Spidola Neimanis

Heat sensitivity of chloroplasts and leaves: Leakage of protons from thylakoids and reversible activation of cyclic electron transport

In illuminated intact spinach chloroplasts, warming to and beyond 40 °C increased the proton permeability of thylakoids before linear electron transport through Photosystem II was inhibited. Simultaneously, antimycin A-sensitive cyclic electron transport around Photosystem II was activated with oxygen or CO2, but not with nitrite as electron acceptors. Between 40 to 42 °C, activation of cyclic electron transport balanced the loss of protons so that a sizeable transthylakoid proton gradient was maintained. When the temperature of darkened spinach leaves was slowly increased to 40°C, reduction of the quinone acceptor of Photosystem II, QA, increased particularly when respiratory CO2 production and autoxidation of plastoquinones was inhibited by decreasing the oxygen content of the atmosphere from 21 to 1%. Simultaneously, Photosystem II activity was partially lost. The enhanced dark QA reduction disappeared after the leaf temperature was decreased to 20 °C. No membrane energization was detected by light-scattering measurements during heating the leaf in the dark. In illuminated spinach leaves, light scattering and nonphotochemical quenching of chlorophyll fluorescence increased during warming to about 40 °C while Photosystem II activity was lost, suggesting extra energization of thylakoid membranes that is unrelated to Photosystem II functioning. After P700 was oxidized by far-red light, its reduction in the dark was biphasic. It was accelerated by factors of up to 10 (fast component) or even 25 (slow component) after short heat exposure of the leaves. Similar acceleration was observed at 20 °C when anaerobiosis or KCN were used to inhibit respiratory oxidation of reductants. Methyl viologen, which accepts electrons from reducing side of Photosystem II, completely abolished heat-induced acceleration of P700+ reduction after far-red light. The data show that increasing the temperature of isolated chloroplasts or intact spinach leaves to about 40 °C not only inhibits linear electron flow through Photosystem II but also activates Photosystem I-driven cyclic electron transport pathways capable of contributing to the transthylakoid proton gradient. Heterogeneity of the kinetics of P700+ reduction after far-red oxidation is discussed in terms of Photosystem I-dependent cyclic electron transport in stroma lamellae and grana margins.

Transport as the basis of the kok effect. Levels of some photosynthetic intermediates and activation of light-regulated enzymes during photosynthesis of chloroplasts and green leaf protoplasts

Abstract Experiments were performed with intact chloroplasts and leaf cell protoplasts isolated from spinach. The light-dependent decrease in (H+) in the chloroplast stroma counteracts carbon reduction and is offset at low light intensities by a large decrease in NADP and a significant increase in [ ATP ] [ ADP ] ratios. Excess accumulation of NADPH and/or ATP permits 3-phosphogly cerate reduction to occur. With increasing light intensity, NADP levels and [ ATP ] [ ADP ] ratios increased. High rates of photosynthesis were observed at high and at low [ ATP ] [ ADP ] ratios. Levels of dihydroxyacetone phosphate were dramatically increased in the light. In chloroplasts, this permitted conversion to ribulose bisphosphate which on carboxylation yields 3-phosphoglycerate. The light-dependent alkalization of the chloroplast stroma is known to be responsible for phosphogly cerate retention in the chloroplasts. A high chloroplast ratio of phosphogly cerate to dihydroxyacetone phosphate aids carbon reduction. Measured ratios of dihydroxyacetone phosphate to phosphogly cerate were averages between low chloroplast ratios and high cytosolic ratios. They were far higher, even under low-intensity illumination, than dark ratios. Since cytosolic NADH levels are known to increase much less in the light than cytosolic dihydroxyacetone phosphate levels, the large increase in the ratio of didydroxyacetone phosphate to phosphogly cerate must considerably increase cytosolic phosphorylation potentials even at very low light intensities. It is proposed that this increase is communicated to the mitochondrial adenylate system, and inhibits dark respiratory activity, giving rise to the Kok effect. The extent of stroma alkalization, the efficiency of metabolite shuttles across the chloroplast envelope, and rates of cytosolic ATP consumption are proposed to be factors determining whether and to what extent the Kok effect can be observed. Light activation of chloroplast enzymes was slow at low and fast at high light intensities. This contrasts to low NADP levels at low and usually higher levels at high light intensities. Maximum enzyme activation was observed far below light saturation of photosynthesis, and light activation of enzymes was often less pronounced at very high than at intermediate light intensities.

Light-dependent pH changes in leaves of C3 plants. I, Recording pH changes in various cellular compartments by fluorescent probes

Chloroplasts, mesophyll protoplasts, cytoplasts, vacuoplasts, vacuoles and leaves were stained with pH-indicating fluorescent dyes of differing pK values. Fluorescence microscopy was used to obtain information on the intracellular and intercellular distribution of the probes. The kinetics of blue or green fluorescence emitted from chloroplasts, protoplasts, cytoplasts and leaves was measured during illumination with red light. The intensity of light used for fluorescence excitation was so low that it had little effect on photosynthesis. In leaves, fluorescence signals emitted from chloroplasts were small and usually insignificant compared to signals originating from the cytosol. Both indicated light-dependent alkalization and reversal of alkalization on darkening. Vacuolar signals were opposite in sign to cytosolic signals. They indicated acidification of the vacuole in the light-dark transient and reversal of this effect on darkening.

Photosystem I-dependent cyclic electron transport is important in controlling Photosystem II activity in leaves under conditions of water stress

Leaves of the C3 plant Brassica oleracea were illuminated with red and/or far-red light of different photon flux densities, with or without additional short pulses of high intensity red light, in air or in an atmosphere containing reduced levels of CO2 and/or oxygen. In the absence of CO2, far-red light increased light scattering, an indicator of the transthylakoid proton gradient, more than red light, although the red and far-red beams were balanced so as to excite Photosystem II to a comparable extent. On red background light, far-red supported a transthylakoid electrical field as indicated by the electrochromic P515 signal. Reducing the oxygen content of the gas phase increased far-red induced light scattering and caused a secondary decrease in the small light scattering signal induced by red light. CO2 inhibited the light-induced scattering responses irrespective of the mode of excitation. Short pulses of high intensity red light given to a background to red and/or far-red light induced appreciable additional light scattering after the flashes only, when CO2 levels were decreased to or below the CO2 compensation point, and when far-red background light was present. While pulse-induced light scattering increased, non-photochemical fluorescence quenching increased and F0 fluorescence decreased indicating increased radiationless dissipation of excitation energy even when the quinone acceptor QA in the reaction center of Photosystem II was largely oxidized. The observations indicate that in the presence of proper redox poising of the chloroplast electron transport chain cyclic electron transport supports a transthylakoid proton gradient which is capable of controlling Photosystem II activity. The data are discussed in relation to protection of the photosynthetic apparatus against photoinactivation.

Changes in apoplastic pH and membrane potential in leaves in relation to stomatal responses to CO2, malate, abscisic acid or interruption of water supply.

Abstract. Low CO2 concentrations open CO2-sensitive stomata whereas elevated CO2 levels close them. This CO2 response is maintained in the dark. To elucidate mechanisms underlying the dark CO2 response we introduced pH- and potential-sensitive dyes into the apoplast of leaves. After mounting excised leaves in a gas-exchange chamber, changes in extracellular proton concentration and transmembrane potential differences as well as transpiration and respiration were simultaneously monitored. Upon an increase in CO2 concentration transient changes in apoplastic pH (occasionally brief acidification, but always followed by alkalinization) and in membrane potential (brief hyperpolarization followed by depolarization) accompanied stomatal closure. Alkalinization and depolarization were also observed when leaves were challenged with abscisic acid or when water flow was interrupted. During stomatal opening in response to CO2-free air the apoplastic pH increased while the membrane potential initially depolarized before it transiently hyperpolarized. To examine whether changes in apoplastic malate concentrations represent a closing signal for stomata, malate was fed into the transpiration stream. Although malate caused apoplastic alkalinization and membrane depolarization reminiscent of the effects observed with CO2 and abscisic acid, this dicarboxylate closed the stomata only partially and less effectively than CO2. Apoplastic alkalinization was also observed and stomata closed partially when KCl was fed to the leaves. Respiration increased on feeding of malate or KCl, or while abscisic acid closed the stomate. From these results we conclude that CO2 signals modulate the activity of plasma-membrane ion channels and of plasmalemma H+-ATPases during changes in stomatal aperture. Responses to potassium malate and KCl are not restricted to guard cells and neighbouring cells.

Photosynthesis under osmotic stress : Differential recovery of photosynthetic activities of stroma enzymes, intact chloroplasts, protoplasts, and leaf slices after exposure to high solute concentrations.

The reversibility of the inhibition of photosynthetic reactions by water stress was examined with four systems of increasing complexity—stromal enzymes, intact chloroplasts, mesophyll protoplasts, and leaf slices. The inhibition of soluble chloroplast enzymes by high solute concentrations was instantly relieved when solutes were properly diluted. In contrast, photosynthesis was not restored but actually more inhibited when isolated chloroplasts exposed to hypertonic stress were transferred to conditions optimal for photosynthesis of unstressed chloroplasts. Upon transfer, chloroplast volumes increased beyond the volumes of unstressed chloroplasts, and partial envelope rupture occurred. In protoplasts and leaf slices, considerable and rapid, but incomplete restoration of photosynthesis was observed during transfer from hypertonic to isotonic conditions. Chloroplast envelopes did not rupture in situ during water uptake. It is concluded that inhibition of photosynthesis by severe water stress is at the biochemical level brought about in part by reversible inhibition of chloroplast enzymes and in part by membrane damage which requires repair mechanisms for reversibility. Both soluble enzymes and membranes appear to be affected by the increased concentration of internal solutes, which is caused by dehydration.

Assimilatory power as a driving force in photosynthesis

Intact spinach chloroplasts were permitted to photoreduce added 3-phosphoglycerate until oxygen evolution was replaced by oxygen uptake. The chloroplast suspensions were then analyzed for dihydroxyacetone phosphate and residual 3-phosphoglycerate. Ratios of dihydroxyacetone phosphate to phosphoglycerate served to calculate assimilatory power ([ATP][ADP][Pi]) × ([NADPH][NADP]). Extrapolation yielded maximum values of assimilatory power PA of about 4000 (M−1) as long as the chloroplasts continued to oxidize dihydroxyacetone phosphate via ribulose bisphosphate oxygenase. When the oxygen concentration was reduced to 25 μM, maximum values of PA approached 25 000 (M−1) at light saturation of phosphoglycerate reduction. Maximum PA declined as chloroplasts aged. When the light intensity was reduced, PA decreased and intact chloroplasts oxidized dihydroxyacetone phosphate which had previously been exported into the medium. This observation explains the transient inhibition of photosynthesis of leaves after a sudden reduction in light intensity. Maximum PA calculated for broken chloroplasts by multiplication of NADPHNADP ratios and maximum phosphorylation potentials [ATP][ADP][Pi] measured separately in thylakoid suspensions at light saturation was as high as 2.5·106 (M−1). The discrepancy between PA in the stroma of intact chloroplasts and the values calculated for thylakoid suspensions is explained by inefficient cyclic electron flow which is incapable of raising phosphorylation potentials to high levels when NADP is reduced in chloroplasts. In leaves, maximum PA in chloroplasts was even lower than in isolated chloroplasts. Turnover of ATP and NADPH in situ prevents PA from reaching high levels even when net assimilation is zero. PA was higher in leaves at low light intensities when carbon reduction was slow than at high light intensities when it was fast. This can explain the Kok effect. The apparent paradox that photosynthetic flux is increased as the driving force PA is decreased is explained by regulation of enzymes of the Calvin cycle. Maximum rates of electron flow and phosphorylation and therefore also of photosynthesis are possible only when levels of PA are kept low. Rapid use of PA requires high activities of the enzymes of the Calvin cycle and may explain the necessity of enzyme activation.

Fractional control of photosynthesis by the QB protein, the cytochrome f/b 6 complex and other components of the photosynthesic apparatus.

In order to obtain information on fractional control of photosynthesis by individual catalysts, catalytic activities in photosynthetic electron transport and carbon metabolism were modified by the addition of inhibitors, and the effect on photosynthetic flux was measured using chloroplasts of Spinacia oleracea L. In thylakoids with coupled electron transport, light-limited electron flow to ferricyanide was largely controlled by the QB protein of the electron-transport chain. Fractional control by the cytochrome f/b6 complex was insignificant under these conditions. Control by the cytochrome f/b6 complex dominated at high energy fluence rates where the contribution to control of the QB protein was very small. Uncoupling shifted control from the cytochrome f/b6 complex to the QB protein. Control of electron flow was more complex in assimilating chloroplasts than in thylakoids. The contributions of the cytochrome f/b6 complex and of the QB protein to control were smaller in intact chloroplasts than in thylakoids. Thus, even though the transit time for an electron through the electron-transport chain may be below 5 ms in leaves, oxidation of plastohydroquinone was only partially responsible for limiting photosynthesis under conditions of light and CO2 saturation. The energy fluence rate influenced control coefficients. Fractional control of photosynthesis by the ATP synthetase, the cytochrome f/b6 complex and by ribulose-1,5-bisphosphate carboxylase increased with increasing fluence rates, whereas the contributions of the QB protein and of enzymes sensitive to SH-blocking agents decreased. The results show that the burdens of control are borne by several components of the photosynthetic apparatus, and that burdens are shifted as conditions for photosynthesis change.

Co-operative activation of chloroplast fructose-1,6-bisphosphatase by reductant, pH and substrate

Abstract Intact chloroplasts capable of high rates of photosynthesis fail to reduce CO2 when illuminated in the absence of oxygen. While anaerobiosis limits proton gradient formation leading to ATP deficiency (Ziem-Hanck, U. and Heber, U. (1980) Biochim. Biophys. Acta 591, 266–274), light activation of fructose-1,6-bisphosphatase was also inhibited by anaerobiosis, whereas light activation of NADP-malate dehydrogenase was stimulated by anaerobiosis, indicating that reductant was still available for light activation. The chloroplast pool of NADP was largely reduced during illumination under anaerobiosis and electron transport to oxaloacetate was not inhibited by anaerobic conditions. Significant light activation of fructose-bisphosphatase was observed in anaerobic chloroplasts with 3-phosphoglycerate as substrate, but not with dihydroxyacetone phosphate (3-phosphoglycerate supports electron transport and hence proton gradient formation). In the absence of added substrates, illumination of anaerobic chloroplasts resulted in some light activation of fructose-bisphosphatase when the pH of the medium was increased. Under these conditions, light activation was stimulated by dihydroxyacetone phosphate. Dihydroxyacetone phosphate added together with oxaloacetate allowed light activation of fructose-bisphosphatase in anaerobic chloroplasts, while neither substrate added alone was effective. Formation of a transthylakoid proton gradient can therefore substitute for an alkaline suspension medium by causing an alkaline shift of the stromal pH on illumination. The data are interpreted as indicating that fructose-bisphosphatase, but not NADP-malate dehydrogenase, requires an alkaline pH and the presence of substrate for rapid reductive light activation and they bear on the interpretation of the lag observed in photosynthesis in chloroplasts and leaves on illumination after a prolonged dark period.

Coupled cyclic electron transport in intact chloroplasts and leaves of C3 plants: Does it exist? if so, what is its function?

Transthylakoid proton transport based on Photosystem I-dependent cyclic electron transport has been demonstrated in isolated intact spinach chloroplasts already at very low photon flux densities when the acceptor side of Photosystem I (PS I) was largely closed. It was under strict redox control. In spinach leaves, high intensity flashes given every 50 s on top of far-red, but not on top of red background light decreased the activity of Photosystem II (PS II) in the absence of appreciable linear electron transport even when excitation of PS II by the background light was extremely weak. Downregulation of PS II was a consequence of cyclic electron transport as shown by differences in the redox state of P700 in the absence and the presence of CO2 which drained electrons from the cyclic pathway eliminating control of PS II. In the presence of CO2, cyclic electron transport comes into play only at higher photon flux densities. At H+/e=3 in linear electron transport, it does not appear to contribute much ATP for carbon reduction in C3 plants. Rather, its function is to control the activity of PS II. Control is necessary to prevent excessive reduction of the electron transport chain. This helps to protect the photosynthetic apparatus of leaves against photoinactivation under light stress.