Henrik Laasch

On the mechanism of photoinhibition in chloroplasts : relationship between changes in fluorescence and activity of photosystem II

Summary Spinach ( Spinacia oleracea L.) leaves were subjected to photoinhibitory treatments. Thylakoid membranes isolated from such leaves showed inhibition of the optimal quantum yield and of the capacity of photosystem (PS)II-dependent electron transport to the same extent. The decline in PS II capacity was linearly related to the decrease in the ratio of variable to maximum fluorescence (F v /F M ) of the leaves. Chlorophyll fluorescence properties (in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, DCMU) and PS II activity were studied in thylakoid membranes released from photoinhibited spinach chloroplasts (or mesophyll protoplasts of Valerianella locusta L.). PS II electron transport capacity was linearly related to F v /F M and to the DCMU binding capacity. The maximum complementary area above the fluorescence induction curve (A max ) declined with progressing photoinhibition to the same extent as F v . Area growth analysis indicated a predominant photoinhibition of PS IIα, but no transformation of a to β units. The results support the view that photoinhibition is based on transformation of active reaction centers to photochemically inactive fluorescence quenchers, which convert excitation energy to heat. Indications for increased thermal deactivation in the antenna system of PS II were not observed.

Non-photochemical quenching of chlorophyll a fluorescence in isolated chloroplasts under conditions of stressed photosynthesis

Non-photochemical quenching of chlorophyll a fluorescence after short-time light, heat and osmotic stress was investigated with intact chloroplasts from Spinacia oleracea L. The proportions of non-photochemical fluorescence quenching (qN) which are related (qE) and unrelated (qI) to the transthylakoid proton gradient (ΔpH) were determined. Light stress resulted in an increasing contribution of qIto total qN.The linear dependence of q. Eand ΔpH, as seen in controls, was maintained. The mechanisms underlying this type of quenching are obviously unaffected by photoin-hibition. In constrast, qEwas severely affected by heat and osmotic stress. In low light, the response of qEto changes in ΔpH was enhanced, whereas it was reduced in high light. The data are discussed with reference to the hypothesis that qEis related to thermal dissipation of excitation energy from photosystem II. It is shown that qEis not only controlled by ΔpH, but also by external factors.

Energy-Dependent Chlorophyll Fluorescence Quenching in Chloroplasts Correlated with Quantum Yield of Photosynthesis

Abstract Chlorophyll a fluorescence quenching was studied in intact, CO2 fixing chloroplasts isolated from spinach. Energy-dependent quenching (qᴇ), which is correlated with the light-induced pro ton gradient across the thylakoid membrane presumably reflects an increase in the rate-constant of thermal dissipation of excitation energy in the photosynthetic pigment system . The extent of qᴇ was found to be linearly related to the decrease of quantum yield of photosynthesis. We suggest that this relationship indicates a dynamic property of the membrane to adjust thermal dissipation of absorbed light energy to the energy requirement of photosynthesis.

Photosynthetic control, “energy-dependent” quenching of chlorophyll fluorescence and photophosphorylation under influence of tertiary amines

The effects of the tertiary amines tetracaine, brucine and dibucaine on photophosphorylation and control of photosynthetic electron transport in isolated chloroplasts of Spinacia oleracea were investigated. Tertiary amines inhibited photophosphorylation while the related electron transport decreased to the rates, observed under non-phosphorylating conditions. Light induced quenching of 9-aminoacridine fluorescence and uptake of 14C-labelled methylamine in the thylakoid lumen declined in parallel with photophosphorylation, indicating a decline of the transthylakoid proton gradient. In the presence of ionophoric uncouplers such as nigericin, no effect of tertiary amines on electron transport was seen in a range of concentration where photophosphorylation was inhibited. Under the influence of the tertiary amines tested, pH-dependent feed-back control of photosystem II, as indicated by energy-dependent quenching of chlorophyll fluorescence, was unaffected or even increased in a range of concentration where 9-aminoacridine fluorescence quenching and photophosphorylation were inhibited. The data are discussed with respect to a possible involvement of localized proton flow pathways in energy coupling and feed-back control of electron transport.

Differential sensitivity to dibucaine of photosynthetic control of electron transport and photophosphorylation in chloroplasts

Abstract The effect of dibucaine on the photosynthetic control of electron transport, as related to acidification of the thylakoid lumen, and photophosphorylation in isolated chloroplasts of Spinacia oleracea was studied and compared to that of a typical uncoupler (nigericin) and energy transfer inhibitor (3′- O -napthyl-ADP). In some respects, dibucaine resembled an uncoupler: it inhibited photophosphorylation and decreased quenching of 9-aminoacridine fluorescence as well as uptake of [ 14 C]methylamine and inhibited the intrathylakoid accumulation of H + , as measured by a glass electrode. The preactivated thylakoid ATPase was stimulated by dibucaine, although this stimulatory effect was to some extent superimposed by an inhibition of the enzyme. However, as opposed to what is expected for an uncoupler, dibucaine did not release the pH-dependent control of electron transport. In addition, pH-dependent high-energy quenching of chlorophyll fluorescence, normally inhibited by uncouplers, was not affected by dibucaine. It is concluded that dibucaine selectively reduces the driving force of photophosphorylation, while pH-dependent control of electron transport remains largely unaffected. The data are discussed regarding current models on coupling of photophosphorylation and on regulation of electron transport.

Photoinhibition of Photosynthesis Studies on Mechanisms of Damage and Protection in Chloroplasts

The energy of visible light, which is the driving force of photosynthesis, is also potentially damaging to the photosynthetic apparatus. The related phenomena have been termed photoinhibition of photosynthesis (for a review see ref. 1). By means of various protective mechanisms plants seem to be able to minimize light-induced damage. Under favorable environmental conditions little or no photoinhibition can be detected. However, if absorption of light and turnover of the absorbed light energy are highly unbalanced, damage may occur. Such situation may arise, e.g., when shade-adapted plants are exposed to full sunlight. Even a light flux as permanently received by the plant during growth can induce damage, when energy utilization in photosynthetic or photorespiratory carbon metabolism is severely restricted. This may be caused by various environmental stress factors (1). The protecting effect of carbon metabolism has been experimentally demonstrated for whole leaves (1) and for intact chloroplasts (2,3).

Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) 1. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P700 upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded Hinf I fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT → TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.

A new method for the determination of the transthylakoid pH gradient in isolated chloroplasts: the pH-dependent activity of violaxanthin de-epoxidase

Abstract The activity of the enzyme violaxanthin de-epoxidase, VDase, in osmotically shocked chloroplasts from Spinacia oleracea cv. Monatol was employed for the determination of the transthylakoid pH gradient, ΔpH. VDase activity was monitored as zeaxanthin formation by absorbance measurements at 505 and 560 nm. For the calibration of VDase activity, the cation permeability of the membranes was increased with gramicidin D and formation of zeaxanthin was started by a pH jump towards acidification. The magnitude of the ΔpH determined this way, was independent of the intrathylakoid volume. The considerable independence of VDase activity of the concentration of permeable cations indicated that VDase was an indicator of pH in the bulk aqueous phase of the thylakoid lumen. ATP formation, assayed simultaneously with VDase activity, was driven here by linear electron flow. The velocity of ATP formation, V p , was linearly related to ΔpH. When ΔpH was adjusted by light, its relationship to V p compared to that observed after a determination of ΔpH by 14C-methylamine uptake. When nigericin was employed for the adjustment of ΔpH, the relationships of ΔpH and V p were different, depending on the method of ΔpH determination applied. The advantages and limitations of the new method are discussed.

Detecting localized proton currents in photophosphorylation by procaine inhibition of the transthylakoid pH-gradient

The relationship between the transthylakoid pH-gradient, delta pH, and the velocity of photophosphorylation, Vp, in thylakoid membranes from spinach was investigated using the local anesthetic amine procaine as inhibitor of delta pH. When delta pH was driven by Photosystem (PS) II+I-dependent electron flow, passing through the cytochrome b6/f complex, inhibition by procaine was accompanied by an increase of ATP formation. It appeared that procaine allowed for values of Vp similar to those in controls (without procaine) at a significantly lower delta pH than in the controls. In contrast, when delta pH was driven by cyclic electron flow around PS I or by PS-II+I-dependent electron flow via a bypass around the cytochrome b6/f complex, or by PS II alone, procaine simultaneously caused an inhibition of delta pH and a decrease of ATP formation. Inhibition of delta pH by procaine did not induce an electrical membrane potential gradient that otherwise may have energetically compensated for the observed decline of delta pH. The electron flow capacity was unaffected by procaine. However, inhibition of delta pH did not significantly relax pH-dependent control of electron flux. Procaine accelerated ATP hydrolysis by pre-activated thylakoid ATPase to rates which were observed in the presence of uncouplers and had no direct effect on the activation state of the ATPase. The shift in the relationship between delta pH and Vp towards lower delta pH persisted in thermodynamic equilibrium between the phosphorylation potential and delta pH. The data indicated that the unconventional effect of procaine on photophosphorylation may be related to effects on proton translocation at the cytochrome b6/f complex and that a localized protonic coupling may occur between cytochrome b6/f and thylakoid-ATP-synthase complexes.

Inhibition of the transthylakoid gradient of electrochemical proton potential by the local anesthetic dibucaine

The effects of the local anesthetic dibucaine on coupling between electron transport and ATP synthesis-hydrolysis by the coupling-factor complex (CF0CF1 ATPase) were investigated in thylakoid membranes from Spinacia oleracea L. cv. Monatol. Evidence is presented that inhibition of ATP synthesis was produced by a specific uncoupling mechanism which was based on dibucaine-membrane surface interactions rather than on the interaction of dibucaine with the ATPase complex. Dibucaine reduced the osmotic space of thylakoid vesicles. At low pH of the medium it stimulated ATP hydrolysis beyond the rates obtained with optimum concentrations of ‘classical’ uncouplers. After addition of dibucaine, there was displacement of membrane-bound Mg2+ and strong thylakoid stacking in the presence of only low Mg2+ concentrations. Inhibition of ATP synthesis and transmembrane pH gradient increased with medium pH. Hydrolysis of ATP by isolated CF1 and the CF0CF1 complex was only slightly affected by dibucaine. The data are discussed assuming the involvement of localized proton channels on the membrane surface in protonic coupling of electron transport and ATP synthesis. A hypothesis for the mechanisms of action of local anesthetics at the thylakoid membrane is presented.