A klororespiráció terminális oxidázának keresése ciano-bakteriális modellrendszerben

A projekt keretében kíséreletet tettünk a klororespiráció terminális oxidázának azonosítására és a légzési kölcsönhatás fiziológiai jelentőségének tisztázására; fotoszintetikus működést befolyásoló stresszhatásokat tanulmányoztunk. A klororespiráció jelenlétére zöldalgákban a termolumineszcencia ún. afterglow (AG TL) jele további megerősítést adott. Elsőként figyeltünk meg AG-TL-t cianobakteriális sejtekben, jelezve a légzési kölcsönhatás fontosságát. A fotoszintézissel versengő terminális oxidáz azonosítása nem sikerült, ez más laborokban sem vezetett eredményre. Ennek oka nem ismert. A stresszhatások területén: (i) PAC-spektroszkópia segítségével azonosítottuk a Cd minden valószínűség szerint elsődleges hatóhelyét ? kimutattuk a szénsav-anhidráz enzim érintettségét; (ii) gránumos tilakoidokban három egymástól jól elkülöníthető termo-optikai szerkezetváltozást azonosítottunk; (iii) a magas hőmérsékleti stressz vizsgálatára bevezettük a hő-pulzus módszerét, amely jelentősen megkönnyítette a primér hőstressz-hatás azonosítását és a helyreállás folyamatának követését; (iv) halofil cianobaktériumon tanulmányoztuk a magas só és magas pH együttes környezeti stresszhatás kivédésének mechanizmusát: következtetni tudtunk egy Na+ pumpa működésére és vázoltuk a PSII károsodásának menetét; (v) a membrán fizikai állapotának változásait P-NMR vizsgálatokkal végeztük, és megállapítottuk, hogy a nem-lamelláris fázis fontos szerepet játszik egyes fotoszinetikus funkciókban. | In the frame of the project we attempted to identify the terminal oxidase of chlororespiration and studied the physiological significance of the interaction between photosynthesis and respiration; and studied various stress-effects on photosynthesis. Operation of chlororespiration in a green alga was confirmed with the so-called afterglow thermoluminescence (AG TL) signal. We were the first who observe the AG TL in a cyanobacterium, indicating the importance of the interaction between photosynthesis and respiration. Efforts to identify the terminal oxidase competing with photosynthesis failed. This enigmatic question awaits further studies. On the field of stress-effects: (i) we identified the most likely primary target of Cd, by using PAC-spectroscopy we showed the involvement of carbonic anhydrase; (ii) in granal thylakoids we separated three well discernible thermo-optically induced structural changes; (iii) to study high temperature stress in higher plants, we introduced a heat-pulse method, which made easier to determine the primary stress effects and their recovery; (iv) combined stress effects, high salt and high pH, were studied in Spirulina: it was concluded that a sodium pump maintained the pH homeostasis, in high salt stress conditions PSII was damaged already at low light; (v) P-NMR was used to monitor the physical status of the membranes, leading to the conclusion that non-lamellar lipid phases play an important role in certain photosynthetic functions

Photosynthetic electron transport activity in heat-treated barley leaves: The role of internal alternative electron donors to photosystem II

AbstractElectron transport processes were investigated in barley leaves in which the oxygen-evolution was fully inhibited by a heat pulse (48 °C, 40 s). Under these circumstances, the K peak (∼F400 μs) appears in the chl a fluorescence (OJIP) transient reflecting partial QA reduction, which is due to a stable charge separation resulting from the donation of one electron by tyrozine Z. Following the K peak additional fluorescence increase (indicating QA− accumulation) occurs in the 0.2–2 s time range. Using simultaneous chl a fluorescence and 820 nm transmission measurements it is demonstrated that this QA− accumulation is due to naturally occurring alternative electron sources that donate electrons to the donor side of photosystem II. Chl a fluorescence data obtained with 5-ms light pulses (double flashes spaced 2.3–500 ms apart, and trains of several hundred flashes spaced by 100 or 200 ms) show that the electron donation occurs from a large pool with t1/2 ∼30 ms. This alternative electron donor is most probably ascorbate

Far-red fluorescence:A direct spectroscopic marker for LHCII oligomer formation in non-photochemical quenching

AbstractTime-resolved fluorescence on oligomers of the main light-harvesting complex from higher plants indicate that in vitro oligomerization leads to the formation of a weakly coupled inter-trimer chlorophyll–chlorophyll (Chl) exciton state which converts in tens of ps into a state which is spectrally broad and has a strongly far-red enhanced fluorescence spectrum. Both its lifetime and spectrum show striking similarity with a 400ps fluorescence component appearing in intact leaves of Arabidopsis when non-photochemical quenching (NPQ) is induced. The fluorescence components with high far-red/red ratio are thus a characteristic marker for NPQ conditions in vivo. The far-red emitting state is shown to be an emissive Chl–Chl charge transfer state which plays a crucial part in the quenching

Evidence for a fluorescence yield change driven by a light-induced conformational change within photosystem II during the fast chlorophyll a fluorescence rise

AbstractExperiments were carried out to identify a process co-determining with QA the fluorescence rise between F0 and FM. With 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), the fluorescence rise is sigmoidal, in its absence it is not. Lowering the temperature to −10°C the sigmoidicity is lost. It is shown that the sigmoidicity is due to the kinetic overlap between the reduction kinetics of QA and a second process; an overlap that disappears at low temperature because the temperature dependences of the two processes differ. This second process can still relax at −60°C where recombination between QA− and the donor side of photosystem (PS) II is blocked. This suggests that it is not a redox reaction but a conformational change can explain the data. Without DCMU, a reduced photosynthetic electron transport chain (ETC) is a pre-condition for reaching the FM. About 40% of the variable fluorescence relaxes in 100ms. Re-induction while the ETC is still reduced takes a few ms and this is a photochemical process. The fact that the process can relax and be re-induced in the absence of changes in the redox state of the plastoquinone (PQ) pool implies that it is unrelated to the QB-occupancy state and PQ-pool quenching. In both +/−DCMU the process studied represents ~30% of the fluorescence rise. The presented observations are best described within a conformational protein relaxation concept. In untreated leaves we assume that conformational changes are only induced when QA is reduced and relax rapidly on re-oxidation. This would explain the relationship between the fluorescence rise and the ETC-reduction

Modulation of non-bilayer lipid phases and the structure and functions of thylakoid membranes: effects on the water-soluble enzyme violaxanthin de-epoxidase

The role of non-bilayer lipids and non-lamellar lipid phases in biological membranes is an enigmatic problem of membrane biology. Non-bilayer lipids are present in large amounts in all membranes; in energy-converting membranes they constitute about half of their total lipid content-yet their functional state is a bilayer. In vitro experiments revealed that the functioning of the water-soluble violaxanthin de-epoxidase (VDE) enzyme of plant thylakoids requires the presence of a non-bilayer lipid phase. P-31-NMR spectroscopy has provided evidence on lipid polymorphism in functional thylakoid membranes. Here we reveal reversible pH- and temperature-dependent changes of the lipid-phase behaviour, particularly the flexibility of isotropic non-lamellar phases, of isolated spinach thylakoids. These reorganizations are accompanied by changes in the permeability and thermodynamic parameters of the membranes and appear to control the activity of VDE and the photoprotective mechanism of non-photochemical quenching of chlorophyll-a fluorescence. The data demonstrate, for the first time in native membranes, the modulation of the activity of a water-soluble enzyme by a non-bilayer lipid phase