Christa Critchley

Effects of salt stress on the growth, ion content, stomatal behaviour and photosynthetic capacity of a salt-sensitive species, Phaseolus vulgaris L.

Phaseolus vulgaris (cv. Hawkesbury Wonder) was grown over a range of NaCl concentrations (0–150 mM), and the effects on growth, ion relations and photosynthetic performance were examined. Dry and fresh weight decreased with increasing external NaCl concentration while the root/shoot ratio increased. The Cl- concentration of leaf tissue increased linearly with increasing external NaCl concentration, as did K+ concentration, although to a lesser degree. Increases in leaf Na+ concentration occurred only at the higher external NaCl concentrations (≧100 mM). Increases in leaf Cl- were primarily balanced by increases in K+ and Na+. X-ray microanalysis of leaf cells from salinized plants showed that Cl- concentration was high in both the cell vacuole and chloroplast-cytoplasm (250–300 mM in both compartments for the most stressed plants), indicating a lack of effective intracellular ion compartmentation in this species. Salinity had little effect on the total nitrogen and ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) content per unit leaf area. Chlorophyll per unit leaf area was reduced considerably by salt stress, however. Stomatal conductance declined substantially with salt stress such that the intercellular CO2 concentration (Ci) was reduced by up to 30%. Salinization of plants was found to alter the δ13C value of leaves of Phaseolus by up to 5‰ and this change agreed quantitatively with that predicted by the theory relating carbon-isotope fractionation to the corresponding measured intercellular CO2 concentration. Salt stress also brought about a reduction in photosynthetic CO2 fixation independent of altered diffusional limitations. The initial slope of the photosynthesis versus Ci response declined with salinity stress, indicating that the apparent in-vivo activity of RuBP carboxylase was decreased by up to 40% at high leaf Cl- concentrations. The quantum yield for net CO2 uptake was also reduced by salt stress.

Modification of the chloride requirement for photosynthetic O2 evolution: The role of the 23 kDa polypeptide

In thylakoid preparations from spinach and the halophyte Avicennia marina a correlation is observed between functional O2 evolution at low chloride concentrations and the presence of the 23 kDa protein. Addition of spinach 23 kDa protein to polypeptide‐depleted halophyte inside‐out thylakoid vesicles lowers their chloride requirement for optimal O2 evolution activity from 250 to 5 mM. It is suggested that the specific role for the 23 kDa protein is to increase the affinity of the water oxidation site for chloride.

Further studies on the role of chloride in photosynthetic O2 evolution in higher plants

Abstract (1) Thylakoid preparations from two salt-tolerant higher plant species, Avicennia eucalyptifolia and Salicornia quinqueflora , were shown to require very high salt concentrations for Photosystem II electron transport at alkaline pH. (2) High concentrations (250–500 mM) of tetramethylammonium chloride were found to be as effective as NaCl in stimulating maximal O 2 -evolution activity, supporting the evidence that the effect is mediated by chloride and not by cations. (3) Light-response curves of O 2 evolution at high and low concentrations of NaCl were indistinguishable in the light-limited region, providing further evidence for the O 2 -evolving complex as the site of chloride action. (4) A considerable shift in pH optimum towards the alkaline region for O 2 evolution in the presence of increasing concentrations of NaCl, and pH-jump experiments in the presence and absence of NaCl indicate possible competitiveness or at least some form of interdependence of Cl − and OH − binding to the O 2 -evolving complex. (5) The results are discussed in the light of recent evidence concerning the alkaline inhibition of O 2 evolution in non-halophytic thylakoids, and a comparison is made between the two systems.

Studies on oxygen evolution of inside-out thylakoid vesicles from mangroves: Chloride requirement, pH dependence and polypeptide composition

Inside-out thylakoid vesicles from the halophyte Avicennia marina were isolated by the aqueous polymer phase partition method. Oxygen-evolution activity measured with ferricyanide and phenyl-p-benzoquinone was absolutely dependent on added chloride, since the vesicles were almost completely depleted of the 23 and 16 kDa polypeptides of the O2-evolving complex. Addition of the spinach 23 kDa protein to the mangrove inside-out vesicles lowered their chloride requirement for O2 evolution at least 50-fold. In the absence of added chloride, the mangrove vesicles were very sensitive to inhibition by light, which could be prevented by high chloride or low chloride plus added purified spinach 23 kDa protein. The preparations were also inactivated by neutral or alkaline pH (greater than 7.2) in the absence of high chloride concentrations. This inactivation was not significantly influenced by addition of spinach 23 kDa protein. Chloride binding and alkaline inhibition may therefore be closely related, either directly via the manganese centers or, more likely, via pKa changes in as yet unidentified proteins.

A manganese-chloride cluster as the functional centre of the O2 evolving enzyme in photosynthetic systems

Photosynthetic O2 evolution O2 evolving complex mechanism Manganese Chloride Metal cluster

Structure and energy-linked activities in reconstituted bacteriorhodopsin-yeast ATPase proteoliposomes

Abstract Bacteriorhodopsin-F1·F0 (mitochondrial oligomycin-sensitive ATPase complex) proteoliposomes have poor proton pumping and photophosphorylation activities when reconstituted by cholate dialysis. A considerable proportion of the bacteriorhodopsin is not incorporated by cholate dialysis, the particles being too large to be combined into liposomes. Much better reconstitution is achieved where the purple membranes are first fragmented by sonication. Optimal incorporation occurs where bacteriorhodopsin and the phospholipids are sonicated together, suggesting that some perturbation of the liposomes is necessary for successful integration. Since F1·F0 is denatured by sonication a two-step reconstitution procedure has been developed wherein bacteriorhodopsin is first incorporated by sonication, then F1·F0 by cholate dialysis. The vesicles have high phosphorylation rates and also catalyze postillumination [32P]ATP formation where pyridine is present during first stage illumination. F1·F0 can also be incorporated into sonicated bacteriorhodopsin vesicles by “direct incorporation.” This depends on the presence of negatively charged amphiphiles such as cholate or phosphatidylserine in the membranes, and is stimulated by divalent metal cations. Optimum conditions for the various reconstitution procedures are described.

Ca2+ requirement for photosynthetic oxygen evolution of spinach and mangrove photosystem II membrane preparations

CaCl2 significantly increased the photosynthetic oxygen‐evolution activity of photosystem II membrane preparations isolated from spinach and the mangrove Avicennia marina, but not that of spinach thylakoids. This increase is composed of two parts; one due to Cl−, the other to Ca2+. Under Cl−‐sufficient conditions photosystem II particle preparations required only 2 mM Ca2+ for maximum oxygen evolution, irrespective of the presence of the 23‐ and 18‐kDa polypeptides. It is suggested that there is no immediate interaction between Ca2+ and the 23‐ or 18‐kDa polypeptides and that instead Ca2+ acts directly on the oxygen‐evolving centre, perhaps via another, as yet unidentified protein.

Photosynthesis and plasmamembrane permeability properties of Prochloron

Photosynthetic carbon fixation of freshly isolated cells of Prochloron, the symbiont of Lissoclinum patella, proceeded at high rates (80–180 μmol O2·mgChl-1·h-1) in buffered seawater and showed a typical light response, saturating at about 300 μE·m-2·s-1. However, in NaCl solutions osmotically equivalent to seawater CO2-dependent O2 evolution ceased or was severely inhibited. Hypotonic or hypertonic conditions induce degrees of swelling or shrinkage, respectively, apparently causing similar increases in the plasmamembranes permeability to ferricyanide. Initially high, but rapidly declining, rates of electron transport were observed when the cells were suspended in distilled water. This inhibition was not caused by rupture of the cells, indicating instead diffusive loss of some essential factor(s) which normally exchange easily and rapidly between the cells and/or the host environment. Such rapid exchange may be part of the mechanism of this symbiosis and, if not adequately understood, may frustrate attempts to culture Prochloron away from its host.

Protection of photosynthetic O2 evolution against heat inactivation: the role of chloride, pH and coupling status

Heat inactivation of photosynthetic O2 evolution was studied in isolated thylakoids from spinach (Spinacia oleracea) and mangrove (Avicennia marina) leaves. Different temperatures, salt, pH and uncoupler effects were investigated. From these results and others in the literature it was concluced that chloride loss from the membrane and, more specifically, the oxygen-evolving complex of photosystem II, may be the cause of inhibition of oxygen evolution during heat inactivation.

Electron microscopic structure and oxygen evolution activity of thylakoids from Avicennia marina prepared under different osmotic and ionic conditions

Stacking of thylakoid membranes in vitro was assessed using electron microscopy. Grana stacks of spinach thylakoids formed when 5 mol m-3 MgCl2 was present, but no stacking of thylakoids from the mangrove Avicennia marina occurred in the presence of 10 mol m-3 ? MgCl2 . Isolation of mangrove thylakoids with a high osmotic strength medium did not induce grana formation if the medium consisted only of sorbitol or glycinebetaine. Addition of cations to the high osmotic strength medium did induce some loose-grana formation, with divalent cations being more effective than monovalent cations. Glycinebetaine was a better osmoticum than sorbitol for grana formation provided divalent cations had been added. Oxygen evolution activity of the preparations was influenced by the amount of membrane stacking, with the preparations with the greatest amount of stacked membrane having the highest activity. Isolation with sorbitol or glycinebetaine based media did not alter this pattern, nor did assay in sorbitol or glycinebetaine. Mangrove thylakoids have a requirement for both a high osmotic strength and divalent cations for grana formation in vitro which may be related to the low water potential of the plant environment in vivo.