Heterogeneity and Photoinhibition of Photosystem II Studied with Thermoluminescence

Thermoluminescence (TL) signals were recorded from grana stacks, margins, and stroma lamellae from fractionated, dark-adapted thylakoid membranes of spinach (Spinacia oleracea L.) in the absence and in the presence of 2,6-dichlorphenylindophenol (DCMU). In the absence of DCMU, the TL signal from grana fractions consisted of a homogenous B-band, which originates from recombination of the semi-quinone Q_B⁻ with the S₂ state of the water-splitting complex and reflects active photosystem II (PSII). In the presence of DCMU, the B-band was replaced by the Q-band, which originates from an S₂Q_A⁻ recombination. Margin fractions mainly showed two TL-bands, the B- and C-bands, at approximately 50°C in the absence of DCMU, and Q- and C-bands in the presence of DCMU. The C-band is ascribed to a Tyr_D⁺-Q_A⁻ recombination. In the absence of DCMU, the fractions of stromal lamellae mainly gave rise to a TL emission at 42°C. The intensity of this band was independent of the number of excitation flashes and was shifted to higher temperatures (52°C) after the addition of DCMU. Based on these observations, this band was considered to be a C-band. After photoinhibitory light treatment of uncoupled thylakoid membranes, the TL intensities of the B- and Q-bands decreased, whereas the intensity at 45°C (C-band) slightly increased. It is proposed that the 42 to 52°C band that was observed in marginal and stromal lamellae and in photoinhibited thylakoid membranes reflects inactive PSII centers that are assumed to be equivalent to inactive PSII Q_B-nonreducing centers.     

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.     

Differences in the Interactions between the Subunits of Photosystem II Dependent on D1 Protein Variants in the Thermophilic Cyanobacterium Thermosynechococcus elongatus

The main cofactors involved in the oxygen evolution activity of Photosystem II (PSII) are located in two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is encoded by either the psbA₁ or the psbA₃ gene, the expression of which is dependent on environmental conditions. It has been shown that the energetic properties of the PsbA1-PSII and those of the PsbA3-PSII differ significantly (Sugiura, M., Kato, Y., Takahashi, R., Suzuki, H., Watanabe, T., Noguchi, T., Rappaport, F., and Boussac, A. (2010) Biochim. Biophys. Acta 1797, 1491–1499). In this work the structural stability of PSII upon a PsbA1/PsbA3 exchange was investigated. Two deletion mutants lacking another PSII subunit, PsbJ, were constructed in strains expressing either PsbA1 or PsbA3. The PsbJ subunit is a 4-kDa transmembrane polypeptide that is surrounded by D1 (i.e. PsbA1), PsbK, and cytochrome b₅₅₉ (Cyt b₅₅₉) in existing three-dimensional models. It is shown that the structural properties of the PsbA3/ΔPsbJ-PSII are not significantly affected. The polypeptide contents, the Cyt b₅₅₉ properties, and the proportion of PSII dimer were similar to those found for PsbA3-PSII. In contrast, in PsbA1/ΔPsbJ-PSII the stability of the dimer is greatly diminished, the EPR properties of the Cyt b₅₅₉ likely indicates a decrease in its redox potential, and many other PSII subunits are lacking. These results shows that the 21-amino acid substitutions between PsbA1 and PsbA3, which appear to be mainly conservative, must include side chains that are involved in a network of interactions between PsbA and the other PSII subunits.     

The lack of LHCII proteins modulates excitation energy partitioning and PSII charge recombination in Chlorina F2 mutant of barley

Analysis of the partitioning of absorbed light energy within PSII into fractions utilized by PSII photochemistry (Ø_PSII), thermally dissipated via ΔpH-and zeaxanthin-dependent energy quenching (Ø_NPQ) and constitutive non-photochemical energy losses (Ø_NO) was performed in wild type and F2 mutant of barley. The estimated energy partitioning of absorbed light to various pathways indicated that the fraction of Ø_PSII was slightly higher, while the proportion of thermally dissipated energy through Ø_NPQ was 38% lower in F2 mutant than in WT. In contrast, Ø_NO, i.e. the fraction of absorbed light energy dissipated by additional quenching mechanism(s) was 34% higher in F2 mutant. The increased proportion of Ø_NO correlated with narrowing the temperature gap (ΔT_M) between S_2/3Q_B− and S₂Q_A− charge recombinations in F2 mutant as revealed by thermoluminescence measurements. We suggest that this would result in increased probability for an alternative non-radiative P680+Q_A− radical pair recombination pathway for energy dissipation within the reaction centre of PSII (reaction center quenching) and that this additional quenching mechanism might play an important role in photoprotection when the capacity for the primary, zeaxanthin-dependent non-photochemical quenching (Ø_NPQ) and state transitions pathways are restricted in the absence of LHCII polypeptides in F2 mutant.Key words: Energy partitioning, Non-photochemical quenching, Hordeum vulgare L., PSII photochemistry, Q_A, Q_B, Thermoluminescence     

Photoinhibition and recovery in a herbicide-resistant mutant from<Emphasis Type="Italic"> Glycine max</Emphasis> (L.) Merr. cell cultures deficient in fatty acid unsaturation

Photoinhibition and recovery were studied in two photosynthetic cell suspensions from soybean (Glycine max L. Merr): the wild type (WT) and the herbicide-resistant D1 mutant STR7. This mutant also showed an increase in saturated fatty acids from thylakoid lipids. STR7 was more sensitive to photoinhibition under culture conditions. In vivo photoinhibition experiments in the presence of chloramphenicol, in vitro studies in isolated thylakoid membranes, and immunoblot analysis indicated that the process of light-induced degradation of the D1 protein was not involved in the response of STR7 to light. At growth temperature (24°C), the recovery rate of photoinhibited photosystem II (PSII) was slower in STR7 relative to WT. Photoinhibition and recovery were differentially affected by temperature in both cell lines. The rates of photoinhibition were faster in STR7 at any temperature below 27°C. The rates of PSII recovery from STR7 were more severely affected than those of WT at temperatures lower than 24°C. The photoinhibition and recovery rates of WT at 17°C mimicked those of STR7 at 24°C. In organelle translation studies indicated that synthesis and elongation of D1 were substantially similar in both cell lines. However, sucrose gradient fractionation of chloroplast membranes demonstrated that D1 and also other PSII proteins such as D2, OEE33, and LCHII had a reduced capability to incorporate into PSII to yield a mature assembled complex in STR7. This effect may become the rate-limiting step during the recovery of photoinhibited PSII and may explain the increased sensitivity to high light found in STR7. Our data may hint at a possible role of fatty acids from membrane lipids in the assembly and dynamics of PSII.Abbreviations DCBQ 2,6-Dichloro p-benzoquinone - DM Dodecyl--d-maltoside - DTT Dithiothreitol - HL High light - LHCII Light-harvesting complex II - LL Low light - OEE Oxygen-evolving extrinsic proteins - PAGE Polyacrylamide gel electrophoresis - PG Phosphatidylglycerol - PSII Photosystem II - Q_A and Q_B Secondary quinone electron acceptors from PSII - RC Reaction center - SDS Sodium dodecyl sulfate - WT Wild type     

A Determinant of Substrate Specificity Predicted from the Acyl-Acyl Carrier Protein Desaturase of Developing Cat''s Claw Seed

Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Δ9) plus cis-vaccenic acid (18:1Δ11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Δ9–18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Δ9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Δ9–18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Δ9–18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Δ9–18:0-ACP desaturase.     

Activation of a Reserve Pool of Photosystem II in Chlamydomonas reinhardtii Counteracts Photoinhibition

The effect of strong irradiance (2000 micromole photons per square meter per second) on PSII heterogeneity in intact cells of Chlamydomonas reinhardtii was investigated. Low light (LL, 15 micromole photons per square meter per second) grown C. reinhardtii are photoinhibited upon exposure to strong irradiance, and the loss of photosynthetic functioning is due to damage to PSII. Under physiological growth conditions, PSII is distributed into two pools. The large antenna size (PSII_α) centers account for about 70% of all PSII in the thylakoid membrane and are responsible for plastoquinone reduction (Q_b-reducing centers). The smaller antenna (PSII_β) account for the remainder of PSII and exist in a state not yet able to photoreduce plastoquinone (Q_b-nonreducing centers). The exposure of C. reinhardtii cells to 60 minutes of strong irradiance disabled about half of the primary charge separation between P680 and pheophytin. The PSII_β content remained the same or slightly increased during strong-irradiance treatment, whereas the photochemical activity of PSII_α decreased by 80%. Analysis of fluorescence induction transients displayed by intact cells indicated that strong irradiance led to a conversion of PSII_β from a Q_b-nonreducing to a Q_b-reducing state. Parallel measurements of the rate of oxygen evolution revealed that photosynthetic electron transport was maintained at high rates, despite the loss of activity by a majority of PSII_α. The results suggest that PSII_β in C. reinhardtii may serve as a reserve pool of PSII that augments photosynthetic electron-transport rates during exposure to strong irradiance and partially compensates for the adverse effect of photoinhibition on PSII_α.     

Studies on the Photoactivation of the Water-Oxidizing Enzyme: II. Characterization of Weak Light Photoinhibition of PSII and Its Light-Induced Recovery

Inactivation of the water splitting enzyme complex in leaves or isolated chloroplasts results in increased susceptibility of photosystem II (PSII) to damage by light. Photoinhibition under this condition occurs in very weak light. The site of damage is exclusive of the water splitting complex yet still on the oxidizing side of PSII, as the Q_B locus is unaffected while photoreduction of silicomolybdate is inhibited. The kinetics of loss in PSII activity are more complex than apparent first-order, and the quantum efficiency is low. We observe no evidence of deletion from thylakoid membranes of any PSII polypeptide as a consequence of photoinhibition, although recovery from the photoinhibition is dependent upon both light and 70S protein synthesis. Enhanced synthesis of two proteins occurs during recovery, only one of which (D2) appears to be causally related to the recovery. We present a model which describes the relationship of weak light photoinhibition and its recovery to photoactivation of the S-state water oxidizing complex.     

The Regulation of Photosynthetic Electron Transport during Nutrient Deprivation in Chlamydomonas reinhardtii

The light-saturated rate of photosynthetic O₂ evolution in Chlamydomonas reinhardtii declined by approximately 75% on a per-cell basis after 4 d of P starvation or 1 d of S starvation. Quantitation of the partial reactions of photosynthetic electron transport demonstrated that the light-saturated rate of photosystem (PS) I activity was unaffected by P or S limitation, whereas light-saturated PSII activity was reduced by more than 50%. This decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). In addition to a decline in the light-saturated rate of O₂ evolution, there was reduced efficiency of excitation energy transfer to the reaction centers of PSII (because of dissipation of absorbed light energy as heat and because of a transition to state 2). These findings establish a common suite of alterations in photosynthetic electron transport that results in decreased linear electron flow when C. reinhardtii is limited for either P or S. It was interesting that the decline in the maximum quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers were regulated specifically during S-limited growth by the SacI gene product, which was previously shown to be critical for the acclimation of C. reinhardtii to S limitation (J.P. Davies, F.H. Yildiz, and A.R. Grossman [1996] EMBO J 15: 2150–2159).     

Structural basis of cyanobacterial photosystem II Inhibition by the herbicide terbutryn

Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the Q_B site in the D1 subunit and thus block the electron transfer from Q_A to Q_B. Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the Q_B site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of Q_A, which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334–342). This discovery is discussed in the context of proton transfer to the lumen.     

Mutation of Residue Threonine-2 of the D2 Polypeptide and Its Effect on Photosystem II Function in Chlamydomonas reinhardtii

The D2 polypeptide of the photosystem II (PSII) complex in the green alga Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which residue Thr-2 has been replaced by either alanine or serine. Both mutants grew photoautotrophically at wild-type rates, and noninvasive biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The susceptibility of mutant PSII to photoinactivation as measured by the light-induced loss of PSII activity in whole cells in the presence of the protein-synthesis inhibitors chloramphenicol or lincomycin was similar to that of wild type. These results indicate that phosphorylation at Thr-2 is not required for PSII function or for protection from photoinactivation. In control experiments the phosphorylation of D2 in wild-type C. reinhardtii was examined by ³²P labeling in vivo and in vitro. No evidence for the phosphorylation of D2 in the wild type could be obtained. [¹⁴C]Acetate-labeling experiments in the presence of an inhibitor of cytoplasmic protein synthesis also failed to identify phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.     

Internal CO(2) Supply during Photosynthesis of Sun and Shade Grown CAM Plants in Relation to Photoinhibition

Leaves of Kalanchoë pinnata were exposed in the dark to air (allowing the fixation of CO₂ into malic acid) or 2% O₂, 0% CO₂ (preventing malic acid accumulation). They were then exposed to bright light in the presence or absence of external CO₂ and light dependent inhibition of photosynthetic properties assessed by changes in 77 K fluorescence from photosystem II (PSII), light response curves and quantum yields of O₂ exchange, rates of electron transport from H₂O through Q_B (secondary electron acceptor from the PSII reaction center) in isolated thylakoids, and numbers of functional PSII centers in intact leaf discs. Sun leaves of K. pinnata experienced greater photoinhibition when exposed to high light in the absence of CO₂ if malic acid accumulation had been prevented during the previous dark period. Shade leaves experienced a high degree of photoinhibition when exposed to high light regardless of whether malic acid had been allowed to accumulate in the previous dark period or not. Quantum yields were depressed to a greater degree than was 77 K fluorescence from PSII following photoinhibition.     

Pre-Diabetes Augments Neuropeptide Y1- and α1-Receptor Control of Basal Hindlimb Vascular Tone in Young ZDF Rats

Background

Peripheral vascular disease in pre-diabetes may involve altered sympathetically-mediated vascular control. Thus, we investigated if pre-diabetes modifies baseline sympathetic Y₁-receptor (Y₁R) and α₁-receptor (α₁R) control of hindlimb blood flow (Q_fem) and vascular conductance (VC).

Methods

Q_fem and VC were measured in pre-diabetic ZDF rats (PD) and lean controls (CTRL) under infusion of BIBP3226 (Y₁R antagonist), prazosin (α₁R antagonist) and BIBP3226+prazosin. Neuropeptide Y (NPY) concentration and Y₁R and α₁R expression were determined from hindlimb skeletal muscle samples.

Results

Baseline Q_fem and VC were similar between groups. Independent infusions of BIBP3226 and prazosin led to increases in Q_fem and VC in CTRL and PD, where responses were greater in PD (p<0.05). The percent change in VC following both drugs was also greater in PD compared to CTRL (p<0.05). As well, Q_fem and VC responses to combined blockade (BIBP3226+prazosin) were greater in PD compared to CTRL (p<0.05). Interestingly, an absence of synergistic effects was observed within groups, as the sum of the VC responses to independent drug infusions was similar to responses following combined blockade. Finally, white and red vastus skeletal muscle NPY concentration, Y₁R expression and α₁R expression were greater in PD compared to CTRL.

Conclusions

For the first time, we report heightened baseline Y₁R and α₁R sympathetic control of Q_fem and VC in pre-diabetic ZDF rats. In support, our data suggest that augmented sympathetic ligand and receptor expression in pre-diabetes may contribute to vascular dysregulation.     

Photoinhibition and light-dependent turnover of the D1 reaction-centre polypeptide of photosystem II are enhanced by mineral-stress conditions

The function of photosystem II (PSII) and the turnover of its D1 reaction-center protein were studied in spinach (Spinacia oleracea L.) plants set under mineral stress. The mineral deficiencies were induced either by supplying the plants with an acidic nutrient solution or by strongly reducing the supply of magnesium alone or together with sulfur. After exposure for 8–10 weeks to the different media, the plants were characterized by a loss of chlorophyll and an increase in starch content, indicating a disturbance in the allocation of assimilates. Depending on the severity of the mineral deficiencies the plants lost their ability to adapt even to moderate iradiances of 400 mol photons·m^–2·s^–1 and became photoinhibited, as indicated by the decrease in F_v/F_m (the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centers are closed). The loss of PSII function was induced by changes on the acceptor side of PSII. Fast fluorescence decay showed a loss of PSII centers with bound Q_B, the secondary quinone acceptor of PSII, and a fast reoxidation kinetic of q _a ^- , the primary quinone acceptor of PSII, in the photoinactivated plants. No appreciable change could be observed in the amount of PSII centers with unbound Q_B and in Q_B-nonreducing PSII centers. Immunological studies showed that the contents of the D1 and D2 proteins of the PSII reaction center and of the 33-kDa protein of the water-splitting complex were diminished in the photoinhibited plants, and the occurrance of a new polypetide of 14 kDa that reacted with an antibody against the C-termius of the D1 protein. As shown by pulse-labelling experiments with [¹⁴C]leucine both degradation and synthesis of the D1 protein were enhanced in the mineral-deficient plants when compared to non-deficient plants. A stimulation of D1-protein turnover was also observed in pH 3-grown plants, which were not inhibited at growth-light conditions. Obviously, stimulation of D1-protein turnover prevented photoinhibition in these plants. However, in the Mg- and Mg/S-deficient plants even a further stimulation of D1-protein turnover could not counteract the increased rate of photoinactivation.Abbreviations amp_(f,m,s) amplitude of the fast, (medium and slow) exponential component of fluorescence decay - F_m yield of maximum fluorescenc when all reaction centers are closed - F_o yield of intrinsic fluorescence at open PSII reaction centers in the dark - F_v yield of variable fluorescence, (difference between F_m and F_o) - LHC light-harvesting complex - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII Dedicated to Professor Dr. Dres. hc. Achim Trebst on the occasion of his 65th birthdayThis work was supported by grants from the BMFT and the Ministerium für Umwelt, Raumordnung and Landwirtschaft, Nordrhein-Westfalen. The authors thank H. Wietoska and M. Bronzel for skilful technical assistance.     

Effect of Cold Treatments on the Binding Stability of Photosystem II Extrinsic Proteins and an Associated Increase in Susceptibility to Photoinhibition

When pea plants (Pisum sativum L. cv Feltham First) are subjected to freezing conditions (−18°C) followed by a thaw to 18°C, there is a significant inhibition of water-splitting capacity judged by the rate of light-induced reduction of 2,6-dichlorophenol indophenol using isolated thylakoid membrane fragments enriched in photosystem II (PSII). The freeze-thaw-induced inhibition of water-splitting activity has been correlated with the loss of the 17- and 23-kilodalton extrinsic protein of PSII and with a weakening of the binding of the 33-kilodalton protein. There was no apparent loss of bound manganese. Addition of 10 millimolar CaCl₂, however, allowed a full recovery of the water-splitting activity of these modified PSII-enriched particles. The freeze-thaw-induced changes in the organization and functional capacity of PSII was found to increase its susceptibility to photoinhibition in agreement with the concepts presented in the accompanying paper, that oxidative damage can occur within the PSII reaction center as a consequence of extending the lifetime of P680⁺.     

Reversible Redox-Dependent Inactivation of Photosystem II as Photosynthesis Regulation in Chlorella

Inactivation of photosystem II (PSII) in the alga Chlorella pyrenoidosa Chick induced by photoinhibition (high light illumination at an intensity 10 times higher than photosynthesis-saturating light) or by incubation at a supraoptimum temperature (41°C) in darkness, resulted in a decrease in the relative yield of variable fluorescence due to a selective suppression of the slow phase of its rise. This indicates that low-activity PSII complexes, with a low efficiency of Q_A ^– formation are inactivated first. We suppose that the transition of normal PSII complexes to a low-activity state precedes the complete loss of their photochemical activity. The existence of some common stages of PSII inactivation, when induced by photoinhibition or incubation at supraoptimum temperature in darkness, is discussed. We suggest a scheme of the sequential stages in the regulation of photosynthetic light reactions involving a reversible redox-dependent PSII inactivation.     

Photosystem II function and herbicide binding sites during photoinhibition of spinach chloroplasts in-vivo and in-vitro

The time courses of some Photosystem II (PS II) parameters have been monitored during in-vivo and in-vitro photoinhibition of spinach chloroplasts, at room temperature and at 10 °C or 0 °C. Exposing leaf discs of low-light grown spinach at 25 °C to high light led to photoinhibition of chloroplasts in-vivo as manifested by a parallel decrease in the number of functional PS II centres, the variable chlorophyll fluorescence at 77K (F _v /F _m ), and the number of atrazine-binding sites. When the photoinhibitory treatment was given at 10 °C, the former two parameters declined in parallel but the loss of atrazine-binding sites occurred more slowly and to a lesser extent. During in-vitro photoinhibition of chloroplast thylakoids at 25 °C, the loss of functional PS II centres proceeded slightly more rapidly than the loss of atrazine-binding sites, and this difference in rate was further increased when the thylakoids were photoinhibited at 0 °C. During the recovery phase of leaf discs (up to 9 h) the increases in F _v /F _m preceded that of the number of functional PS II centres, while only a further decline in the number of atrazine-binding sites was observed. The recovery of variable chlorophyll fluorescence and the concentration of functional PS II centres occurred more rapidly at 25 °C than at 10 °C. These results suggest that the photoinhibition of PS II function is a relatively temperature-independent early photochemical event, whereas the changes in the concentration of herbicide-binding sites appear to be a more complex biochemical process which can occur with a delayed time course.Abbreviations BSA bovine serum albumin - Chl chlorophyll - D1 32kDa herbicide-binding polypeptide in photosystem II and product of the psbA gene - D2 34kDa polypeptide in photosystem II which is the product of the psbD gene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolin-dophenol - F ₀, F _v , F _m chlorophyll fluorescence with reaction centres open, variable and maximum fluorescence, respectively - LDS lithium dodecyl sulfate - MES 2-(N-morpholino) ethanesulfonic acid - PSII photosystem II - Q_A, Q_B first and second quinone-type PS II acceptor, respectively     

Slow degradation of the d1 protein is related to the susceptibility of low-light-grown pumpkin plants to photoinhibition

Photoinhibition of photosystem II (PSII) electron transport and subsequent degradation of the D1 protein were studied in pumpkin (Cucurbita pepo L.) leaves developed under high (1000 μmol m⁻² s⁻¹) and low (80 μmol m⁻² s⁻¹) photon flux densities. The low-light leaves were more susceptible to high light. This difference was greatly diminished when illumination was performed in the presence of chloramphenicol, indicating that a poor capacity to repair photodamaged PSII centers is decisive in the susceptibility of low-light leaves to photoinhibition. In fact, the first phases of the repair cycle, degradation and removal of photodamaged D1 protein from the reaction center complex, occurred slowly in low-light leaves, whereas in high-light leaves the degradation of the D1 protein more readily followed photoinhibition of PSII electron transport. A modified form of the D1 protein, with slightly slower electrophoretic mobility than the original D1, accumulated in the appressed thylakoid membranes of low-light leaves during illumination and was subsequently degraded only slowly.