Protein synthesis in isolated spinach chloroplasts: comparison of light-driven and ATP-driven synthesis

A comparison has been made of the optimal concentrations of Mg²⁺ and K⁺ ions necessary for both light-driven protein synthesis in intact spinach chloroplasts and for ATP-driven protein synthesis in broken chloroplasts, and the products of the two systems have been compared by polyacrylamide gel electrophoresis. Light-driven incorporation of amino acids into polypeptides in intact chloroplasts assayed in buffer systems containing sucrose or sorbitol as the osmoticum is inhibited by the addition of Mg²⁺, the effect being most marked at low concentrations (less than 40 mm) of KCl. On the other hand, chloroplasts suspended in 0.2 m KCl as osmoticum require Mg²⁺ (3 mm) for optimal light-driven protein-synthesizing activity. Incorporation of amino acids by broken chloroplasts in the dark, supplemented with ATP and GTP, requires 9 mm Mg²⁺ for maximum activity. A requirement for monovalent cations is best filled by K⁺ (approx 30 mm) in the case of the light-driven, intact chloroplast system whereas, in the ATP-driven, broken chloroplast system, NH_4⁺ (approx 80 mm) gave the highest activity.Autoradiographs of Na dodecyl sulfate-polyacrylamide gels of the products from both the light-driven, intact chloroplasts and from the ATP-driven, broken chloroplasts reveal qualitatively similar patterns. There are at least four radioactive polypeptides in the soluble protein fraction the dominant product being coincident with the large subunit of Fraction 1 protein. In the membrane fraction at least nine discrete products can be resolved.     

Starch degradation in isolated spinach chloroplasts

A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with ¹⁴C-starch is described. These intact chloroplasts were incubated aerobically in the dark for 30 minutes. Radioactivity in starch declined and glyceric acid 3-phosphate and maltose were the major radioactive products. It is proposed that starch is degraded within the chloroplast to glyceric acid 3-phosphate and to maltose.     

Carbon dioxide assimilation by leaves, isolated chloroplasts, and ribulose bisphosphate carboxylase from spinach

The relationship between rate of photosynthesis and CO(2) concentration has been reinvestigated using isolated spinach (Spinacia oleracea) chloroplasts. The apparently low CO(2) concentration required for half-maximal photosynthesis is shown to result partly from a ceiling imposed by electron transport. In double reciprocal plots of rate against CO(2) concentration, this ceiling results in departures from linearity at high CO(2) concentrations. If these rate limitations are disregarded in extrapolation the "true" CO(2) concentration required for half maximal carboxylation by intact chloroplasts is approximately 46 mum (CO(2)).When assayed under comparable conditions, ribulose bisphosphate carboxylase from these chloroplasts also shows an apparent Km (CO(2)) of approximately 46 mum, suggesting that its characteristics are not modified by extraction. An improved assay for ribulose bisphosphate carboxylase yielded rates of carboxylation considerably higher than those previously reported, the highest maximal velocities recorded approaching 1000 mumoles CO(2) fixed mg(-1) chlorophyll hr(-1) at 20 C. With such Km and V(max), values the carboxylase would be able to achieve, at concentrations of CO(2) less than atmospheric, rates of CO(2) fixation equal to those displayed by the parent tissue or by the average plant under favorable conditions in its natural environment.     

Photosynthetic intermediates, the warburg effect, and glycolate synthesis in isolated spinach chloroplasts

Increasing levels of CO(2) have been shown to stimulate the rate of photosynthesis, eliminate the oxygen inhibition of photosynthesis (Warburg effect), and decrease glycolate formation in isolated spinach chloroplasts. Ribose 5-phosphate and fructose 1,6-diphosphate at concentrations of 5 to 10 mum also stimulate the rate of plastid photosynthesis and eliminate the Warburg effect. In contrast to the effect of high CO(2) levels, these sugar phosphates have little effect on glycolate formation. Evidence is presented to show that the level of intermediates of the photosynthetic carbon reduction cycle may influence the Warburg effect in vivo. It is postulated that the formation of glycolate is not the causal factor of the Warburg effect.     

Linoleate and alpha-linolenate synthesis by isolated spinach (Spinacia oleracea) chloroplasts.

Diacylgalactosylglycerol synthesis was a prerequisite for the incorporation of [1-14C]-acetate into linoleate and alpha-linolenate of isolated spinach (Spinacia oleracea) chloroplasts. Oleate at position 1 of diacylgalactosylglycerol was desaturated to linoleate and alpha-linolenate both in the light and in the dark. Some desaturation of palmitate was also observed after prolonged incubations.     

Tocopherol and plastoquinone synthesis in spinach chloroplasts subfractions

Subfractions isolated from intact purified spinach chloroplasts are able to prenylate the aromatic moiety of α-tocopherol and plastoquinone-9 precursors. The biosynthesis of α-tocopherol and plastoquinone-9 is a compartmentalized process. The chloroplast envelope membranes are the only site of the enzymatic prenylation in α-tocopherol synthesis whereas the thylakoid membrane is also involved in the prenylation and methylation sequence of plastoquinone-9 biosynthesis. A very active kinase which forms phytyl-PP is localized in the stroma. Phytol but not geranylgeraniol is the polyprenol precursor of the side chain of α-tocopherol in spinach chloroplasts.     

Phytol synthesis from geranylgeraniol in spinach chloroplasts

The reduction of /2-¹⁴C/-geranylgeranylpyrophosphate to phytylpyrophosphosphate is shown for the first time in chloroplasts. The esterification of exogenous /2-¹⁴C/-geranylgeranylpyrophosphate with endogenous chlorophyllide and the stepwise reduction of the pigment bound geranylgeraniol to phytol was also proved for spinach chloroplasts for the first time.     

Synthesis of mono- and digalactosyldiacylglycerol in isolated spinach chloroplasts

Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose-labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U-¹⁴C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-¹⁴C]acetate or sn-[U-¹⁴C]glycerol-3-P in the presence of Mg²⁺ and unlabeled UDP-Gal resulted in high ¹⁴C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase.     

Photoreduction of sulfur dioxide by spinach leaves and isolated spinach chloroplasts

Labeled sulfur dioxide was found to be extensively absorbed by spinach (Spinacea oleracea L.) leaves. Labeled sulfides detected in leaf blades following fumigations with sulfur dioxide in light indicated that photoreduction of sulfur dioxide had occurred. Measurable proportions of this labeled sulfur was localized within the chloroplast fraction. Suspensions of isolated chloroplasts supplied with labeled sulfur dioxide contained labeled sulfides following a 30-minute illumination period in water-cooled reaction vessels. With reference to recent studies of the chloroplast sulfur reduction pathway, probable points of entry for sulfur dioxide and the subsequent release of hydrogen sulfide are discussed.     

Level of photosynthetic intermediates in isolated spinach chloroplasts

The level of intermediates of the photosynthetic carbon cycle was measured in intact spinach chloroplasts in an attempt to determine the cause of the induction lag in CO₂ assimilation. In addition, transient changes in the level of the intermediates were determined as affected by a light-dark period and by the addition of an excess amount of bicarbonate during a period of steady photosynthesis. Assayed enzymically were: ribulose 1,5-diphosphate, pentose monophosphates (mixture of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, hexose monophosphates (mixture of glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate), glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerate acid 3-phosphate, a mixture of fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).     

Reconstitution of amino acid synthesis by combining spinach chloroplasts with other leaf organelles

By adding leaf peroxisomes to purified intact chloroplasts, glycine synthesis was reconstituted. On adding leaf mitochondria, serine synthesis was also reconstituted. However, aromatic amino acid synthesis which was effected by purified chloroplasts was not enhanced on adding peroxisomes or mitochondria although the rate in whole leaves was considerably higher.     

Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts

The prenylquinone content and biosynthetic capabilities of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts were analyzed. Both envelope membranes contain prenylquinones, and in almost similar amounts (on a protein basis). However, the outer envelope membrane contains more alpha-tocopherol than the inner one although this prenylquinone is the major one in both fractions. On the contrary, plastoquinone-9 is present in higher amounts in the inner envelope membrane than in the outer one. In addition, it has been demonstrated that all the enzymes involved in the last steps of alpha-tocopherol and plastoquinone-9 biosynthesis, i.e., homogentisate decarboxylase polyprenyltransferase, S-adenosyl-methionine:methyl-6-phytylquinol methyltransferase, S-adenosyl-methionine: alpha-tocopherol methyltransferase, homogentisate decarboxylase solanesyltransferase, S-adenosyl-methionine:methyl-6-solanesylquinol methyltransferase, and possibly 2,3-dimethylphytylquinol cyclase, are localized on the inner envelope membrane. These results demonstrate that the inner membrane of the chloroplast envelope plays a key role in chloroplast biogenesis, and especially for the synthesis of the two major plastid prenylquinones.