The distribution of metabolites between spinach chloroplasts and medium during photosynthesis in vitro

Lilley, Ross McC.; Chon, Chong Ja; Mosbach, Annemarie; Heldt, Hans W.

doi:10.1016/0005-2728(77)90212-2

Cited by 152 publications

(43 citation statements)

References 16 publications

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“…Correspondingly, the relative chloroplastic to extrachloroplastic content is far higher for 3-P-glycerate (3.80) than for triose-P (0.52). This is reminiscent of the situation in the isolated chloroplast (13) and indicates that triose-P is preferentially exported from the chloroplast in situ in the light. The ATP/ADP ratio is higher in the extrachloroplast than in the chloroplast compartment.…”

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confidence: 61%

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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: 61%

“…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.…”

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confidence: 99%

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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“…Therefore, with isolated chloroplasts, triose-P and glycolate are products of photosynthesis. Low bicarbonate and high 02 favors glycolate synthesis while high bicarbonate and/or low 02 favors synthesis of DHAP (8,11,14) (Fig. 4 and Table I).…”

Section: Discussionmentioning

confidence: 97%

Influence of Varying CO₂ and Orthophosphate Concentrations on Rates of Photosynthesis, and Synthesis of Glycolate and Dihydroxyacetone Phosphate by Wheat Chloroplasts

Usuda¹,

Edwards²

1982

Plant Physiol.

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Intact chloroplasts of wheat (Triticum aestivwm) were isolated from mesophyll protoplasts. With decreasing concentrations of bicarbonate from 10 to 0.3 millimolar (pH 8.0), the optimal concentration of orthophosphate (Pi) for photosynthetic 02 evolution decreased from a value of 0.1 to 0.2 milHimolar to 0 to 0.025 millimolar. The extremely low Pi optimum for photosynthesis at the low bicarbonate levels of0.3 millimolar was increased by lowering the 02 concentration from 253 (21% gas phase) to 72 micromolar (6% gas phase). The

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“…A lthough this may appear to be a very low rate, it can decrease the stromal concentration of PG A (or of Pi, from which PG A is synthesized) by 8 mM within one hour. Thus, the commonly accepted idea of phosphate and phos phate ester homeostasis in chloroplasts, which is based on the anion exchange characteristics of the phosphate translocator [23], requires modification.…”

Section: Leakage O F Phosphoglycerate From the Chloroplastsmentioning

confidence: 99%

Photoinhibitory Damage to Chloroplasts under Phosphate Deficiency and Alleviation of Deficiency and Damage by Photorespiratory Reactions

Heber

Viil

Neimanis

et al. 1989

Zeitschrift Für Naturforschung C

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D edicated to P rofessor A chim Trebst on the occasion o f his 60th birthdayPhotosynthesis, Photoinhibition, Photorespiration, Electron Transport, Phosphate Deficiency, Phosphate Homeostasis Effects of Pi deficiency on photosynthesis ot isolated spinach chloroplasts were examined. The following observations were made:(1) Chloroplasts isolated in Pi-free media evolved oxygen in the light in the absence of added Pi until acid-extractable P, in the chloroplasts had decreased to 1 to 2.5 m M . This Pj was unavailable for photophosphorylation as shown by the inability of the chloroplasts to respond by oxygen evolution to the addition of PGA. In the state of Prdeficiency, stromal ATP to A DP ratios were in the light close to or below ratios observed in the dark. In the presence of 2 mM PGA, addition of 20 (j.m Pi was insufficient to increase ATP to ADP ratios, but sufficient for appreciable oxygen evolution.(2) More Pi was available for oxygen evolution of phosphate-deficient chloroplasts at low levels of C 0 2 than at high levels. This was due mainly to the suppression of oxygenation of RuBP by high C 0 2 levels which prevented formation of phosphoglycolate and the subsequent release of Pi into the chloroplast stroma.(3) More oxygen was produced by phosphate-deficient chloroplasts at a low light intensity than at a high light intensity. This was due to increased availability of endogenous Pi under low light and to photoinhibition of the chloroplasts by high light. The main product of photosynthesis of phosphate-deficient chloroplasts in the presence of a high bicarbonate concentration was starch, and the main soluble product was PGA.(4) After phosphate-deficient chloroplasts had ceased to evolve oxygen in the light, they be came photosensitive. Part of the loss of the capacity for oxygen evolution is attributed to leakage of PGA, but the main reason for loss of function is photoinactivation of electron transport. Both photosystems of the electron transport chain were damaged by light.(5) Protection against photoinactivation was provided by coupled electron transport. Photo inactivation of phosphate-deficient chloroplasts was less extensive in the presence of low C 0 2 concentrations which permitted oxygenation of RuBP than at high CO: concentrations. Electron transport to C 0 2 and other physiological electron acceptors and to the herbicide methylviologen was also protective. However, electron transport to oxygen in the Mehler reaction failed to provide appreciable protection against high light intensities, because oxygen reduction is slow and reactive oxygen species produced in the light contribute to photoinactivation.

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The distribution of metabolites between spinach chloroplasts and medium during photosynthesis in vitro

Cited by 152 publications

References 16 publications

Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

Influence of Varying CO₂ and Orthophosphate Concentrations on Rates of Photosynthesis, and Synthesis of Glycolate and Dihydroxyacetone Phosphate by Wheat Chloroplasts

Photoinhibitory Damage to Chloroplasts under Phosphate Deficiency and Alleviation of Deficiency and Damage by Photorespiratory Reactions

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The distribution of metabolites between spinach chloroplasts and medium during photosynthesis in vitro

Cited by 152 publications

References 16 publications

Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

Enzymic Determination of Metabolites in the Subcellular Compartments of Spinach Protoplasts

Influence of Varying CO2 and Orthophosphate Concentrations on Rates of Photosynthesis, and Synthesis of Glycolate and Dihydroxyacetone Phosphate by Wheat Chloroplasts

Photoinhibitory Damage to Chloroplasts under Phosphate Deficiency and Alleviation of Deficiency and Damage by Photorespiratory Reactions

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Influence of Varying CO₂ and Orthophosphate Concentrations on Rates of Photosynthesis, and Synthesis of Glycolate and Dihydroxyacetone Phosphate by Wheat Chloroplasts