Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants

Phosphorylation-dependent movement of the light-harvesting complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) takes place in order to balance the function of the two photosystems. Traditionally, the phosphorylatable fraction of LHCII has been considered as the functional unit of this dynamic regulation. Here, a mechanical fractionation of the thylakoid membrane of Spinacia oleracea was performed from leaves both in the phosphorylated state (low light, LL) and in the dephosphorylated state (dark, D) in order to compare the phosphorylation-dependent protein movements with the excitation changes occurring in the two photosystems upon LHCII phosphorylation. Despite the fact that several LHCII proteins migrate to stroma lamellae when LHCII is phosphorylated, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI-LHCI complex. A new model for LHCII phosphorylation driven regulation of relative PSII/PSI excitation thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon LHCII phosphorylation and resulting from the movement of PSI-LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. The grana margins probably give a flexibility for regulation of linear and cyclic electron flow in plant chloroplasts.     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700(+) and Y(D)( .-), respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIbeta) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIalpha) 300, PSI (PSIbeta) in stroma lamellae 214, PSII in grana core (PSIIalpha) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700⁺ and Y_D, respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIβ) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIα) 300, PSI (PSIβ) in stroma lamellae 214, PSII in grana core (PSIIα) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.     

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.     

Digitonin-sensitive LHCII enlarges the antenna of Photosystem I in stroma lamellae of Arabidopsis thaliana after far-red and blue-light treatment

Light drives photosynthesis. In plants it is absorbed by light-harvesting antenna complexes associated with Photosystem I (PSI) and photosystem II (PSII). As PSI and PSII work in series, it is important that the excitation pressure on the two photosystems is balanced. When plants are exposed to illumination that overexcites PSII, a special pool of the major light-harvesting complex LHCII is phosphorylated and moves from PSII to PSI (state 2). If instead PSI is over-excited the LHCII complex is dephosphorylated and moves back to PSII (state 1). Recent findings have suggested that LHCII might also transfer energy to PSI in state 1. In this work we used a combination of biochemistry and (time-resolved) fluorescence spectroscopy to investigate the PSI antenna size in state 1 and state 2 for Arabidopsis thaliana. Our data shows that 0.7 ± 0.1 unphosphorylated LHCII trimers per PSI are present in the stroma lamellae of state-1 plants. Upon transition to state 2 the antenna size of PSI in the stroma membrane increases with phosphorylated LHCIIs to a total of 1.2 ± 0.1 LHCII trimers per PSI. Both phosphorylated and unphosphorylated LHCII function as highly efficient PSI antenna.     

Functional heterogeneity of photosystem II in domain specific regions of the thylakoid membrane of spinach (Spinacia oleracea L.)

A mild sonication and phase fractionation method has been used to isolate five regions of the thylakoid membrane in order to characterize the functional lateral heterogeneity of photosynthetic reaction centers and light harvesting complexes. Low-temperature fluorescence and absorbance spectra, absorbance cross-section measurements, and picosecond time-resolved fluorescence decay kinetics were used to determine the relative amounts of photosystem II (PSII) and photosystem I (PSI), to determine the relative PSII antenna size, and to characterize the excited-state dynamics of PSI and PSII in each fraction. Marked progressive increases in the proportion of PSI complexes were observed in the following sequence: grana core (BS), whole grana (B3), margins (MA), stroma lamellae (T3), and purified stromal fraction (Y100). PSII antenna size was drastically reduced in the margins of the grana stack and stroma lamellae fractions as compared to the grana. Picosecond time-resolved fluorescence decay kinetics of PSII were characterized by three exponential decay components in the grana fractions, and were found to have only two decay components with slower lifetimes in the stroma. Results are discussed in the framework of existing models of chloroplast thylakoid membrane lateral heterogeneity and the PSII repair cycle. Kinetic modeling of the PSII fluorescence decay kinetics revealed that PSII populations in the stroma and grana margin fractions possess much slower primary charge separation rates and decreased photosynthetic efficiency when compared to PSII populations in the grana stack.     

Structural characterization of a complex of photosystem I and light-harvesting complex II of Arabidopsis thaliana

Chloroplasts are central to the provision of energy for green plants. Their photosynthetic membrane consists of two major complexes converting sunlight: photosystem I (PSI) and photosystem II (PSII). The energy flow toward both photosystems is regulated by light-harvesting complex II (LHCII), which after phosphorylation can move from PSII to PSI in the so-called state 1 to state 2 transition and can move back to PSII after dephosphorylation. To investigate the changes of PSI and PSII during state transitions, we studied the structures and frequencies of all major membrane complexes from Arabidopsis thaliana chloroplasts at conditions favoring either state 1 or state 2. We solubilized thylakoid membranes with digitonin and analyzed the complete set of complexes immediately after solubilization by electron microscopy and image analysis. Classification indicated the presence of a PSI-LHCII supercomplex consisting of one PSI-LHCI complex and one LHCII trimer, which was more abundant in state 2 conditions. The presence of LHCII was confirmed by excitation spectra of the PSI emission of membranes in state 1 or state 2. The PSI-LHCII complex could be averaged with a resolution of 16 A, showing that LHCII has a specific binding site at the PSI-A, -H, -L, and -K subunits.     

A stable LHCII-PSI aggregate and suppression of photosynthetic state transitions in the psae1-1 mutant of Arabidopsis thaliana

During photosynthetic state transitions, a fraction of the major light-harvesting complex (LHCII) shuttles between photosystems II (PSII) and I (PSI), depending on whether or not it is phosphorylated. Its phosphorylation state in turn depends on the relative activity of the two photosystems, which is a function of redox state and illumination parameters. In the psae1-1 mutant of Arabidopsis thaliana (L.) Heynh., amounts of the PSI subunits E, C, D, H and L are decreased. A fraction of LHCII is stably associated with PSI when plants are exposed to low light conditions, giving rise to a high-molecular-mass protein-pigment complex detectable in native protein gels. The formation of this abnormal LHCII-PSI complex is associated with an almost complete suppression of state transitions, a drastic increase in the levels of phosphorylated LHCII under all light regimes tested, and a permanent reduction in PSII antenna size. All these observations suggest that the altered polypeptide composition of PSI perturbs the docking of phosphorylated LHCII, making psae1-1 a unique mutant for the study of PSI-LHCII interactions and additional effects of the mutation, such as a decrease in grana stacking and increased adenylate kinase activity.     

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

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

Characterisation of senescence-induced changes in light harvesting complex II and photosystem I complex of thylakoids of Cucumis sativus cotyledons: age induced association of LHCII with photosystem I

Structure and function of chloroplasts are known to after during senescence. The senescence-induced specific changes in light harvesting antenna of photosystem II (PSII) and photosystem I (PSI) were investigated in Cucumis cotyledons. Purified light harvesting complex II (LHCII) and photosystem I complex were isolated from 6-day non-senescing and 27-day senescing Cucumis cotyledons. The chlorophyll a/b ratio of LHCII obtained from 6-day-old control cotyledons and their absorption, chlorophyll a fluorescence emission and the circular dichroism (CD) spectral properties were comparable to the LHCII preparations from other plants such as pea and spinach. The purified LHCII obtained from 27-day senescing cotyledons had a Chl a/b ratio of 1.25 instead of 1.2 as with 6-day LHCII and also exhibited significant changes in the visible CD spectrum compared to that of 6-day LHCII, indicating some specific alterations in the organisation of chlorophylls of LHCII. The light harvesting antenna of photosystems are likely to be altered due to aging. The room temperature absorption spectrum of LHCII obtained from 27-day senescing cotyledons showed changes in the peak positions. Similarly, comparison of 77K chlorophyll a fluorescence emission characteristics of LHCII preparation from senescing cotyledons with that of control showed a small shift in the peak position and the alteration in the emission profile, which is suggestive of possible changes in energy transfer within LHCII chlorophylls. Further, the salt induced aggregation of LHCII samples was lower, resulting in lower yields of LHCII from 27-day cotyledons than from normal cotyledons. Moreover, the PSI preparations of 6-day cotyledons showed Chl a/b ratios of 5 to 5.5, where as the PSI sample of 27-day cotyledons had a Chl a/b ratio of 2.9 suggesting LHCII association with PSI. The absorption, fluorescence emission and visible CD spectral measurements as well as the polypeptide profiles of 27-day cotyledon-PSI complexes indicated age-induced association of LHCII of PSII with PSI obtained from 27-day cotyledons. We modified our isolation protocols by increasing the duration of detergent Triton X-100 treatment for preparing the PSI and LHCII complexes from 27-day cotyledons. However, the PSI complexes isolated from senescing samples invariably proved to have significantly low Chl a/b ratio suggesting an age induced lateral movement and possible association of LHCII with PSI complexes. The analyses of polypeptide compositions of LHCII and PSI holocomplexes isolated from 6-day control and 27-day senescing cotyledons showed distinctive differences in their profiles. The presence of 26-28 kDa polypeptide in PSI complexes from 27-day cotyledons, but not in 6-day control PSI complexes is in agreement with the notion that senescence induced migration of LHCII to stroma lamellae and its possible association with PSI. We suggest that the migration of LHCII to the stroma lamellae region and its possible association with PSI might cause the destacking and flattening of grana structure during senescence of the chloroplasts. Such structural changes in light harvesting antenna are likely to alter energy transfer between two photosystems. The nature of aging induced migration and association of LHCII with PSI and its existence in other senescing systems need to be estimated in the future.     

The domain organization of the plant thylakoid membrane

A model of the photosynthetic membrane from higher plants is presented. The different photosystems, PSI alpha, PSI beta, PSII alpha and PSII beta, are located in separate domains. The photosystems with the largest antenna systems, the alpha systems, are in the grana and the other in the stroma lamellae. In each grana disc PSI alpha is located in a flat annulus surrounding a circular PSII alpha domain. In this the PSII alpha units with the largest antennae are found in the center. The model is consistent with results from recent membrane fractionation experiments.     

Photosystem II in different parts of the thylakoid membrane: a functional comparison between different domains

The electron transport properties of photosystem II (PSII) from five different domains of the thylakoid membrane were analyzed by flash-induced fluorescence kinetics. These domains are the entire grana, the grana core, the margins from the grana, the stroma lamellae, and the Y100 fraction (which represent more purified stroma lamellae). The two first fractions originate from appressed grana membranes and have PSII with a high proportion of O(2)-evolving centers (80-90%) and efficient electron transport on the acceptor side. About 30% of the granal PSII centers were found in the margin fraction. Two-thirds of those PSII centers evolve O(2), but the electron transfer on the acceptor side is slowed. PSII from the stroma lamellae was less active. The fraction containing the entire stroma has only 43% O(2)-evolving PSII centers and slow electron transfer on the acceptor side. In contrast, PSII centers of the Y100 fraction show no O(2) evolution and were unable to reduce Q(B). Flash-induced fluorescence decay measurements in the presence of DCMU give information about the integrity of the donor side of PSII. We were able to distinguish between PSII centers with a functional Mn cluster and without any Mn cluster, and PSII centers which undergo photoactivation and have a partially assembled Mn cluster. From this analysis, we propose the existence of a PSII activity gradient in the thylakoid membrane. The gradient is directed from the stroma lamellae, where the Mn cluster is absent or inactive, via the margins where photoactivation accelerates, to the grana core domain where PSII is fully photoactivated. The photoactivation process correlates to the PSII diffusion along the membrane and is initiated in the stroma lamellae while the final steps take place in the appressed regions of the grana core. The margin domain is seemingly very important in this process.     

The High Efficiency of Photosystem I in the Green Alga Chlamydomonas reinhardtii Is Maintained after the Antenna Size Is Substantially Increased by the Association of Light-harvesting Complexes II

Photosystems (PS) I and II activities depend on their light-harvesting capacity and trapping efficiency, which vary in different environmental conditions. For optimal functioning, these activities need to be balanced. This is achieved by redistribution of excitation energy between the two photosystems via the association and disassociation of light-harvesting complexes (LHC) II, in a process known as state transitions. Here we study the effect of LHCII binding to PSI on its absorption properties and trapping efficiency by comparing time-resolved fluorescence kinetics of PSI-LHCI and PSI-LHCI-LHCII complexes of Chlamydomonas reinhardtii. PSI-LHCI-LHCII of C. reinhardtii is the largest PSI supercomplex isolated so far and contains seven Lhcbs, in addition to the PSI core and the nine Lhcas that compose PSI-LHCI, together binding ∼320 chlorophylls. The average decay time for PSI-LHCI-LHCII is ∼65 ps upon 400 nm excitation (15 ps slower than PSI-LHCI) and ∼78 ps upon 475 nm excitation (27 ps slower). The transfer of excitation energy from LHCII to PSI-LHCI occurs in ∼60 ps. This relatively slow transfer, as compared with that from LHCI to the PSI core, suggests loose connectivity between LHCII and PSI-LHCI. Despite the relatively slow transfer, the overall decay time of PSI-LHCI-LHCII remains fast enough to assure a 96% trapping efficiency, which is only 1.4% lower than that of PSI-LHCI, concomitant with an increase of the absorption cross section of 47%. This indicates that, at variance with PSII, the design of PSI allows for a large increase of its light-harvesting capacities.     

植物光合机构的状态转换

植物光合机构的状态转换是一种通过光系统Ⅱ的捕光天线色素蛋白复合体（LHCⅡ）的可逆磷酸化调节激发能在两个光系统间的分配来适应环境中光质等短期变化的机制．一般植物光合机构的LHCⅡ磷酸化主要受电子递体质醌和细胞色素b6f复合体氧化还原状态的调节,从而影响其在两种光系统间的移动。植物光合机构的状态转换也可以通过两种光系统相互接近导致激发能满溢来平衡两个光系统的激发能分配。外界离子浓度骤变可以引起盐藻LHCⅡ磷酸化,其调节过程与电子递体的氧化还原状态无关。绿藻的状态转换可以调节细胞内的ATP供求关系。     

Thylakoid Protein Phosphorylation in Higher Plant Chloroplasts Optimizes Electron Transfer under Fluctuating Light

Several proteins of photosystem II (PSII) and its light-harvesting antenna (LHCII) are reversibly phosphorylated according to light quantity and quality. Nevertheless, the interdependence of protein phosphorylation, nonphotochemical quenching, and efficiency of electron transfer in the thylakoid membrane has remained elusive. These questions were addressed by investigating in parallel the wild type and the stn7, stn8, and stn7 stn8 kinase mutants of Arabidopsis (Arabidopsis thaliana), using the stn7 npq4, npq4, npq1, and pgr5 mutants as controls. Phosphorylation of PSII-LHCII proteins is strongly and dynamically regulated according to white light intensity. Yet, the changes in phosphorylation do not notably modify the relative excitation energy distribution between PSII and PSI, as typically occurs when phosphorylation is induced by “state 2” light that selectively excites PSII and induces the phosphorylation of both the PSII core and LHCII proteins. On the contrary, under low-light conditions, when excitation energy transfer from LHCII to reaction centers is efficient, the STN7-dependent LHCII protein phosphorylation guarantees a balanced distribution of excitation energy to both photosystems. The importance of this regulation diminishes at high light upon induction of thermal dissipation of excitation energy. Lack of the STN7 kinase, and thus the capacity for equal distribution of excitation energy to PSII and PSI, causes relative overexcitation of PSII under low light but not under high light, leading to disturbed maintenance of fluent electron flow under fluctuating light intensities. The physiological relevance of the STN7-dependent regulation is evidenced by severely stunted phenotypes of the stn7 and stn7 stn8 mutants under strongly fluctuating light conditions.Several proteins of PSII and its light-harvesting antenna (LHCII) are reversibly phosphorylated by the STN7 and STN8 kinase-dependent pathways according to the intensity and quality of light (Bellafiore et al., 2005; Bonardi et al., 2005). The best-known phosphorylation-dependent phenomenon in the thylakoid membrane is the state transition: a regulatory mechanism that modulates the light-harvesting capacity between PSII and PSI. According to the traditional view, “state 1” prevails when plants are exposed to far-red light (state 1 light), which selectively excites PSI. Alternatively, thylakoids are in “state 2” when plants are exposed to blue or red light (state 2 light), favoring PSII excitation. In state 1, the yield of fluorescence from PSII is higher in comparison with state 2 (for review, see Allen and Forsberg, 2001). State transitions are dependent on the phosphorylation of LHCII proteins (Bellafiore et al., 2005) and their association with PSI proteins, particularly PSI-H (Lunde et al., 2000). Under state 2 light, both the PSII core and LHCII proteins are strongly phosphorylated, whereas the state 1 light induces dephosphorylation of both the PSII core and LHCII phosphoproteins (Piippo et al., 2006; Tikkanen et al., 2006). In nature, however, such extreme changes in light quality rarely occur. The intensity of light, on the contrary, fluctuates frequently in all natural habitats occupied by photosynthetic organisms, thus constantly modulating the extent of thylakoid protein phosphorylation in a highly dynamic manner (Tikkanen et al., 2008a).The regulation of PSII-LHCII protein phosphorylation by the quantity of light is much more complex than the regulatory circuits induced by the state 1 and state 2 lights. Whereas changes in light quality induce a concurrent increase or decrease in the phosphorylation levels of both the PSII core (D1, D2, and CP43) and LHCII (Lhcb1 and Lhcb2) proteins, the changes in white light intensity may influence the kinetics of PSII core and LHCII protein phosphorylation in higher plant chloroplasts even in opposite directions (Tikkanen et al., 2008a). Indeed, it is well documented that low light (LL; i.e. lower than that generally experienced during growth) induces strong phosphorylation of LHCII but relatively weak phosphorylation of the PSII core proteins. Exposure of plants to high light (HL) intensities, on the contrary, promotes the phosphorylation of PSII core proteins but inhibits the activity of the LHCII kinase, leading to dephosphorylation of LHCII proteins (Rintamäki et al., 2000; Hou et al., 2003).Thylakoid protein phosphorylation induces dynamic migrations of PSII-LHCII proteins along the thylakoid membrane (Bassi et al., 1988; Iwai et al., 2008) and modulation of thylakoid ultrastructure (Chuartzman et al., 2008). According to the traditional state transition theory, the phosphorylation of LHCII proteins decreases the antenna size of PSII and increases that of PSI, which is reflected as a quenched fluorescence emission from PSII. Alternatively, subsequent dephosphorylation of LHCII increases the antenna size of PSII and decreases that of PSI, which in turn is seen as increased PSII fluorescence (Bennett et al., 1980; Allen et al., 1981; Allen and Forsberg, 2001). This view was recently challenged based on studies with thylakoid membrane fractions, revealing that modulations in the relative distribution of excitation energy between PSII and PSI by LHCII phosphorylation specifically occur in the areas of grana margins, where both PSII and PSI function under the same antenna system, and the energy distribution between the photosystems is regulated via a more subtle mechanism than just the robust migration of phosphorylated LHCII (Tikkanen et al., 2008b). It has also been reported that most of the PSI reaction centers are located in the grana margins in a close vicinity to PSII-LHCII-rich grana thylakoids (Kaftan et al., 2002), providing a perfect framework for the regulation of excitation energy distribution from LHCII to both PSII and PSI.When considering the natural light conditions, the HL intensities are the only known light conditions that in higher plant chloroplasts specifically dephosphorylate only the LHCII proteins but not the PSII core proteins. However, such light conditions do not lead to enhanced function of PSII. Instead, the HL conditions strongly down-regulate the function of PSII via nonphotochemical quenching of excitation energy (NPQ) and PSII photoinhibition (for review, see Niyogi, 1999). On the other hand, after dark acclimation of leaves and relaxation of NPQ, PSII functions much more efficiently when plants/leaves are transferred to LL despite strong phosphorylation of LHCII, as compared with the low phosphorylation state of LHCII upon transfer to HL conditions.The delicate regulation of thylakoid protein phosphorylation in higher plant chloroplasts according to prevailing light intensity is difficult to integrate with the traditional theory of state transitions (i.e. the regulation of the absorption cross-section of PSII and PSI by reversible phosphorylation of LHCII). Moreover, besides LHCII proteins, reversible phosphorylation of the PSII core proteins may also play a role in dynamic light acclimation of plants. Recently, we demonstrated that the PSII core protein phosphorylation is a prerequisite for controlled turnover of the PSII reaction center protein D1 upon photodamage (Tikkanen et al., 2008a). This, however, does not exclude the possibility that the strict regulation of PSII core protein phosphorylation is also connected to the regulation of light harvesting and photosynthetic electron transfer. Moreover, the interactions between PSII and LHCII protein phosphorylation, nonphotochemical quenching, and cyclic electron flow around PSI in the regulation of photosynthetic electron transfer reactions remain poorly understood. To gain a deeper insight into such regulatory networks, we explored the effect of strongly fluctuating white light on chlorophyll (chl) fluorescence in Arabidopsis (Arabidopsis thaliana) mutants differentially deficient in PSII-LHCII protein phosphorylation and/or the regulatory systems of NPQ.     

EPR characterization of photosystem II from different domains of the thylakoid membrane

We report electron paramagnetic resonance (EPR) studies on photosystem II (PSII) from higher plants in five different domains of the thylakoid membrane prepared by sonication and two-phase partitioning. The domains studied were the grana core, the entire grana stack, the grana margins, the stroma lamellae and the purified stromal fraction, Y100. The electron transport properties of both donor and acceptor sides of PSII such as oxygen evolution, cofactors Y D, Q A, the CaMn 4-cluster, and Cytb 559 were investigated. The PSII content was estimated on the basis of oxidized Y D and Q A (-) Fe (2+) signal from the acceptor side vs Chl content (100% in the grana core fraction). It was found to be about 82% in the grana, 59% in the margins, 35% in the stroma and 15% in the Y100 fraction. The most active PSII centers were found in the granal fractions as was estimated from the rates of electron transfer and the S 2 state multiline EPR signal. In the margin and stroma fractions the multiline signal was smaller (40 and 33%, respectively). The S 2 state multiline could not be induced in the Y100 fraction. In addition, the oxidized LP Cytb 559 prevailed in the stromal fractions while the HP form dominated in the grana core. The margins and entire grana fractions have Cytb 559 in both potential forms. These data together with previous analyses indicate that the sequence of activation of the PSII properties can be represented as: PSII content > oxygen evolution > reduced Cytb 559 > dimerization of PSII centers in all fractions of the thylakoid membrane with the gradual increase from stromal fractions via margin to the grana core fraction. The results further support the existence of a PSII activity gradient which reflects lateral movement and photoactivation of PSII centers in the thylakoid membrane. The possible role of the PSII redox components in this process is discussed.     

Molecular remodeling of photosystem II during state transitions in Chlamydomonas reinhardtii

State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits.     

The structural and functional domains of plant thylakoid membranes

In plants, the stacking of part of the photosynthetic thylakoid membrane generates two main subcompartments: the stacked grana core and unstacked stroma lamellae. However, a third distinct domain, the grana margin, has been postulated but its structural and functional identity remains elusive. Here, an optimized thylakoid fragmentation procedure combined with detailed ultrastructural, biochemical, and functional analyses reveals the distinct composition of grana margins. It is enriched with lipids, cytochrome b₆f complex, and ATPase while depleted in photosystems and light‐harvesting complexes. A quantitative method is introduced that is based on Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE) and dot immunoblotting for quantifying various photosystem II (PSII) assembly forms in different thylakoid subcompartments. The results indicate that the grana margin functions as a degradation and disassembly zone for photodamaged PSII. In contrast, the stacked grana core region contains fully assembled and functional PSII holocomplexes. The stroma lamellae, finally, contain monomeric PSII as well as a significant fraction of dimeric holocomplexes that identify this membrane area as the PSII repair zone. This structural organization and the heterogeneous PSII distribution support the idea that the stacking of thylakoid membranes leads to a division of labor that establishes distinct membrane areas with specific functions.     

Immunocytochemical and Cytochemical Localization of Photosystems I and II

Cytochemical and immunocytochemical methods were used to localize photosystems I and II in barley (Hordeum vulgare L. cv Himalaya) chloroplasts. PSI activity, monitored by diaminobenzidine oxidation, was associated with the lumen side of the thylakoids of both grana and stroma lamellae. The P₇₀₀ chlorophyll a protein, the reaction center of PSI, was localized on thin sections of barley chloroplasts using monospecific antibodies to this protein and the peroxidase-antiperoxidase procedure. Results obtained by immunocytochemistry were similar to those of the diaminobenzidine oxidation: both grana and stroma lamellae contained immunocytochemically reactive material. Both the grana and stroma lamellae were also labeled when isolated thylakoids were reacted with the P₇₀₀ chlorophyll a protein antiserum and then processed by the peroxidase-antiperoxidase procedure. PSII activity was localized cytochemically by monitoring the photoreduction of thiocarbamyl nitroblue tetrazolium, a reaction sensitive to the PSII inhibitor, DCMU. PSII reactions occurred primarily on the grana lamellae, with weaker reactions on the stroma lamellae.     

Fragmentation and separation analysis of the photosynthetic membrane from spinach

Membrane vesicles, originating from grana, grana core (appressed grana regions), grana margins and stroma lamellae/end membranes, were analysed by counter current distribution (CCD) using aqueous dextran-polyethylene glycol two-phase systems. Each vesicle population gave rise to distinct peaks in the CCD diagram representing different vesicle subpopulations. The grana vesicles and grana core vesicles each separated into 3 different subpopulations having different chlorophyll a/b ratios and PSI/PSII ratios. Two of the grana core subpopulations had a chlorophyll a/b ratio of 2.0 and PSI/PSII ratio of 0.10 and are among the most PSII enriched thylakoid vesicle preparation obtained so far by a non detergent method. The margin vesicles separated into 3 different populations, with about the same chlorophyll a/b ratios, but different fluorescence emission spectra. The stroma lamellae/end membrane vesicles separated into 4 subpopulations. Plastoglobules, connected to membrane vesicles, were highly enriched in 2 of these subpopulations and it is proposed that these 2 subpopulations originate from stroma lamellae while the 2 others originate from end membranes. Fragmentation and separation analysis shows that the margins of grana constitute a distinct domain of the thylakoid and also allows the estimation of the chlorophyll antenna sizes of PSI and PSII in different thylakoid domains.