Fructose-and sedoheptulosebisphosphatase. The sites of a possible control of CO2 fixation by light-dependent changes of the stromal Mg2+ concentration

Portis, Archie R.; Chon, Chong Ja; Mosbach, Annemarie; Heldt, Hans W.

doi:10.1016/0005-2728(77)90181-5

Cited by 106 publications

(29 citation statements)

References 25 publications

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“…Similar levels are found for 3-P-glycerate, RuBP, Pi, and hexose-P in isolated chloroplasts and in chloroplasts contained in protoplasts. However, the Fru-1,6-bisP and triose-P are both three to seven times higher in the chloroplasts from protoplasts and, as a result (17) the ATP/ADP quotient is altered. The lower triose-P and Fru-1,6-bisP in isolated chloroplasts might be explained as follows.…”

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confidence: 90%

Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

Wirtz¹,

Stitt²,

Heldt³

1980

Plant Physiol.

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141

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A method for determining the subcellular metabolite levels in spinach protoplasts is described. The protoplasts are disrupted by centrifugation through a nylon net, releasing intact chloroplasts which pass through a layer of silicone oil into perchloric acid while the remaining cytoplasmic components remain over the oil and are simultaneously quenched as acid is centrifuged into them. Cross-contamination is measured and corrected for using ribulose 1,5-bisphosphate as a chloroplastic marker and phosphoenolpyruvate carboxylase as a cytoplasmic marker. A method for separation of intact protoplasts from the medium by silicone oil centrifugation is described, which allows a correction to be made for the effect of free chloroplasts and broken protoplasts. Methods for inhibiting chloroplast photosynthesis, without inhibiting protoplasts, are presented. It is demonstrated that ribulose 1,5-bisphosphate, ATP, ADP, AMP, inorganic phosphate, hexose phosphate, triose phosphate, fructose 1,6-bisphosphate, and 3-phosphoglycerate can be reliably recovered in the subcellular fractions isolated from protoplasts, and measured by enzymic substrate analysis.give reliable measurements of the subcellular metabolite levels. The method has been specifically developed for the enzymic analysis of the subcellular levels of metabolites in unlabeled extracts but can obviously be extended to, and used in conjunction with, radioactive analysis techniques.A method for subcellular metabolite analysis must meet the following criteria. (a) The cellular compartments must be separated into fractions with acceptably low levels of cross-contamination. The extent of cross-contamination must be determined and corrected for by the use of suitable markers. (b) Separation and quenching of the fractions must be rapid enough to prevent any significant changes in the metabolite levels during, or as a result of, the fractionation procedures. (c) Protoplast preparations inevitably contain some free chloroplasts. The activity of these chloroplasts must be inhibited, without altering protoplast metabolism. The end products of chloroplast photosynthesis which accumulate in the medium are triose-P and, to a lower extent, 3-P-glycerate (2, 13). These are relatively small, rapidly turning Isolated intact chloroplasts are widely used to investigate photosynthesis. However the isolation of chloroplasts can produce changes in their metabolism. Interactions with the cytoplasm and other organelles are lost, and changes in the rates of transport of metabolites across the bounding envelope membrane are to be expected. Moreover, important processes which occur in the cytoplasm and other organelles cannot be studied; these include sucrose synthesis, interactions with respiration, photorespiration, and aspects of nitrogen metabolism and lipid metabolism. On the other hand, isolated cells (15) or protoplasts (4) provide an easily manipulable whole cell system but pose the problem of resolving the internal compartmental organization.When protoplasts are forced through...

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confidence: 90%

Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

Wirtz¹,

Stitt²,

Heldt³

1980

Plant Physiol.

Self Cite

141

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“…This clearly indicates that its reoxidation is limited by ATP availability. Furthermore, an ATP/ADP ratio of approximately 0.25 was observed at steady state in intact chloroplasts performing high rates of CO 2 assimilation (Portis et al, 1977). In contrast, it is well established that the effect of light is to increase the ATP concentration in both intact leaves and isolated protoplasts (e.g., see Santarius and Heber, 1965;Stitt et al, 1982;Furbank and Horton, 1987) under steady-state conditions.…”

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confidence: 99%

In Vivo Changes of the Oxidation-Reduction State of NADP and of the ATP/ADP Cellular Ratio Linked to the Photosynthetic Activity in Chlamydomonas reinhardtii

et al. 2003

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The ATP/ADP and NADP/NADPH ratios have been measured in whole-cell extract of the green alga Chlamydomonas reinhardtii, to understand their availability for CO 2 assimilation by the Calvin cycle in vivo. Measurements were performed during the dark-light transition of both aerobic and anaerobic cells, under illumination with saturating or low light intensity. Two different patterns of behavior were observed: (a) In anaerobic cells, during the lag preceding O 2 evolution, ATP was synthesized without changes in the NADP/NADPH ratio, consistently with the operation of cyclic electron flow. (b) In aerobiosis, illumination increased the ATP/ADP ratio independently of the intensity used, whereas the amount of NADPH was decreased at limiting photon flux and regained the dark-adapted level under saturating photon flux. Moreover, under these conditions, the addition of low concentrations of uncouplers stimulated photosynthetic O 2 evolution. These observations suggest that the photosynthetic generation of reducing equivalents rather than the rate of ATP formation limits the photosynthetic assimilation of CO 2 in C. reinhardtii cells. This situation is peculiar to C. reinhardtii, because neither NADPH nor ATP limited this process in plant leaves, as shown by their increase upon illumination in barley (Hordeum vulgare) leaves, independent of light intensity. Experiments are presented and were designed to evaluate the contribution of different physiological processes that might increase the photosynthetic ATP/NADPH ratio-the Mehler reaction, respiratory ATP supply following the transfer of reducing equivalents via the malate/oxaloacetate shuttle, and cyclic electron flow around PSI-to this metabolic situation.The assimilation of CO 2 in oxygenic photosynthesis depends upon the generation of NADPH and ATP by the light-driven electron transport from water to NADP. This process uses the two photochemical reactions catalyzed by photosystem II (PSII) and photosystem I (PSI) in series and also comprises the cytochrome b 6 f complex, which is reduced by PSII via plastoquinol and oxidized by PSI via plastocyanin. It is coupled to the synthesis of ATP via the generation of an electrochemical proton gradient across the photosynthetic membranes. The stoichiometry of one ATP per NADPH (or one ATP per two electrons; for review, see Witt, 1979) has been established in thylakoids isolated from higher plants, and more recent experiments have confirmed this stoichiometry (see, e.g. and Rumberg, 1999;Sacksteder et al., 2000). So, the measurements with isolated thylakoids consistently show lower ATP per two electrons ratios than that required for CO 2 assimilation. A fraction of the lightdriven electron transport must therefore be involved in the generation of ATP, without using NADP as the terminal electron acceptor.Different pathways have been proposed to perform such a role: (a) The first one is the Mehler reaction (Egneus et al., 1975), i.e. the reduction of molecular oxygen by photosynthetic electron transfer at the level of the reducing...

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“…Stromal Mg2+ concentrations rose by I to 3 mm in the light, roughly doubling dark levels of free Mg2+ ( 16,23). As key stromal enzymes are controlled by, among other factors, Mg2" (3,23,24), lightinduced fluxes of Mg2' into the stroma provided an attractive link between the biophysically based "light reactions" in the thylakoid membranes, and the "dark reactions" in the stroma.However, whether the Mg2+ efflux alone would be enough to balance the H+ influx across the thylakoids is uncertain. …”

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Direct Measurement of K⁺ Channels in Thylakoid Membranes by Incorporation of Vesicles into Planar Lipid Bilayers

Tester¹,

Blatt²

1989

Plant Physiol.

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Light-driven electron transfer reactions cause the active accumulation of protons inside thylakoids, yet at steady state the electrical potential difference across the thylakoid membrane is very small; therefore, there must be a flux of other ions to balance the charge that would otherwise be built up by the net movement of HW. This paper presents direct measurements of ion movements through channels in the thylakoid membrane. These were made possible by fusing thylakoid vesicles from spinach (Spinacia oleracea L.) into planar lipid bilayers, using techniques developed originally to study sarcoplasmic reticulum. No Mg2+ current was found, but voltage-dependent channels have been characterized, these being somewhat selective for K+ over Cl. The data are consistent with a role for these channels in charge balance during light-driven H' movements.Inside chloroplasts are two major compartments, the stroma and the thylakoids, which are separated by an extensive membrane system. Light-driven electron transfer reactions in the thylakoid membrane provide an energy gradient for the production of ATP by a vectorial proton-translocating ATP synthase in the membrane (20). Unlike in mitochondria, this gradient is largely chemical (ApH), with the electrical potential difference (PD)2 being very small at steady state ( 14). Historically, it was assumed that the main ion counterbalancing H+ accumulation was K+, largely because of the abundance of this ion in chloroplasts (33). However, various measurements showed a clear increase upon illumination of Mg2" concentration in the stroma, the aqueous solution outside the thylakoid, with the Mg2+ ions coming from the intrathylakoidal lumen (1,4,6,12,16,23). Stromal Mg2+ concentrations rose by I to 3 mm in the light, roughly doubling dark levels of free Mg2+ ( 16,23). As key stromal enzymes are controlled by, among other factors, Mg2" (3,23,24), lightinduced fluxes of Mg2' into the stroma provided an attractive link between the biophysically based "light reactions" in the thylakoid membranes, and the "dark reactions" in the stroma.However, whether the Mg2+ efflux alone would be enough to balance the H+ influx across the thylakoids is uncertain.

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Fructose-and sedoheptulosebisphosphatase. The sites of a possible control of CO2 fixation by light-dependent changes of the stromal Mg2+ concentration

Cited by 106 publications

References 25 publications

Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

In Vivo Changes of the Oxidation-Reduction State of NADP and of the ATP/ADP Cellular Ratio Linked to the Photosynthetic Activity in Chlamydomonas reinhardtii

Direct Measurement of K⁺ Channels in Thylakoid Membranes by Incorporation of Vesicles into Planar Lipid Bilayers

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Fructose-and sedoheptulosebisphosphatase. The sites of a possible control of CO2 fixation by light-dependent changes of the stromal Mg2+ concentration

Cited by 106 publications

References 25 publications

Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

In Vivo Changes of the Oxidation-Reduction State of NADP and of the ATP/ADP Cellular Ratio Linked to the Photosynthetic Activity in Chlamydomonas reinhardtii

Direct Measurement of K+ Channels in Thylakoid Membranes by Incorporation of Vesicles into Planar Lipid Bilayers

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Direct Measurement of K⁺ Channels in Thylakoid Membranes by Incorporation of Vesicles into Planar Lipid Bilayers