit cytochrome b6/f activity. However, kinase activity was inhibited by these analogues in all preparations including those which could not phosphorylate LHCII. We thus propose that the redox activation of LHCII phosphorylation is mediated by kinase interaction with cytochrome b6/f while the deactivation may be related to a distinct quinone binding site of the enzyme molecule.     

Redox-controlled thylakoid protein phosphorylation. News and views

Thylakoid protein phosphorylation regulates state transition and PSII protein turnover under light-dependent redox control via a signal transduction system. The redox-dependent activation/deactivation of the membrane-bound protein kinase(s), mostly localized in the grana partitions, differs for the various phosphoproteins. Reduction of the plastoquinone pool may be sufficient to activate phosphorylation of few of these proteins. Phosphorylation of LHCII, requires the presence of the cytochrome bf complex in an 'activating mode' characterized by the reduction of its high potential path components and ability to interact with a reduced plastoquinol without oxidizing it. Activation and maintenance of this kinase activity is considered to involve alternate interactions with a cytochrome bf in its activating mode and with the substrate PSII(LHCII). The segregation of the thylakoid components into grana and stroma partitions appears to be mandatory for the kinase activation process. The protein substrate specificity and kinetics differs for various kinases. The thylakoid redox-controlled kinase(s) have not yet been isolated. Preparations highly enriched in kinase activity capable to phosphorylate LHCII and PSII core proteins, contain two kinase active bands, resolved by denaturing electrophoresis and renaturation, and having apparent molecular masses of about 53 and 66 kDa. The roughly estimated abundance of these putative kinase(s) in the grana partitions may be compatible with a ratio of kinase(s): PSII(LHCII) dimers:cytochrome bf dimers in the range of 1:60:30 and a ratio of kinase:phosphorylation sites of about 1:2000. Only about 10–20% of these sites are phosphorylated during state transition. The low turnover rate of the LHCII kinase(s) (< 5) may be due to hindrance of the required random lateral migration within the grana domain rich in tightly packed PSII(LHCII) and cytochrome bf complexes.     

The rate and extent of phosphorylation of the two light-harvesting chlorophyll a/b binding protein complex (LHC-II) polypeptides in isolated spinach thylakoids

The level of phosphorylation of the 24 kDa and the 25 kDa light-harvesting chlorophyll a/b binding protein complex (LHC) II polypeptides in isolated spinach thylakoids has been determined by quantitative SDS-polyacrylamide gel electrophoresis. The time-course of phosphorylation, after correction for the molar abundance of these two polypeptides, shows that (a) the rate of phosphorylation of the 24 kDa polypeptide is at least 3-fold faster compared with the 25 kDa polypeptide, (b) the final extent of phosphorylation for both the polypeptides is very similar, and (c) the final extent of phosphorylation is typically between 0.15 and 0.25 mol phosphate per mol polypeptide. The low extent of phosphorylation is not simply a consequence of inactivation of the kinase and / or LHC II substrate or ATP depletion. These observations suggest that there are at least three different sub-populations of LHC II in isolated thylakoids: (i) phosphorylated ‘mobile’, (ii) phosphorylated ‘PS II-coupled’ and (iii) non-phosphorylated. Furthermore, the reported differences in the specific activity of phosphorylation for the ‘mobile’ and the ‘PS II-coupled’ LHC II sub-populations (Kyle, D.J. et al. (1984) Biochim. Biophys. Acta 765, 89–96) are no longer observed following correction for the non-phosphorylated LHC-II population.     

Low temperature development of winter rye leaves alters the detergent solubilization of thylakoid membranes

Thylakoids isolated from leaves of winter rye (Secale cereale L. cv Puma) grown at either 20 or 5°C were extracted with the nonionic detergents Triton X-100 and octyl glucoside. Less total chlorophyll was extracted from 5°C thylakoids by these detergents under all conditions, including pretreatment with cations. Thylakoids from either 20 or 5°C leaves were solubilized in 0.7% Triton X-100 and centrifuged on sucrose gradients to purify the light harvesting complex (LHCII). Greater yields of LHCII were obtained by cation precipitation of particles derived from 20°C thylakoids than from 5°C thylakoids. When 20 and 5°C thylakoids were phosphorylated and completely solubilized in sodium dodecyl sulfate, no differences were observed in the ³²Pi-labeling characteristics of the membrane polypeptides. However, when phosphorylated thylakoids were extracted with octyl glucoside, extraction of LHCII associated with the 5°C thylakoids was markedly reduced in comparison with the extraction of LHCII from 20°C membranes. Since 20 and 5°C thylakoids exhibited significant differences in the Chl content and Chl a/b ratios of membrane fractions produced after solubilization with either Triton X-100 or octyl glucoside, and since few differences between the proteins of the two membranes could be observed following complete denaturation in sodium dodecyl sulfate, we conclude that the integral structure of the thylakoid membrane is affected during rye leaf development at low temperature.     

Polypeptide composition,assembly and phosphorylation patterns of the photosystem II antenna system of Chlamydomonas reinhardtii

In recent years major progress has been made in describing the gene families that encode the polypeptides of the light-harvesting antenna system of photosystem II (PSII). At the same time, advances in the biochemical characterization of these antennae have been hampered by the high degree of similarity between the apoproteins. To help interpret the molecular results, we have re-examined the composition, the assembly and the phosphorylation patterns of the light-harvesting antenna of PSII (LHCII) in the green alga Chlamydomonas reinhardtii Dang, using a non-Tris SDS-PAGE system capable of resolving polypeptides that differ by as little as 200 daltons. Research to date has suggested that in C. reinhardtii the LHCII comprises just four polypeptides (p11, p13, p16 and p17), and CP29 and CP26 just one polypeptide each (p9 and p10, respectively), i.e. a total of six polypeptides. We report here that these antenna systems contain at least 15 polypeptides, 10 associated with LHCII, 3 with CP29, and 2 with CP26. All of these polypeptides have been positively identified by means of appropriate antibodies. We also demonstrate substantial heterogeneity to the pattern of in-vitro phosphorylation, with major differences found among members of closely spaced and immunologically related polypeptides. Most intriguing is the fact that the polypeptides that cross-react with the anti-type 2 LHCII antibodies of higher plants (p16, and to a lesser extent p11) are not phosphorylated, whereas in higher plants these are the most highly phosphorylated polypeptides. Also, unlike in higher plants, CP29 is heavily phosphorylated. Phosphorylation does not appear to have any effect on the mobility of polypeptides on fully denaturing SDS-PAGE gels. To learn more about the accumulation and organization of the light-harvesting polypeptides, we have also investigated a chlorophyll b-less mutant, cbn1-48. The LHCII is almost completely lost in this mutant, along with at least some LHCI. But the accumulation of CP29 and CP26 and their binding to PSII core complexes, is relatively unaffected. As expected, the loss of antenna polypeptides is accompanied by a reduction of the size of large reaction-center complexes. Following in-vitro phosphorylation the number of phosphorylated proteins is greatly increased in the mutant thylakoids compared to wildtype thylakoids. We present a model of the PSII antenna system to account for the new polypeptide complexity we have demonstrated.This work was supported by National Institute of Health grant GM22912 to L.A.S. We would like to thank Anastasios Melis for helpful discussions.     

Characteristics of Photosystem I Complexes in the End Membranes of Pea Granal Thylakoids

Two fractions of the light fragments enriched in the photosystem I (PSI) complexes were obtained from pea (Pisum sativum L.) thylakoids by digitonin treatment and subsequent differential centrifugation. The ratio of chlorophyll a to chlorophyll b, chlorophyll/P700 spectra of low-temperature fluorescence, and excitation spectra of long-wave fluorescence were measured. These characteristics were shown to be different due to variation in the size and composition of the light-harvesting antenna of PSI complexes present in the particles obtained. The larger antenna size of one of the fractions was related to the incorporation of the pool of light-harvesting complex II (LHCII). A comparison with the data available allowed us to identify these particles as fragments of intergranal thylakoids and end membranes of granal thylakoids. The suggestion that an increase in the PSI light-harvesting antenna in intergranal thylakoids is related to the attachment of phosphorylated LHCII is discussed.     

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.     

Ascorbate-mediated LHCII protein phosphorylation--LHCII kinase regulation in light and in darkness

A freeze-thaw cycle of isolated thylakoids in darkness in the presence of ascorbate was employed as a novel experimental system to activate the light-harvesting complex (LHC) II kinase. Under these conditions ascorbate reduces Q(A), the primary quinone electron acceptor of photosystem (PS) II, and the subsequent reduction of plastoquinone and the cytochrome (cyt) b(6)f complex results in the activation of the LHCII kinase. Using this activation system, several facets of regulation of LHCII protein phosphorylation were unravelled. (i) Myxothiazol inhibited the activation of LHCII protein phosphorylation, thus being a potent inhibitor of electron flow not only in cyt bc complexes but in darkness also in cyt b(6)f complexes. (ii) Oxygen, the only electron acceptor in darkness, was required for LHCII kinase activation demonstrating that after a full reduction of the cyt b(6)f complex, an additional plastoquinol oxidation cycle in the quinol oxidation (Qo) site is required for LHCII kinase activation. (iii) In the absence of electron flow, when the intersystem electron carriers are reduced, the activated LHCII kinase has a half-life of 40 min, whereas the fully activated LHCII kinase becomes deactivated in a time scale of seconds upon oxidation of the cyt b(6)f complex, indicating that the kinase constantly reads the redox poise of the cyt b(6)f complex. (iv) The LHCII kinase is more tightly bound to the thylakoid membrane than the PS II core protein kinase(s). It is concluded that oxidation of plastoquinol at the Qo site of the reduced cyt b(6)f complex is required for LHCII kinase activation, while rapid reoccupation of the Qo site with plastoquinol is crucial for sustenance of the active state of the LHCII kinase.     

Preheating accelerates mitogen-activated protein (MAP) kinase inactivation post-heat shock via a heat shock protein 70-mediated increase in phosphorylated MAP kinase phosphatase-1

Heat shock (HS) activates mitogen-activated protein (MAP) kinases. Although prior exposure to nonlethal HS makes cells refractory to the lethal effect of a subsequent HS, it is unclear whether this also occurs in MAP kinase activation. This study was undertaken to evaluate the effect of a heat pretreatment on MAP kinase activation by a subsequent HS and to elucidate its possible mechanism. Preheating did not make BEAS-2B cells refractory to extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) activation by a second HS but accelerated their inactivation after HS. The rapid inactivation of ERK and JNK was dependent on de novo protein synthesis and associated with the up-regulation of heat shock protein 70 (HSP70). Moreover, the inhibition of phosphatase activity reversed this rapid inactivation. MAP kinase phosphatase-1 (MKP-1) expression was increased by HS, and the presence of its phosphorylated form (p-MKP-1) correlated with the observed rapid ERK and JNK inactivation. Blocking induction of p-MKP-1 with antisense MKP-1 oligonucleotides suppressed the rapid inactivation of ERK and JNK in preheated cells. HSP70 overexpression caused the early phosphorylation of MKP-1. Moreover, MKP-1 phosphorylation and the rapid inactivation of ERK were inhibited by blocking HSP70 induction in preheated cells. In addition, MKP-1 was insolubilized by HS, and HSP70 associated physically with MKP-1, suggesting that a chaperone effect of HSP70 might have caused the early phosphorylation of MKP-1. These results indicate that preheating accelerated MAP kinase inactivation after a second HS and that this is related to a HSP70-mediated increase in p-MKP-1.