A chlorophyll-protein complex lacking in photosystem I mutants of Chlamydomonas reinhardtii

Sodium dodecyl sulfate gel electrophoresis of unheated, detergent-solubilized thylakoid membranes of Chlamydomonas reinhardtii gives two chlorophyll-protein complexes. Chlorophyll-protein complex I (CP I) is the blue-green in color and can be dissociated by heat into "free" chlorophyll and a constituent polypeptide (polypeptide 2; mol wt 66,000). Similar experiments with spinach and Chinese cabbage show that the higher plant CP I contains an equivalent polypeptide but of slightly lower molecular weight (64,000). Both polypeptide 2 and its counterpart in spinach are soluble in a 2:1 (vol/vol) mixture of chloroform-methanol. Chemical analysis reveals that C. reinhardtii CP I has a chlorophyll a to b weight ratio of about 5 and that it contains approximately 5% of the total chlorophyll and 8-9% of the total protein of the thylakoid membranes. Thus, it can be calculated that each constituent polypeptide chain is associated with eight to nine chlorophyll molecules. Attempts to measure the molecular weight of CP I by calibrated SDS gels were unsuccessul since the complex migrates anomalously in such gels. Two Mendelian mutants of C. reinhardtii, F1 and F14, which lack P700 but have normal photosystem I activity, do not contain CP I or the 66,000-dalton polypeptide in their thylakoid membranes. Our results suggest that CP I is essential for photosystem I reaction center activity and that P700 may be associated with the 66,000-dalton polypeptide.     

Effect of dichromate on photosystem II activity in xanthophyll-deficient mutants of Chlamydomonas reinhardtii

The photosystem II activity and energy dissipation was investigated when algal Chlamydomonas reinhardtii genotypes were exposed to dichromate toxicity effect. The exposure during 24 h to dichromate effect of two C. reinhardtii mutants having non-functional xanthophylls cycle, as npq1 zeaxanthin deficient and npq2 zeaxanthin accumulating, induced inhibition of PSII electron transport. After dichromate-induced toxicity, PSII functions of C. reinhardtii mutants were investigated under different light intensities. To determine dichromate toxicity and light intensity effect on PSII functional properties we investigated the change of energy dissipation via PSII electron transport, non-photochemical regulated and non-regulated energy dissipation according to Kramer et al. (Photosynth Res 79:209–218, 2004). We showed the dependency between dichromate toxicity and light-induced photoinhibition in algae deficient in xanthophyll cycle. When algal mutants missing xanthophylls cycle were exposed to dichromate toxicity and to high light intensity energy dissipation via non-regulated mechanism takes the most important pathway reaching the value of 80%. Therefore, the mutants npq1 and npq2 having non-functional xanthophylls cycle were more sensitive to dichromate toxic effects.     

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

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

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis     

The carboxy-terminal extension of the D1-precursor protein is dispensable for a functional photosystem II complex in Chlamydomonas reinhardtii

The D1-precursor protein of the photosystem II reaction centre contains a carboxy-terminal extension whose proteolytic removal is necessary for oxygen-evolving activity. To address the question of the role of the carboxy-terminal extension in the green alga Chlamydomonas reinhardtii, we truncated D1 by converting codon Ser345 of the psbA gene into a stop codon. Particle gun transformation of an in vitro modified psbA gene fragment also carrying mutations conferring herbicide resistance yielded a homoplasmic transformant containing the stop codon. Since oxygen evolution capacity is not affected in this mutant as compared with herbicide-resistant control cells, the carboxy-terminal extension is dispensable for a functional photosystem II complex under normal growth conditions.     

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.     

Antenna structure and excitation dynamics in photosystem I. II. Studies with mutants of Chlamydomonas reinhardtii lacking photosystem II.

Using time-resolved single photon counting, fluorescence decay in photosystem I (PS I) was analyzed in mutant strains of Chlamydomonas reinhardtii that lack photosystem II. Two strains are compared: one with a wild-type PS I core antenna (120 chlorophyll a/P700) and a second showing an apparent reduction in core antenna size (60 chlorophyll a/P700). These data were calculated from the lifetimes of core antenna excited states (75 and 45 ps, respectively) and from pigment stoichiometries. Fluorescence decay in wild type PS I is composed of two components: a fast 75-ps decay that represents the photochemically limited lifetime of excited states in the core antenna, and a minor (less than 10%) 300-800 ps component that has spectral characteristics of both peripheral and core antenna pigments. Temporal and spectral properties of the fast PS I decay indicate that (a) excitations are nearly equilibrated among the range of spectral forms present in the PS I core antenna, (b) an average excitation visits a representative distribution of core antenna spectral forms on all pigment-binding subunits regardless of the origin of the excitation, (c) reduction in core antenna size does not alter the range of antenna spectral forms present, and (d) transfer from peripheral antennae to the PS I core complex is rapid (less than 5 ps).     

Further identification of the exoplasmic face particles on the freeze-fractured thylakoid membranes: a study using double and triple mutants from Chlamydomonas reinhardtii lacking various photosystem II subunits and the cytochrome b6/f complex.

About 20% of the exoplasmic face (EF) particles present in the freeze-fractured thylakoid membranes of the wild type strain of Chlamydomonas reinhardtii remain in mutants lacking photosystem II (PSII) because of the absence of either one of the two PSII subcomplexes CP43 or D1/D2/CP47. We show that about half of these residual EF particles can be accounted for by PSII subcomplexes still present in such mutants, and by cytochrome (cyt) b6/f complexes. Analysis of double mutants lacking both types of protein complexes points to an association of cyt b6/f complexes with PSII subcomplexes in some of these EF particles and to a requirement in cyt b6/f complexes for the translocation of each of the two PSII subcomplexes (the CP43 subunit and the D1/D2/CP47 subcomplex) from the unstacked to the stacked regions of the thylakoid membranes.     

Analysis of the genes of the OEE1 and OEE3 proteins of the photosystem II complex from Chlamydomonas reinhardtii

The sequences of the nuclear genes of the 33 kDa (OEE1) and the 16 kDa (OEE3) polypeptides of the oxygen evolving complex of Chlamydomonas reinhardtii have been established. Comparison between the OEE1 protein sequences of C. reinhardtii and higher plants and cyanobacteria reveals 67 and 47% homology. In contrast, C. reinhardtii and higher plants have only 28% overall homology for OEE3 which is mostly limited to the central portion of the protein. The transit peptides of the C. reinhardtii proteins consist of 52 (OEE1) and, most likely, 51 (OEE1) amino acids. They have a basic amino terminal region and, at least in the case of OEE1, a hydrophobic segment at their carboxy terminal end typical of thylakoid lumen proteins. Comparison of the genomic and cDNA clones indicates that the OEE1 and OEE3 genes contain five and four introns, respectively, some of which are located within the coding sequences of the transit peptides.     

Changes in the carotenoid composition of chloroplast membranes from Chlamydomonas reinhardtii double mutants with alterations of various sites in photosystem II

The variable fluorescence and polypeptide and carotenoid compositions of the chlorophyll b-deficient mutant C-48 of the unicellular green alga Chlamydomonas reinhardtii and its double mutants without chlorophyll b and with inactive photosystem II were compared with those of the wild-type algal cells. Studying variable fluorescence demonstrated the alterations at the donor side (AC-121), the acceptor side (AC-234) or immediately in the photosystem II reaction centre (AC-184, AC-864). Gel electrophoresis showed that the absence of chlorophyll b in all mutants was due to the lack of 26, 28 and 31 kDa polypeptides in the light-harvesting chlorophyll a/b-protein complex II (LHC II). As a result of the second mutation, the chlorophyll a-protein complex of photosystem II did not form in chloroplast membranes. The disassembly of this complex in the mutants AC-121, AC-234 and AC-864 was related to the deficiency of both polypeptides of the reaction centre (30 and 32 kDa) and polypeptides of the water-oxidizing system (18, 23 and 34 kDa). Besides the loss of these polypeptides, the contents of polypeptides with molecular masses of 47 and 51 kDa decreased in the double mutant AC-184. Substantial changes were revealed in the carotenoid composition of the double mutants. We observed the considerable accumulation of carotenes that accompanied alterations in the donor (mutant AC-121) or acceptor (mutant AC-234) sides of PS II. In the first case, beta-carotene predominantly accumulated (87%); in the second case, it was alpha-carotene (52%). Alterations in the PS II reaction centre (mutants AC-184, AC-864) caused accumulation of xanthophylls, mainly lutein (38-41%). We suppose that alterations in different parts of the PS II chloroplast membrane lead to substantial changes in the carotenoid composition.     

Efficient assembly of photosystem II in Chlamydomonas reinhardtii requires Alb3.1p, a homolog of Arabidopsis ALBINO3

Alb3 homologs Oxa1 and YidC have been shown to be required for the integration of newly synthesized proteins into membranes. Here, we show that although Alb3.1p is not required for integration of the plastid-encoded photosystem II core subunit D1 into the thylakoid membrane of Chlamydomonas reinhardtii, the insertion of D1 into functional photosystem II complexes is retarded in the Alb3.1 deletion mutant ac29. Alb3.1p is associated with D1 upon its insertion into the membrane, indicating that Alb3.1p is essential for the efficient assembly of photosystem II. Furthermore, levels of nucleus-encoded light-harvesting proteins are vastly reduced in ac29; however, the remaining antenna systems are still connected to photosystem II reaction centers. Thus, Alb3.1p has a dual function and is required for the accumulation of both nucleus- and plastid-encoded protein subunits in photosynthetic complexes of C. reinhardtii.     

Molecular and biophysical analysis of herbicide-resistant mutants of Chlamydomonas reinhardtii: structure-function relationship of the photosystem II D1 polypeptide.

Plants and green algae can develop resistance to herbicides that block photosynthesis by competing with quinones in binding to the chloroplast photosystem II (PSII) D1 polypeptide. Because numerous herbicide-resistant mutants of Chlamydomonas reinhardtii with different patterns of resistance to such herbicides are readily isolated, this system provides a powerful tool for examining the interactions of herbicides and endogenous quinones with the photosynthetic membrane, and for studying the structure-function relationship of the D1 protein with respect to PSII electron transfer. Here we report the results of DNA sequence analysis of the D1 gene from four mutants not previously characterized at the molecular level, the correlation of changes in specific amino acid residues of the D1 protein with levels of resistance to the herbicides atrizine, diuron, and bromacil, and the kinetics of fluorescence decay for each mutant, which show that changes at two different amino acid residues dramatically slow PSII electron transfer. Our analyses, which identify a region of 57 amino acids of the D1 polypeptide involved in herbicide binding and which define a D1 binding niche for the second quinone acceptor, QB of PSII, provide a strong basis of support for structural and functional models of the PSII reaction center.     

Excitation pressure regulates the activation energy for recombination events in the photosystem II reaction centres of Chlamydomonas reinhardtii.

Using in vivo thermoluminescence, we examined the effects of growth irradiance and growth temperature on charge recombination events in photosystem II reaction centres of the model green alga Chlamydomonas reinhardtii. We report that growth at increasing irradiance at either 29 or 15 degrees C resulted in comparable downward shifts in the temperature peak maxima (T(M)) for S2QB- charge pair recombination events, with minimal changes in S2QA- recombination events. This indicates that such growth conditions decrease the activation energy required for S2QB- charge pair recombination events with no concomitant change in the activation energy for S2QA- recombination events. This resulted in a decrease in the DeltaT(M) between S2QA- and S2QB- recombination events, which was reversible when shifting cells from low to high irradiance and back to low irradiance at 29 degrees C. We interpret these results to indicate that the redox potential of QB was modulated independently of QA, which consequently narrowed the redox potential gap between QA and QB in photosystem II reaction centres. Since a decrease in the DeltaT(M) between S2QA- and S2QB- recombination events correlated with growth at increasing excitation pressure, we conclude that acclimation to growth under high excitation pressure narrows the redox potential gap between QA and QB in photosystem II reaction centres, enhancing the probability for reaction center quenching in C. reinhardtii. We discuss the molecular basis for the modulation of the redox state of QB, and suggest that the potential for reaction center quenching complements antenna quenching via the xanthophyll cycle in the photoprotection of C. reinhardtii from excess light.     

The light-harvesting complex of photosystem I in Chlamydomonas reinhardtii: protein composition, gene structures and phylogenic implications

Light-harvesting chlorophyll a/b-binding proteins (LHCI) associated with photosystem I (PSI) and the genes encoding these proteins have been characterized in the unicellular green alga Chlamydomonas reinhardtii, extending previous studies of the PSII-LHCII [Teramoto et al. (2001) Plant Cell Physiol. 42: 849]. In order to assign LHCI proteins in the thylakoid membranes, the PSI-LHCI supercomplex that retains all of the major LHCI proteins was purified. Seven distinct LHCI proteins were resolved from the purified supercomplex by a high-resolution SDS polyacrylamide gel electrophoresis, and their N-terminal amino acid sequences were determined. One LHCI protein (band e) was newly found, although the other six LHCI proteins corresponded to those previously reported. Genomic clones encoding these seven LHCI proteins were newly isolated and the nucleotide sequences were determined. A comprehensive characterization of all members of Lhc gene family in this alga revealed that LHCI proteins are more highly diverged than LHCII, suggesting functional differentiation of the protein components in LHCI. Neighbor joining trees were constructed for LHC proteins from C. reinhardtii and those of Arabidopsis thaliana or Galdieria sulphuraria to assess evolutionary relationships. Phylogenetic analysis revealed that (1). green algal LHCI and LHCII proteins are more closely related to one another than to LHCI proteins in red algae, (2). green algae and higher plants possess seven common lineages of LHC proteins, and (3). Type I and III LHCI proteins are conserved between green algae and higher plants, while Type II and IV are not. These findings are discussed in the context of evolution of multiple diverse antenna complexes.     

High field EPR study of the pheophytin anion radical in wild type and D1-E130 mutants of photosystem II in Chlamydomonas reinhardtii

The intermediate electron acceptor in photosystem II is a pheophytin molecule. The radical anion of this molecule was studied using high field electron paramagnetic resonance in a series of Chlamydomonas reinhardtii mutants. Glutamic acid 130 of the D1 polypeptide is thought to hydrogen bond the ring V carbonyl group of this radical. Mutations at this site, designed to weaken or remove this hydrogen bond, strongly affected the g tensor of the radical. The upward shift of the g(x) component followed the decreasing hydrogen bonding capacity of the amino acid introduced. This behavior is similar to that of tyrosyl and semiquinone radicals. It is also consistent with the optical spectra of the pheophytin in similar mutants. Density functional calculations were used to calculate the g tensors and rationalize the observed trend in the variation of the g(x) value for pheophytin and bacteriopheophytin radical. The theoretical results support the experimental observations and demonstrate the sensitivity of g values to the electrostatic protein environment for these types of radicals.     

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.     

Functional genomics of plant photosynthesis in the fast lane using Chlamydomonas reinhardtii

Oxygenic photosynthesis by algae and plants supports much of life on Earth. Several model organisms are used to study this vital process, but the unicellular green alga Chlamydomonas reinhardtii offers significant advantages for the genetic dissection of photosynthesis. Recent experiments with Chlamydomonas have substantially advanced our understanding of several aspects of photosynthesis, including chloroplast biogenesis, structure-function relationships in photosynthetic complexes, and environmental regulation. Chlamydomonas is therefore the organism of choice for elucidating detailed functions of the hundreds of genes involved in plant photosynthesis.     

Characterization of photosystem II mutants of Chlamydomonas reinhardii lacking the psbA gene

Summary We have examined 78 chloroplast mutants of Chlamydomonas reinhardii lacking photosystem II activity. Most of them are unable to synthesize the 32 Kdalton protein. Analysis of 22 of these mutants reveals that they have deleted both copies of the psbA gene (which codes for the 32 Kdalton protein) in their chloroplast genome. Although these mutants are able to synthesize and to integrate the other photosystem II polypeptides in the thylakoid membranes, they are unable to assemble a stable functional photosystem II complex. The 32 Kprotein appears therefore to play an important role not only in photosystem II function, but also in stabilizing this complex.     

Role of sulfoquinovosyl diacylglycerol for the maintenance of photosystem II in Chlamydomonas reinhardtii.

The physiological role of sulfoquinovosyl diacylglycerol (SQDG) in photosynthesis was investigated with a SQDG defective mutant (hf-2) of Chlamydomonas reinhardtii that did not have any detectable amount of SQDG. The mutant showed a lower rate of photosystem II (PSII) activity by approximately 40% and also a lower growth rate than those of the wild-type. Results of genetical analysis of hf-2 strongly suggest that the SQDG defect and the lowered PSII activity are due to a single gene mutation. The supplementation of SQDG to hf-2 cells restored the lowered PSII activity to the same level as that of wild-type cells, and also enabled the mutant to grow even in the presence of 135 nm 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Moreover, the incubation of isolated thylakoid membranes of hf-2 with SQDG raised the lowered PSII activity. Chemical modifications of SQDG impaired the recovery of PSII activity. The results suggest that SQDG is indispensable for PSII activity in Chlamydomonas by maintaining PSII complexes in their proper state.