Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

Polymorphism and Mendelian inheritance of photosystem II 23-kilodalton polypeptide

Soluble proteins from leaves of Nicotiana glauca Grah., N. langsdorffii Weinm., their reciprocal hybrids and amphiploid hybrid (N. glaucaxN. langsdorffii) were resolved by two-dimensional gel electrophoresis. Among a group of well-resolved polypeptides, in the isoelectric-point range of 5–5.5 and relative-molecular-mass (M_r) range of 18–23 kilodaltons (kDa), species-specific variation was observed. Polypeptides designated L and l are specific to N. langsdorffii, and G and g to N. glauca, while C is common to both species. Polypeptides L, G and C are localized in the chloroplasts and associated with thylakoid membranes. Polypeptide L is more acidic than polypeptide G, and both polypeptides have an M_r of 23 kDa. They were isolated from two-dimensional gels and their first 13 N-terminal amino-acid sequences were determined. These were found to be identical to the 13N-terminal amino acids of the photosystem II (PSII) 23-kDa polypeptide from spinach (T. Jansen et al. (1987) FEBS Lett. 216, 234–240) and, except for one change, to those from pea (R. Wales et al. (1989) Plant Molec. Biol., in press). Polypeptides G and L cross-react with antiserum against the PSII 23-kDa polypeptide from pea. Therefore, polypeptides G and L are extrinsic PSII 23-kDa polypeptides. They appear jointly and in equal amounts in the reciprocal hybrids. Since chloroplasts in Nicotiana are maternally inherited, these results demonstrate that polypeptides G and L are encoded by nuclear genes, are polymorphic variants of the PSII 23-kDa polypeptide, and are inherited in a Mendelian manner.Abbreviations kDa kilodalton - LS large subunit of Rubisco - M_r relative molecular mass - NEPHGE non-equilibrium pH gradient gel electrophoresis - PSII photosystem II - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SS small subunit of Rubisco     

Measurement of stoichiometry of photosystem II to photosystem I reaction centers

A wide range of values for the photosystem II to photosystem I stoichiometry have been reported. It is likely that some of this variation is due to measurement artifacts, which are discussed. Careful measurements of photosystem II reactions by absorption change at 325 nm, and flash yields of oxygen evolution, of protons from oxidation of water and of reduction of dichloroindophenol give equivalent results. Stoichiometries other than 1:1 are routinely found, and they vary with growth conditions as well as plant type. Two atrazine binding sites are found for every photosystem II reaction center that is active in oxygen evolution.     

Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to accumulate glycinebetaine was established. The wild type and transgenic plants were exposed to heat treatment (25–50°C) for 4 h in the dark and under growth light intensity (300 μmol m⁻² s⁻¹). The analyses of oxygen-evolving activity and chlorophyll fluorescence demonstrated that photosystem II (PSII) in transgenic plants showed higher thermotolerance than in wild type plants in particular when heat stress was performed in the light, suggesting that the accumulation of glycinebetaine leads to increased tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement on thermostability of the oxygen-evolving complex and the reaction center of PSII. The enhanced tolerance was caused by acceleration of the repair of PSII from heat-enhanced photoinhibition. Under heat stress, there was a significant accumulation of H₂O₂, O₂⁻ and catalytic Fe in wild type plants but this accumulation was much less in transgenic plants. Heat stress significantly decreased the activities of catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase in wild type plants whereas the activities of these enzymes either decreased much less or maintained or even increased in transgenic plants. In addition, heat stress increased the activity of superoxide dismutase in wild type plants but this increase was much greater in transgenic plants. Furthermore, transgenic plants also showed higher content of ascorbate and reduced glutathione than that of wild type plants under heat stress. The results suggest that the increased thermotolerance induced by accumulation of glycinebetaine in vivo was associated with the enhancement of the repair of PSII from heat-enhanced photo inhibition, which might be due to less accumulation of reactive oxygen species in transgenic plants.     

Photoactivation of the latent water-oxidizing complex in photosystem II membranes isolated from dark-grown spruce seedlings

Photosystem II membranes (D-PSII) were isolated from dark-grown spruce seedlings. All major PSII proteins except the 17- and 23-kDa extrinsic proteins were present in D-PSII. O₂ evolution and Mn content in D-PSII were negligible, while PSII-donor activity showed a value comparable to that of NH₂OH-treated PSII membranes (NH₂OH-L-PSII) from light-grown seedlings. Light incubation of D-PSII with 1 m M MnCl₂, 50 m M CaCl₂ and 100 μ M DCIP at pH 5.3 resulted in activation of the latent water-oxidizing complex. Accomplishment of photoactivation of PSII membranes from dark-grown spruce seedlings clearly indicates that only ligation of Mn²⁺ to the apo-water oxidizing complex is required for expression of O₂ evolution, and that protein synthesis is not involved in the photoactivation process. There was no essential difference between 'photoactivation' of naturally Mn-free PSII membranes and 'photoreactivation' of artificially Mn-depleted PSII membranes on kinetics, pH dependence, Mn²⁺-concentration dependence. However, kinetics and pH dependence of photoactivation were appreciably different in spruce PSII membranes and in PSII membranes of angiosperms such as wheat and spinach.     

Isolation and characterization of two middle repetitive DNA sequences of nuclear tobacco genome

Summary Two DNA sequences, R8.1 and R8.3, representing two distinct classes of tobacco genomic repeated DNA, were cloned and characterized by Southern blot analysis. Both R8.1 and R8.3 were found to be homologous to the Nicotiana tomentosiformis component of the allotetraploid Nicotiana tabacum genome, and each of them represents about 0.3% of nuclear DNA. The R8.1 and R8.3 differ in the mode of distribution in chromosomes, as revealed by in situ DNA/DNA hybridization.     

Low-temperature photochemistry in photosystem II from Thermosynechococcus elongatus induced by visible and near-infrared light

The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ., can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ., S1TyrZ. and S2TyrZ., respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD., PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ. interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 A units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.     

Polypeptides of photosystem II and their role in oxygen evolution

The linear, four-step oxidation of water to molecular oxygen by photosystem II requires cooperation between redox reactions driven by light and a set of redox reactions involving the S-states within the oxygen-evolving complex. The oxygenevolving complex is a highly ordered structure in which a number of polypeptides interact with one another to provide the appropriate environment for productive binding of cofactors such as manganese, chloride and calcium, as well as for productive electron transfer within the photoact. A number of recent advances in the knowledge of the polypeptide structure of photosystem II has revealed a correlation between primary photochemical events and a core complex of five hydrophobic polypeptides which provide binding sites for chlorophyll a, pheophytin a, the reaction center chlorophyll (P680), and its immediate donor, denoted Z. Although the core complex of photosystem II is photochemically active, it does not possess the capacity to evolve oxygen. A second set of polypeptides, which are water-soluble, have been discovered to be associated with photosystem II; these polypeptides are now proposed to be the structural elements of a special domain which promotes the activities of the loosely-bound cofactors (manganese, chloride, calcium) that participate in oxygen evolution activity. Two of these proteins (whose molecular weights are 23 and 17 kDa) can be released from photosystem II without concurrent loss of functional manganese; studies on these proteins and on the membranes from which they have been removed indicate that the 23 and 17 kDa species from part of the structure which promotes retention of chloride and calcium within the oxygen-evolving complex. A third water-soluble polypeptide of molecular weight 33 kDa is held to the photosystem II core complex by a series of forces which in some circumstances may include ligation to manganese. The 33 kDa protein has been studied in some detail and appears to promote the formation of the environment which is required for optimal participation by manganese in the oxygen evolving reaction. This minireview describes the polypeptides of photosystem II, places an emphasis on the current state of knowledge concerning these species, and discusses current areas of uncertainty concerning these important polypeptides.Abbreviations A 23187 ionophore that exchanges divalent cations with H⁺ - Chl chlorophyll - cyt cytochrome - DCPIP dichlorophenolindophenol - DPC diphenylcarbazide - EGTA ethyleneglycoltetraacetic acid - P680 the chlorophyll a reaction center of photosystem II - pheo pheophytin - PQ plastoquinone - PS photosystem - Q_A and Q_B primary and secondary plastoquinone electron acceptors of photosystem II - Sn (n=0, 1, 2, 3, 4) charge accumulating state of the oxygen evolving system - Signals II_vf, II_f and II_s epr-detectable free radicals associated with the oxidizing side of photosystem II - Z primary electron donor to the photosystem II reaction center The survey of literature for this review ended in September, 1984.     

The appearance of quenching centres in photosystem II

The variable fluorescence quenching found in the presence of DCMU with isolated chloroplasts which have been exposed previously to a prolonged low light intensity (Sinclair and Spence 1988), is accompanied by a loss of the sigmoidal appearance of the fluorescence induction transient. About 80% of the fluorescence decrease is due to the PS II units and 50% of the centres are inactivated by light exposure. Light incubation slows the PS II partial reaction while the PS I partial reaction is unaffected. We propose that in the light, normal PS II centres change into quenching centres which degrade excitation energy to thermal energy. This change can be reversed by 30 min of darkness. A higher flash intensity is needed to saturate the steady state O₂ flash yield from light-incubated chloroplasts indicating a light-induced decrease of the average photosynthetic unit size as would happen if PS II units were preferentially inactivated. These light-induced changes may relate to an adaptation in leaves to increasing light intensity.Abbreviations Chl Chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-Dichlorophenol-Indophenol - EDTA ethylaminediaminetetraacetic acid - F_v Level of variable fluorescence emission - F_o Initial level of fluorescence - Hepes buffer N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]     

Molecular analysis of the Chlamydomonas nuclear gene encoding PsbW and demonstration that PsbW is a subunit of photosystem II,but not photosystem I

PsbW is a nuclear-encoded protein located in the thylakoid membrane of the chloroplast. Studies in higher plants have provided substantial evidence that PsbW is a core component of photosystem II. However, recent data have been presented to suggest that PsbW is also a subunit of photosystem I. Such a sharing of subunits between the two photosystems would represent a novel phenomenon. To investigate this, we have cloned and characterized the psbW gene from the green alga Chlamydomonas reinhardtii. The gene is split by five introns and encodes a polypeptide of 115 residues comprising the 6.1 kDa mature PsbW protein preceded by a 59 amino acid bipartite transit sequence. Using antibodies raised to PsbW we have examined: (1) C. reinhardtii mutants lacking either photosystem and (2) purified photosystem preparations. We find that PsbW is a subunit of photosystem II, but not photosystem I.     

Multiple anion effects on photosystem II in chloroplast membranes

We investigated the activity of several anions at various sites on photosystem II, in particular those associated with the Cl^- effect (anion binding-site I) and the HCO₃ ^- effect (anion binding-site II). Chlorophyll a fluorescence changes were used to monitor partial photosystem II reactions either in the oxygen-evolving mechanism or involving endogenous quinone electron acceptors. We find that anions such as NO₃ ^-, HCO₃ ^-, HCO₂ ^-, F^-, NO₂ ^-, and acetate can, depending on conditions, bind to either anion binding-site I, anion binding-site II, or both sites simultaneously. The anions N₃ ^- and Au(CN)₂ ^- are exceptions. In their presence, oxygen-consumption reactions are enhanced. The results demonstrate that an exclusive site or mode of action of an anion on photosystem II cannot be determined by measuring the Hill reaction alone. Anion interactions with photosystem II are shown to be very complex and, therefore, caution is advisable in interpreting related experiments. Carbonic anhydrase associated with photosystem II was also investigated as a possible target for some anion effects. In Cl^--depleted thylakoids, NO₃ ^-, stimulated both electron transport and carbonic anhydrase activity at low concentrations, while higher concentrations inhibited both. However, carbonic anhydrase was more sensitive to inhibition by NO₃ ^- than was electron flow. Possible interpretations are discussed; the electron transport and carbonic anhydrase activity appear not to be functionally linked.Abbreviations ABSI Anion binding-site(s) I associated with the oxygen-evolving mechanism - ABSII Anion binding-site(s) II, which controls quinone-related reactions on the electron-acceptor side of photosystem II - OAc^- Acetate - Chl Chlorophyll - DCMU 3—(3,4-dichlorophenyl)-1,1-dimethyl urea - DCBQ 2,6-dichloro-p-benzoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - Mes 2-[N-morpholino]ethanesulphonic acid - Mops 3-[N-morpholino]propanesulphonic acid - Tes N-Tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid - Tricine N-Tris[hydroxymethyl]methylglycine     

Precursor of the nuclear-encoded extrinsic 30 kDa protein in photosystem II of Euglena gracilis Z is not a polyprotein

Polyprotein-type precursors have been reported for the nuclear-encoded proteins such as the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the apoproteins of light-harvesting chlorophyll-protein (LHC) in Euglena. We report here that the precursor of the extrinsic 30 kDa protein of photosystem II (PS II) encoded by nuclear DNA is not a polyprotein. The precursor was identified as a 45 kDa protein by immunoprecipitation of in vitro translation products of mRNA and by a pulse-chase experiment. It is probable that the structure of the precursor of the nuclear-encoded protein in Euglena chloroplast is closely related to the feature of assembly, as well as of transport, of the protein in chloroplast.     

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     

Cellular genetic study of a somatic instability in a tobacco mutant: in vitro isolation of valine-resistant spontaneous mutants

Summary A chlorophyll-deficient mutant line of tobacco (Nicotiana tabacum), named tl, displays spontaneously on leaves green, white, and twinned green/white somatic variations at high frequencies (10^–3 to 10^–2). The frequency of cell events leading to the somatic variations has been shown to be closely dependent on the stage of differentiation of cells during plant development. The activity of transposable elements is suspected in the tl genotype, and a study of its mutagenic ability was performed by selecting in vitro new mutant cellular types. The cellular marker chosen was the resistance to toxic doses of valine conferred by a permeation deficiency. A reproducible method allowing efficient selection of valine-resistant mutant clones from haploid tobacco mesophyll protoplast-derived cells was used. In 10 out of 12 experiments, the frequency of spontaneous valine-resistant clones obtained with the wild-type control was null for cell populations tested to the 10⁶. On the other hand, spontaneous valine-resistant clones were repeatedly isolated at variable and sometimes high frequencies (greater than 10^–3) from cell populations of the tl type. Valine resistance of plants regenerated from these clones was transmitted to the progeny as a single monogenic mutation. These results indicate an increased mutagenic ability of the tl genotype, as compared to the wild-type line.     

Knockdown of the PsbP protein does not prevent assembly of the dimeric PSII core complex but impairs accumulation of photosystem II supercomplexes in tobacco

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically found in land plants and green algae. Using PsbP-RNAi tobacco, we have investigated effects of PsbP knockdown on protein supercomplex organization within the thylakoid membranes and photosynthetic properties of PSII. In PsbP-RNAi leaves, PSII dimers binding the extrinsic PsbO protein could be formed, while the light-harvesting complex II (LHCII)-PSII supercomplexes were severely decreased. Furthermore, LHCII and major PSII subunits were significantly dephosphorylated. Electron microscopic analysis showed that thylakoid grana stacking in PsbP-RNAi chloroplast was largely disordered and appeared similar to the stromally-exposed or marginal regions of wild-type thylakoids. Knockdown of PsbP modified both the donor and acceptor sides of PSII; In addition to the lower water-splitting activity, the primary quinone Q_A in PSII was significantly reduced even when the photosystem I reaction center (P700) was noticeably oxidized, and thermoluminescence studies suggested the stabilization of the charged pair, S₂/Q_A⁻. These data indicate that assembly and/or maintenance of the functional MnCa cluster is perturbed in absence of PsbP, which impairs accumulation of final active forms of PSII supercomplexes.