Calmodulin antagonists inhibit electron transport in photosystem II of spinach chloroplasts

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca²⁺ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca²⁺-specific ionophore, Ca²⁺ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.     

EGTA, a calcium chelator, inhibits electron transport in photosystem II of spinach chloroplasts at two different sites

A fifteen minute incubation of spinach chloroplasts with the divalent Ca²⁺ chelator, EGTA, in concentrations 50–250 μM, inhibits electron transport through both photosystems. All photosystem II partial reactions, including indophenol, ferricyanide and the DCMU-insensitive silicomolybdate reduction are inhibited from 70–100%. The photosystem II donor reaction, diphenyl carbazide → indophenol, is also inhibited, indicating that the inhibition site comes after the Mn²⁺ site, and that the first Ca²⁺ effect noted (site II) is not on the water oxidation enzyme, as is commonly assumed, but between the Mn²⁺ site and plastoquinone A pool. The other photosystem II effect of EGTA (Ca²⁺ site I), occurs in the region between plastoquinone A and P700 in the electron transport chain of chloroplasts. About 50% inhibition of the reaction ascorbate + TMPD → methyl viologen is given by incubation with 200 μM EGTA for 15 min. Ca²⁺ site II activity can be restored with 20 mM CaCl₂. Ca²⁺ site I responds to Ca²⁺ and plastocyanin added jointly. More than 90% activity in the ascorbate + TMPD → methylviologen reaction can be restored. Various ways in which Ca²⁺ ions could affect chloroplast structure and function are discussed. Since EGTA is more likely to penetrate chloroplast membranes than EDTA, which is known to remove CF₁, the coupling factor, from chloroplast membranes, and since Mg²⁺ ions are ineffective in restoring activity, it is concluded that Ca²⁺ may function in the electron transport chain of chloroplasts in a hitherto unsuspected manner.     

Site-specific inhibition of photophosphorylation in isolated spinach chloroplasts by HgCl2. II. Evidence for three sites of energy conservation associated with non-cyclic electron transport

Energy transfer inhibition by HgCl₂ has been demonstrated to be selective for certain System I partial reactions. On the basis of different HgCl₂ effects on the System I reactions, reduced 2,6-dichlorophenolindophenol → methylviologen, diaminodurene → methylviologen and N-phenazine methosulfate cyclic, two sites of energy conservation associated with System I are proposed. Furthermore, these sites are in parallel with each other, in series with the site closely associated with Photosystem II and are shared between non-cyclic and cyclic electron transport.     

Cation-induced,inhibitor-resistant photosystem II reactions in cyanobacterial membranes

Ferricyanide-supported oxygen evolution in sonic vesicles from the cyanobacterium $\text{Spirulina}\text{platensis}$ is only partially sensitive to inhibition by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and addition of cations to inhibited membranes stimulates the rate of oxygen evolution. The order of cation effectiveness (M³⁺ > M²⁺ > M⁺) suggests that this stimulation is due at least in part to surface charge screening effects which permit freer access of anionic ferricyanide to the vesicle membrane surface; La³⁺, Ca²⁺, and K⁺ are most effective in this regard. Ferricyanide photoreduction is completely sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and neither mono- nor divalent cations affect this inhibition. Addition of La³⁺, on the other hand, causes a nearly complete restoration of ferricyanide-supported oxygen evolution. We conclude that the membrane surfaces of these vesicles are uniquely different from those o higher plants; sites of ferricyanide reduction associated with the interphotosystem chain are surface localized, and the primary acceptor region of photosystem II is susceptible to a trivalent cation-specific reaction in which ferricyanide may directly oxidize the primary acceptor.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl₂, an inhibitor of cytochrome b₆f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b₆f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

A study on the lateral distribution of the plastoquinone pool with respect to Photosystem II in stacked and unstacked spinach chloroplasts

The quenching of Photosystem II (PS II) chlorophyll fluorescence by oxidised plastoquinone has been used in an attempt to determine their relative distribution in the partition zone and stroma-exposed thylakoid membranes. Thus, the PS II-plastoquinone interaction was determined in stacked (2.5 mM MgCl₂) and largely unstacked (0.25 mM MgCl₂) membranes. A method to correct for spillover or other quenching changes at the different MgCl₂ concentrations, which would compete with the plastoquinone-induced quenching, was devised utilising the quinone dibromothymoquinone. This compound is demonstrated to behave as an ideal (theoretically) PS II quencher at both high and low MgCl₂ concentrations, which indicates that it distributes itself homogeneously between partition zone and stroma-exposed membrane regions. In passing from the stacked to the unstacked configuration, the PS II-plastoquinone interaction decreases less than the PS II-dibromothymoquinone interaction. This is interpreted to mean that plastoquinone is present in both the partition zone and stroma-exposed membranes, with somewhat higher concentrations in the stroma-exposed membranes. Thus, plastoquinone is well placed to transport reducing equivalents from the partition zones to the stroma-exposed membranes.     

An HPLC-based method of estimation of the total redox state of plastoquinone in chloroplasts, the size of the photochemically active plastoquinone-pool and its redox state in thylakoids of Arabidopsis

We have described a direct, high-performance liquid chromatography-based method of estimation of the total level of plastoquinone (PQ) in leaves, the redox state of total (photoactive and non-photoactive) PQ, as well as the redox state of the PQ-pool that is applicable to any illumination conditions. This method was applied to Arabidopsis thaliana leaves but it can be applied to any other plant species. The obtained results show that the level of total PQ was 25 ± 3 molecules/1000 chlorophyll (Chl) molecules in relation to foliar total Chl content. The level of the photoactive PQ, i.e., the PQ-pool, was about 31% of the total PQ present in Arabidopsis leaves that corresponds to about 8 PQ molecules/1000 Chl molecules. The reduction level of the non-photoactive PQ fraction, present outside thylakoids in chloroplasts, was estimated to account for about 49%. The measurements of the redox state of the PQ-pool showed that the pool was reduced during the dark period in about 24%, and during the light period (150 μmol/m²·s) the reduction of the PQ-pool increased to nearly 100%. The obtained results were discussed in terms of the activity of chlororespiration pathways in Arabidopsis and the regulatory role of the redox state of PQ-pool in various physiological and molecular processes in plants.     

The size of the population of weakly coupled chlorophyll pigments involved in thylakoid photoinhibition determined by steady-state fluorescence spectroscopy

On the basis of experiments with singlet quenchers and in agreement with previous data, it is suggested that a population of energetically weakly coupled chlorophylls may play a central role in photoinhibition in vivo and in vitro. In the present study, we have used steady state fluorescence techniques to gain direct evidence for these uncoupled chlorophylls. Due to the presence of their emission maxima, near 650 nm and more prominently in the 670-675 nm interval both chlorophylls b and a seem to be involved. A straightforward mathematical model is developed to describe the data which allows us to conclude that the uncoupled/weakly coupled population size is in the range of 1-3 molecules per photosystem.     

Kinetic study of the plastoquinone pool availability correlated with H2O2 release in seawater and antioxidant responses in the red alga Kappaphycus alvarezii exposed to single or combined high light, chilling and chemical stresses

Under biotic/abiotic stresses, the red alga Kappaphycus alvarezii reportedly releases massive amounts of H₂O₂ into the surrounding seawater. As an essential redox signal, the role of chloroplast-originated H₂O₂ in the orchestration of overall antioxidant responses in algal species has thus been questioned. This work purported to study the kinetic decay profiles of the redox-sensitive plastoquinone pool correlated to H₂O₂ release in seawater, parameters of oxidative lesions and antioxidant enzyme activities in the red alga Kappaphycus alvarezii under the single or combined effects of high light, low temperature, and sub-lethal doses of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which are inhibitors of the thylakoid electron transport system. Within 24 h, high light and chilling stresses distinctly affected the availability of the PQ pool for photosynthesis, following Gaussian and exponential kinetic profiles, respectively, whereas combined stimuli were mostly reflected in exponential decays. No significant correlation was found in a comparison of the PQ pool levels after 24 h with either catalase (CAT) or ascorbate peroxidase (APX) activities, although the H₂O₂ concentration in seawater (R = 0.673), total superoxide dismutase activity (R = 0.689), and particularly indexes of protein (R = 0.869) and lipid oxidation (R = 0.864), were moderately correlated. These data suggest that the release of H₂O₂ from plastids into seawater possibly impaired efficient and immediate responses of pivotal H₂O₂-scavenging activities of CAT and APX in the red alga K. alvarezii, culminating in short-term exacerbated levels of protein and lipid oxidation. These facts provided a molecular basis for the recognized limited resistance of the red alga K. alvarezii under unfavorable conditions, especially under chilling stress.