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.     

Disturbed excitation energy transfer in Arabidopsis thaliana mutants lacking minor antenna complexes of photosystem II

Minor light-harvesting complexes (Lhcs) CP24, CP26 and CP29 occupy a position in photosystem II (PSII) of plants between the major light-harvesting complexes LHCII and the PSII core subunits. Lack of minor Lhcs in vivo causes impairment of PSII organization, and negatively affects electron transport rates and photoprotection capacity. Here we used picosecond-fluorescence spectroscopy to study excitation-energy transfer (EET) in thylakoid membranes isolated from Arabidopsis thaliana wild-type plants and knockout lines depleted of either two (koCP26/24 and koCP29/24) or all minor Lhcs (NoM). In the absence of all minor Lhcs, the functional connection of LHCII to the PSII cores appears to be seriously impaired whereas the “disconnected” LHCII is substantially quenched. For both double knock-out mutants, excitation trapping in PSII is faster than in NoM thylakoids but slower than in WT thylakoids. In NoM thylakoids, the loss of all minor Lhcs is accompanied by an over-accumulation of LHCII, suggesting a compensating response to the reduced trapping efficiency in limiting light, which leads to a photosynthetic phenotype resembling that of low-light-acclimated plants. Finally, fluorescence kinetics and biochemical results show that the missing minor complexes are not replaced by other Lhcs, implying that they are unique among the antenna subunits and crucial for the functioning and macro-organization of PSII.     

Structural organization of photosynthetic apparatus in agranal chloroplasts of maize

We investigated the organization of photosystem II (PSII) in agranal bundle sheath thylakoids from a C(4) plant maize. Using blue native/SDS-PAGE and single particle analysis, we show for the first time that PSII in the bundle sheath (BS) chloroplasts exists in a dimeric form and forms light-harvesting complex II (LHCII).PSII supercomplexes. We also demonstrate that a similar set of photosynthetic membrane complexes exists in mesophyll and agranal BS chloroplasts, including intact LHCI.PSI supercomplexes, PSI monomers, PSII core dimers, PSII monomers devoid of CP43, LHCII trimers, LHCII monomers, ATP synthase, and cytochrome b(6)f complex. Fluorescence functional measurements clearly indicate that BS chloroplasts contain PSII complexes that are capable of performing charge separation and are efficiently sensitized by the associated LHCII. We identified a fraction of LHCII present within BS thylakoids that is weakly energetically coupled to the PSII reaction center; however, the majority of BS LHCII is shown to be tightly connected to PSII. Overall, we demonstrate that organization of the photosynthetic apparatus in BS agranal chloroplasts of a model C(4) plant is clearly distinct from that of the stroma lamellae of the C(3) plants. In particular, supramolecular organization of the dimeric LHCII.PSII in the BS thylakoids strongly suggests that PSII in the BS agranal membranes may donate electrons to PSI. We propose that the residual PSII activity may supply electrons to poise cyclic electron flow around PSI and prevent PSI overoxidation, which is essential for the CO(2) fixation in BS cells, and hence, may optimize ATP production within this compartment.     

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.     

Demonstration of Two Sites of Inhibition of Electron Transport by Protein Phosphorylation in Wheat Thylakoids

The effect of protein phosphorylation on electron transportactivities of thylakoids isolated from wheat leaves was investigated.Protein phosphorylation resulted in a reduction in the apparentquantum yield of whole chain and photosystem II (PSII) electrontransport but had no effect on photosystem I (PSI) activity.The affinity of the D1 reaction centre polypeptide of PSII tobind atrazine was diminished upon phosphorylation, however,this did not reduce the light-saturated rate of PSII electrontransport. Phosphorylation also produced an inhibition of thelight-saturated rate of electron transport from water or durohydroquinoneto methyl viologen with no similar effect being observed onthe light-saturated rate of either PSII or PSI alone. This suggeststhat phosphorylation produces an inhibition of electron transportat a site, possibly the cytochrome b₆/f complex, between PSIIand PSI. This inhibition of whole-chain electron transport wasalso observed for thylakoids isolated from leaves grown underintermittent light which were deficient in polypeptides belongingto the light-harvesting chlorophyll-protein complex associatedwith photosystem II (LHCII). Consequently, this phenomenon isnot associated with phosphorylation of LCHII polypeptides. Apossible role for cytochrome b₆/f complexes in the phosphorylation-inducedinhibition of whole chain electron transport is discussed. Key words: Electron transport, light harvesting, photosystem 2, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

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.     

Impact of PsbTc on forward and back electron flow, assembly, and phosphorylation patterns of photosystem II in tobacco

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn₄Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in ΔpsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electron-accepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in ΔpsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in ΔpsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.     

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.     

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.     

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.     

Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane

Conversion of solar energy into chemical energy in plant chloroplasts concomitantly modifies the thylakoid architecture and hierarchical interactions between pigment–protein complexes. Here, the thylakoids were isolated from light‐acclimated Arabidopsis leaves and investigated with respect to the composition of the thylakoid protein complexes and their association into higher molecular mass complexes, the largest one comprising both photosystems (PSII and PSI) and light‐harvesting chlorophyll a/b‐binding complexes (LHCII). Because the majority of plant light‐harvesting capacity is accommodated in LHCII complexes, their structural interaction with photosystem core complexes is extremely important for efficient light harvesting. Specific differences in the strength of LHCII binding to PSII core complexes and the formation of PSII supercomplexes are well characterized. Yet, the role of loosely bound L‐LHCII that disconnects to a large extent during the isolation of thylakoid protein complexes remains elusive. Because L‐LHCII apparently has a flexible role in light harvesting and energy dissipation, depending on environmental conditions, its close interaction with photosystems is a prerequisite for successful light harvesting in vivo. Here, to reveal the labile and fragile light‐dependent protein interactions in the thylakoid network, isolated membranes were subjected to sequential solubilization using detergents with differential solubilization capacity and applying strict quality control. Optimized 3D‐lpBN‐lpBN‐sodium dodecyl sulfate–polyacrylamide gel electrophoresis system demonstrated that PSII–LHCII supercomplexes, together with PSI complexes, hierarchically form larger megacomplexes via interactions with L‐LHCII trimers. The polypeptide composition of LHCII trimers and the phosphorylation of Lhcb1 and Lhcb2 were examined to determine the light‐dependent supramolecular organization of the photosystems into megacomplexes.     

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.     

Phosphorylation-dephosphorylation of light-harvesting complex II as a response to variation in irradiance is thiol sensitive and thylakoid sufficient: modulation of the sensitivity of the phenomenon by a peripheral component

Downregulation of phosphorylation of chlorophyll a/b-binding proteins (LHCII) of the photosystem II at high irradiance could only be demonstrated with leaf discs but not in isolated thylakoids. The present view suggests this phenomenon to be regulated by stromal thioredoxin. Here, we show that high-light inactivation of LHCII phosphorylation can be reproduced in isolated thylakoids and have explained the apparent absence of inactivation in vitro to be due to the derepressed activity of a peripheral kinase. We investigated this phenomenon with Arachis hypogea thylakoids prepared with (Th:A) or without (Th:B) tricine, where tricine is known for removing peripheral proteins from thylakoids. While LHCII remained phosphorylated at high irradiance in Th:B, the response of Th:A mimicked Arachis leaflets where LHCII was transiently phosphorylated with irradiance. LHCII phosphorylation in Th:A was sensitive to thiol reducing conditions, but in Th:B, the phenomenon became insensitive to thiol reduction following illumination. Washing Th:B with tricine made them resemble Th:A, and conversely, Th:A reconstituted with the Tricine extract resembled Th:B with respect to both irradiance response and thiol sensitivity. In vitro phosphorylation reactions indicated a thiol insensitive kinase activity to be present in the Tricine extract that was capable of phosphorylating histone H1 as well as purified LHCII. This peripherally associated kinase activity explained the sustenance of LHCII phosphorylation as well as its thiol insensitivity at high irradiance in Th:B thylakoids. Contrary to the current view, our results clearly show that irradiance dependent phosphorylation and dephosphorylation of LHCII is a thylakoid sufficient phenomenon, although it remained open to regulation by thiol redox state modulation.     

Reversible changes in structure and function of photosynthetic apparatus of pea (Pisum sativum) leaves under drought stress

The effects of drought on photosynthesis have been extensively studied, whereas those on thylakoid organization are limited. We observed a significant decline in gas exchange parameters of pea (Pisum sativum) leaves under progressive drought stress. Chl a fluorescence kinetics revealed the reduction of photochemical efficiency of photosystem (PS)II and PSI. The non-photochemical quenching (NPQ) and the levels of PSII subunit PSBS increased. Furthermore, the light-harvesting complexes (LHCs) and some of the PSI and PSII core proteins were disassembled in drought conditions, whereas these complexes were reassociated during recovery. By contrast, the abundance of supercomplexes of PSII-LHCII and PSII dimer were reduced, whereas LHCII monomers increased following the change in the macro-organization of thylakoids. The stacks of thylakoids were loosely arranged in drought-affected plants, which could be attributed to changes in the supercomplexes of thylakoids. Severe drought stress caused a reduction of both LHCI and LHCII and a few reaction center proteins of PSI and PSII, indicating significant disorganization of the photosynthetic machinery. After 7 days of rewatering, plants recovered well, with restored chloroplast thylakoid structure and photosynthetic efficiency. The correlation of structural changes with leaf reactive oxygen species levels indicated that these changes were associated with the production of reactive oxygen species.     

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.     

Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii

Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of the green alga Chlamydomonas reinhardtii revealed that the major environmentally dependent changes in phosphorylation are clustered at the interface between the photosystem II (PSII) core and its light-harvesting antennae (LHCII). The photosynthetic membranes that were isolated form the algal cells exposed to four distinct environmental conditions affecting photosynthesis: (i) dark aerobic, corresponding to photosynthetic State 1; (ii) dark under nitrogen atmosphere, corresponding to photosynthetic State 2; (iii) moderate light; and (iv) high light. The surface-exposed phosphorylated peptides were cleaved from the membrane by trypsin, methyl-esterified, enriched by immobilized metal affinity chromatography, and sequenced by nanospray-quadrupole time-of-flight mass spectrometry. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii. Amino-terminal acetylation of seven proteins was concomitantly determined. Sequenced amino termini of six mature LHCII proteins differed from the predicted ones. The State 1-to-State 2 transition induced phosphorylation of the PSII core components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10. Exposure of the algal cells to either moderate or high light caused additional phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to specific hyperphosphorylation of CP29 at seven distinct residues, phosphorylation of another minor LHCII constituent, CP26, at a single threonine, and double phosphorylation of additional subunits of a major LHCII complex including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Environmentally induced protein phosphorylation at the interface of PSII core and the associated antenna proteins, particularly multiple differential phosphorylations of CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions and for LHCII uncoupling from PSII under high light stress to allow thermal energy dissipation.     

Proteolytic activity against the light-harvesting complex and the D1/D2 core proteins of Photosystem II in close association to the light-harvesting complex II trimer

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 degrees C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing "activity gels". Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being "self-digested", also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 degrees C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg(2+), and inhibited by Zn(2+), Cd(2+), EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing "activity gels" or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.     

Multiple sites of retardation of electron transfer in Photosystem II after hydrolysis of phosphatidylglycerol

Phosphatidylglycerol (PG), containing the unique fatty acid Δ3, trans-16:1-hexadecenoic acid, is a minor but ubiquitous lipid component of thylakoid membranes of chloroplasts and cyanobacteria. We investigated its role in electron transfers and structural organization of Photosystem II (PSII) by treating Arabidopsis thaliana thylakoids with phospholipase A₂ to decrease the PG content. Phospholipase A₂ treatment of thylakoids (a) inhibited electron transfer from the primary quinone acceptor Q_A to the secondary quinone acceptor Q_B, (b) retarded electron transfer from the manganese cluster to the redox-active tyrosine Z, (c) decreased the extent of flash-induced oxidation of tyrosine Z and dark-stable tyrosine D in parallel, and (d) inhibited PSII reaction centres such that electron flow to silicomolybdate in continuous light was inhibited. In addition, phospholipase A₂ treatment of thylakoids caused the partial dissociation of (a) PSII supercomplexes into PSII dimers that do not have the complete light-harvesting complex of PSII (LHCII); (b) PSII dimers into monomers; and (c) trimers of LHCII into monomers. Thus, removal of PG by phospholipase A₂ brings about profound structural changes in PSII, leading to inhibition/retardation of electron transfer on the donor side, in the reaction centre, and on the acceptor side. Our results broaden the simple view of the predominant effect being on the Q_B-binding site.     

Decreased Photosystem II Core Phosphorylation in a Yellow-Green Mutant of Wheat Showing Monophasic Fluorescence Induction Curve

In the present work we study the regulation of the distribution of the phosphorylated photosystem II (PSII) core populations present in grana regions of the thylakoids from several plant species. The heterogeneous nature of PSII core phosphorylation has previously been reported (M.T. Giardi, F. Rigoni, R. Barbato [1992] Plant Physiol 100: 1948-1954; M.T. Giardi [1993] Planta 190: 107-113). The pattern of four phosphorylated PSII core populations in the grana regions appears to be ubiquitous in higher plants. In the dark, at least two phosphorylated PSII core populations are always detected. A mutant of wheat (Triticum durum) that shows monophasic room-temperature photoreduction of the primary quinone electron acceptor of PSII as measured by chlorophyll fluorescence increase in the presence and absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and by fluorescence upon flash illumination in intact leaves also lacks the usual distribution of phosphorylated PSII core populations. In this mutant, the whole PSII core population pattern is changed, probably due to altered threonine kinase activity, which leads to the absence of light-induced phosphorylation of CP43 and D2 proteins. The results, correlated to previous experiments in vivo, support the idea that the functional heterogeneity observed by fluorescence is correlated in part to the PSII protein phosphorylation in the grana.     

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-1

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EF_s particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O₂ evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes.