Reto J. Strasser

Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes

The usefulness of a new voltage-sensitive fluorescent dye, the membrane permeant negatively charged oxonol dye diBA-C4-(3)-, was evaluated by measuring the membrane potentials of BICR/M1R-k and L cells with glass microelectrodes and simultaneously recording the fluorescence of the stained cells. The membrane potential of BICR/M1R-k cells was varied between -25 mV and -90 mV by changing the bicarbonate concentration in the medium or by voltage clamping. To avoid any interference by the inserted electrodes with the fluorescence measurement of the cytoplasm, the cells were fused by polyethyleneglycol to form giant cells (homokaryons). These homokaryons also allowed penetration by two glass microelectrodes without causing a serious leakage of the plasma membrane. The slow responding dye diBA-C4-(3)- had a fluorescence response of about 1% per mV. Mathematical analysis of the fluorescence changes after voltage clamping revealed a first-order reaction with a rate constant between 0.1 min-1 and 0.8 min-1, depending on the cell size which was determined by the number of nuclei per homokaryon. A model for the mechanism of the fluorescence changes is proposed.

Violaxanthin de-epoxidase in etiolated leaves.

In etiolated leaves the occurrence of the enzymatic violaxanthin de-epoxidation to zeaxanthin is shown. The carotenoid transformation is provoked by the infiltration of whole leaves with ascorbate at pH 5 and is susceptible to DTT. Identification of the de-epoxidase activity is achieved by in vivo spectroscopy and pigment analysis (TLC).

Energy Pipeline Model of the Photosynthetic Apparatus

The purpose of this paper is to present an overall view of how energy distribution in a photosynthetic apparatus can be measured, calculated and visualized by means of the energy pipeline model. Energy distribution is a term for all phenomena which are correlated to the energetic constellation within the photosynthetic apparatus between the absorption events of photons and the first redox reactions of the electron transport chain. The complex scope of energy distribution correlates fluxes and forces, to pool sizes of pigments as well as to distances between pigments. The fluxes are 1) light absorption fluxes of PSI and PSII indicated as and J1 and J2 energy fluxes within the photosynthetic unit: Excitation fluxes or excitation rates of the pigment pool i are indicated as E. and de-excitation fluxes from one location i to another location j are indicated as Eij The forces which drive the energy fluxes are embodied in the excited pigment which is defined as the exciton density of the pigment i and indicated as Pi*. The energy fluxes are proportional to the exciton density. The de-excitation rate constant kij is the proportionality factor. Eij = Pi*. kij* All equations on energy distribution according to the energy flux theory in biomembranes can be applied to every photosynthetic model. The problem arises when one attempts to correlate experimental signals to the equation of the model. The models symbolize photosynthetic units.

Cerulenin-Induced Modifications in the Fatty Acid Composition Affect Excitation Energy Transfer in Thylakoids of Petunia hybrida Leaves

Cerulenin-induced modifications in the fatty acid composition have been used to investigate the influence of acyl lipids on excitation energy distribution in thylakoid membranes of Petunia hybrida by means of 77 K fluorescence spectroscopy. Although cerulenin has no effect on relative contents of chlorophyll and acyl lipids, changes in the fatty acid composition of all thylakoid acyl lipids have been observed. The main cerulenin effect seems to be an increase in linoleic acid at the expense of saturated and monounsaturated C16- and C18-fatty acids resulting most likely in an increase in acyl lipid species containing both linoleic and linolenic acid. Low temperature (77 K ) fluorescence kinetics reveal a remarkable decrease in the ratio of the variable divided by the maximal fluorescence of photosystem II (F2(v)/F2(M)), taken as indicator for cerulenin-induced changes in this photosystem. Calculations of the excitation energy distribution terms based on a grouped bipartite model of photosynthesis suggest that a decrease in this ratio is caused by changes in energy transfer probabilities responsible for both, photochemical trapping of photosystem II and energetic cooperativity (grouping) between different photosystem II-light harvesting complex-units. Moreover, changes in the conformation responsible for spillover energy transfer are most likely to occur. Correlations between cerulenin-induced modifications of fatty acid composition and energy distribution support the assumption that excitation energy transfer depends on the structural state of the lipid matrix.

The Dynamics of the Photoreduction of Protochlorophyll(Ide) into Chlorophyll(Ide)

The active protochlorophyllide-protein-complex is converted in the light to chlorophyllide. This reaction has been analyzed by several authors. A serie of publications by Sironval, Brouers, Kuiper (1968–1980) report data which support a model for the protochlorophyllide photoreduction. The model proposes: 1) Protochlorophyllide P1 is reduced in the light to chlorophyllide P2. The electron donor is not limiting. 2) The excited protochlorophyllide transfers a part of its excitation energy to chlorophyllide.

State-1 state-2 transition influenced by herbicides which modify fatty acid composition in leaves

Interactions of herbicides with photosynthetic membranes are still not solved in many respects. Three different modes of action have been reported for pyridazinone herbicides: inhibition of (1) photosynthetic electron transport (2) carotenoid biosynthesis and (3) fatty acid desaturation in the galactolipid fraction of chloroplasts (ST. JOHN, HILTON 1976). The pyridazinone BASF 13–338 (=SAN 9785) used in our investigations has no effect on pigmentsynthesis and photosynthetic activity but affects fatty acid desaturation in leaves (TREBST, HARTH 1974). Cerulenin an antibiotic from the fungus Cephalosporium caerulens inhibits fatty acid synthesis generally. Both herbicides act indirectly on photosynthesis because they alter the mobility of photosynthetic units in the membrane.

Thermodynamically Forced State Changes in Chloroplasts

Every living system is thermodynamically open and it can be considered as an energy flux converter. A photon flux keeps a molecular flux in action. The photon input flux is given by the total light absorption flux J = Io-I1 and the photon output flux is given by the fluorescence emission and heat dissipation. The effectively available energy flux J is converted by the system and integrated into the biochemical products which are synthesized from the substrates. The total conformation of the system is symbolized by the system constant K. K is an assembly of all de-excitation and biochemical rate constants. The maximal power which a biochemical system can perform is therefore limited by its conformation K. The effective performance of the system at any given time is the observed biological activity which depends on the absorbed energy flux J, the conformation and the established relative steady state level of the energy conversion flux through the system. We define the relative established steady state level A as the fraction of the actual performance at any given time and the maximal possible performance of the system. For any time the biological behaviour B represents the unused activity-capacity where (1-A) = B. The biological system is therefore always defined by the Trilogy JKB as shown in Fig. 1. Whenever the system changes its conformational term K we define this as a state change. Every change in energy input changes J which changes B and which in turn changes the biological activity v without necessarily changing K. In such a situation all energy fluxes and biological activities will change but this situation would not be called a state change because an unchanged conformation K is at work.

Structure-Function-Relationship in Thylakoids Influenced by the Pyridazinone BAS 13—338 (SAN 9785)

The pyridazinone BAS 13-338 (SAN 9785|) inhibits the desaturation sequence leading to polyunsaturated fatty acids, mainly of glycolipids. Parallel to the inhibition of fatty acid desaturation in the presence of the pyridazinone, changes in energy-distribution parameters have been observed. These data indicate that the amount of polyunsaturated fatty acids in glycolipids is strongly correlated with excitation, trapping, grouping and dissipation, but not with spillover. Functional changes in energy distribution induced by BAS 13-338 are interpreted as a consequence of structural changes in the lipid matrix, which may imply a structure-function relationship between pigment protein complexes and the surrounding lipid environment in thylakoids.

Does a Correlation between the Xanthophyll Cycle and State 1 -State 2 Changes Exist?

The xanthophyll or violaxanthin cycle is widely distributed in higher plants and some algae groups. It consists of the enzymatic de-epoxidation of violaxanthin (V) via antheraxanthin to zeaxanthin (Z) , and the reverse reaction mediated by an epoxi-dase. The de-epoxidase which is thought to be located at the inner surface of the thylakoid membrane needs a low pH and redox equivalents for its activity (1). Under saturating light conditions the V→Z conversion requires about 10 min. in isolated chloroplasts the same conversion occurs in the dark at pH 5 in the presence of ascorbate.

Altered lipid matrix changes energy distribution in thylakoids

Photosynthetic membranes differ from other cellular membranes in that they have a thylakoid specific lipid composition and a high degree of polyunsaturated fatty acids. The typical acyl lipid complement of thylakoids seems to have two functions: (i) organization of the membrane matrix in which the pigment protein complexes are embedded; (ii) indirect structure-function relationships between lipid matrix and pigment protein complexes which influence energy distribution behaviour. The role of high amounts of polyunsaturated fatty acids and indirect structure-function relationships can be studied by inhibiting fatty acid desaturation sequence with pyridazinone herbicides. Depending on the pyridazinone-type used, electron transport, carotenoid synthesis or fatty acid desaturation may be influenced. The pyridazinone 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)pyridazinone (BASF 13-338=SAN 9785), a weak PS II electron transport (1) and potent state change inhibitor (2) has little effect on the organization of pigment forms. However, it inhibits different desaturation steps in the synthesis of polyunsaturated fatty acids leading to an increase of appressed/nonappressed regions of thylakoids (3).