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.     

Effect of sulphate on glutamate synthesis by intact spinach (Spinacia oleracea) chloroplasts.

During the course of NH4+ (or NO2-)-plus-alpha-oxoglutarate-dependent O2 evolution in spinach (Spinacia oleracea) chloroplasts, glutamate was continuously excreted out of the chloroplasts. Under these conditions, for each molecule of NO2- or NH4+ which disappeared, one molecule of glutamate accumulated in the medium and the concentration of glutamate in the stroma space was maintained constant. SO4(2-) (or SO3(2-) behave as inhibitors of NH4+ incorporation into glutamate by intact chloroplasts. This considerable inhibition of glutamate synthesis by SO4(2-) was correlated with a rapid decline in the stromal Pi concentration. The reloading of stromal Pi with either external Pi or PPi4- relieved SO4(2-)-induced inhibition of glutamate synthesis by intact chloroplasts. It was concluded that SO4(2-) induced a rapid efflux of stromal Pi out of the chloroplast, leading to a limitation of ATP synthesis and therefore to an arrest of ATP-dependent glutamine synthetase functioning.     

Stromal protein phosphorylation in spinach (Spinacia oleracea) chloroplasts.

When intact spinach chloroplasts were supplied with [32P]Pi, stromal protein phosphorylation was found to occur in the dark. On illumination the thylakoid protein kinase was activated and the amount of label found in thylakoid proteins quickly exceeded that incorporated into stromal protein, such that the latter was found to account for only 10-15% of the total radioactivity bound to chloroplast proteins after 5 min illumination. The rate of phosphorylation of stromal polypeptides was unchanged by light. After SDS/polyacrylamide-gel electrophoresis, more than 15 labelled polypeptides of stromal origin were observed. A polypeptide with an Mr of approx. 70 000 had the highest specific activity of labelling. Both the large and small subunits of the ribulose-1,5-bisphosphate carboxylase were phosphorylated. The level of phosphorylation of stromal protein was increased by CO2 fixation in intact chloroplasts. This increase was not observed in the absence of NaHCO3 or in the presence of the phosphoribulokinase inhibitor DL-glyceraldehyde. These effects appeared to be largely due to changes in the phosphorylation state of the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Studies with the reconstituted chloroplast system showed that the thylakoid protein kinase(s) played no part in the phosphorylation of stromal protein. The rate and level of phosphorylation of stromal protein was unaffected by the activation state of the thylakoid protein kinase and was unchanged when thylakoids were omitted from the reaction medium. The phosphorylation of stromal proteins is therefore catalysed by a discrete soluble protein kinase.     

Activity of phosphatidylcholine-transfer protein from spinach (Spinacia oleracea) leaves with mitochondria and chloroplasts

A low-molecular-weight protein catalysing the transfer of phosphatidylcholine from liposomes to mitochondria and chloroplasts has been isolated from spinach (Spinacia oleracea) by chromatography on Sephadex G-75.     

Acetyl-coenzyme A carboxylase from avocado (Persea americana) plastids and spinach (Spinacia oleracea) chloroplasts.

1. Protein synthesis has been investigated in different regions of the rat epididymis by measuring incorporation of [35S]methionine in tissue minces incubated in vitro followed by analysis of labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate. Rates of synthesis were highest in the proximal cauda > distal cauda > initial segment > ductuli efferentes > corpus > distal caput > proximal caput. One protein (mol.wt. 23 000) characterized the initial segment, three proteins (mol.wts. 18 500, 19 000 and 32 000) the caput and one protein (mol.wt. 47 000) the cauda. 2. After castration, [35S]methionine incorporation in all regions of the epididymis was reduced to < 10% of that in normal animals but could be restored to control levels within 5 days by testosterone treatment. Other steroids (corticosterone, oestrogen or progesterone) were ineffective. 3. The synthesis of the 18 500, 19 000, and 32 000 mol.wt. proteins in the caput and the 47 000 mol.wt. protein in the cauda were preferentially regulated by androgens, whilst the synthesis of 23 000 and approx. 80 000 mol.wt. proteins in the initial segment was dependent upon factors present in testicular fluid. 4. The androgen-dependent and testicular fluid-dependent proteins were major components of epididymal secretion. Purification and characterization of the 18 500, 19 000, 23 000 and 32 000 mol.wt. proteins showed them to be acidic glycoproteins with a carbohydrate content of 7.6-13.2%. The 47 000 mol.wt. protein, on the other hand, is highly basic. 5. A possible role for these proteins in the acquisition of motility, fertilizing capacity and storage of spermatozoa in the epididymis is discussed.     

Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. The effect of hydrogen peroxide and of Paraquat.

The stroma of spinach chloroplasts contains ascorbic acid and glutathione at millimolar concentrations. [Reduced glutathione]/[oxidized glutathione] and [ascorbate]/[dehydroascorbate] ratios are high under both light and dark conditions and no evidence for a role of oxidized glutathione or dehydroascorbate in the dark-deactivation of fructose bisphosphatase could be obtained. Addition of H2O2 to chloroplasts in the dark decreases the above ratios, an effect that is reversed on illumination. Addition of Paraquat to illuminated chloroplasts caused a rapid oxidation of reduced glutathione and ascorbate, and apparent loss of dehydroascorbate. Paraquat rapidly inactivated fructose bisphosphatase activity, as assayed under physiological conditions.     

The effects of high concentrations of salts on photosynthetic electron transport in spinach (Spinacia oleracea) chloroplasts.

1. Photosynthetic electron transport from water to lipophilic Photosystem II acceptors was stimulated 3--5-fold by high concentrations (greater than or equal to 1 M) of salts containing anions such as citrate, succinate and phosphate that are high in the Hofmeister series. 2. In trypsin-treated chloroplasts, K3Fe(CN)6 reduction insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea was strongly stimulated by high concentrations of potassium citrate, but there was much less stimulation of 2,6-dichloroindophenol reduction in Tris-treated chloroplasts supplied with 1,5-diphenylcarbazide as artificial donor. The results suggest that the main site of action of citrate was the O2-evolving complex of Photosystem II. 3. Photosystem I partial reactions were also stimulated by intermediate concentrations of citrate (up to 2-fold stimulation by 0.6--0.8 M-citrate), but were inhibited at the highest concentrations. The observed stimulation may have been caused by stabilizaton of plastocyanin that was complexed with the Photosystem I reaction centre, 4. At 1 M, potassium citrate protected O2 evolution against denaturation by heat or by the chaotropic agent NaNO3. 5. It is suggested that anions high in the Hofmeister series stimulated and stabilized electron transport by enhancing water structure around the protein complexes in the thylakoid membrane.     

Characterization of elemental sulfur in isolated intact spinach chloroplasts

Incubation of intact spinach (Spinacia oleracea L.) chloroplasts in the presence of ³⁵SO₄²⁻ resulted in the light-dependent formation of a chloroform-soluble sulfur-containing compound distinct from sulfolipid. We have identified this compound as the most stable form (S₈) of elemental sulfur (S⁰, valence state for S = O) by mass spectrometry. It is possible that elemental sulfur (S⁰) was formed by oxidation of bound sulfide, i.e. after the photoreduction of sulfate to sulfide by intact chloroplasts, and released as S₈ under the experimental conditions used for analysis.     

Microdissection and painting of the Y chromosome in spinach (Spinacia oleracea)

Spinach has long been used as a model for genetic and physiological studies of sex determination and expression. Although trisomic analysis from a cross between diploid and triploid plants identified the XY chromosome as the largest chromosome, no direct evidence has been provided to support this at the molecular level. In this study, the largest chromosomes of spinach from mitotic metaphase spreads were microdissected using glass needles. Degenerate oligonucleotide primed polymerase chain reaction was used to amplify the dissected chromosomes. The amplified products from the Y chromosome were identified using the male-specific marker T11A. For the first time, the largest spinach chromosome was confirmed to be a sex chromosome at the molecular level. PCR products from the isolated chromosomes were used in an in situ probe mixture for painting the Y chromosome. The fluorescence signals were mainly distributed on all chromosomes and four pair of weaker punctate fluorescence signal sites were observed on the terminal region of two pair of autosomes. These findings provide a foundation for the study of sex chromosome evolution in spinach.     

Chalcone synthases from spinach (Spinacia oleracea L.)

The two chalcone-synthase forms from leaves ofSpinacia oleracea L. were purified to apparent homogeneity. Antibodies were raised against both proteins in rabbits. The specificity of the antibodies was tested using immunotitration, immunoblotting, and immunoelectrophoresis techniques. The antibodies exhibited exclusive specificity for chalcone synthase and did not discriminate between the two antigens. The homodimeric chalcone synthases had the same subunit molecular weight but differed in their apparent native molecular weights. The peptide maps indicated extensive homology between the proteins. Chalcone-synthase activity was not detected in isolated spinach chloroplasts. Both enzyme forms were present in spinach cell-suspension cultures in which they were induced by light.Abbreviations DEAE diethylaminoethyl - DTE 1,4-dithioerythritol - EDTA ethylenediaminetetraacetic acid - HPLC high-performance liquid chromatography - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Parts of the results were presented at the 14^th International Botanical Congress at Berlin in July 1987     

Role of 5-aminolevulinic acid (ALA) on active oxygen-scavenging system in NaCl-treated spinach (Spinacia oleracea)

ALA is a key precursor in the biosynthesis of porphyrins such as chlorophyll and heme, and was found to induce temporary elevations in the photosynthesis rate, APX, and CAT; furthermore, treatment with ALA at a low concentration might be correlated to the increase of NaCl tolerance of spinach plants. The photosynthetic rate and the levels of active oxygen-scavenging system in the 3rd leaf of spinach (Spinacia oleracea) plants grown by foliar treatment with 0, 0.18, 0.60 and 1.80 mmol/L 5-aminolevulinic acid under 50 and 100 mmol/L NaCl were analyzed. Plants treated with 0.60 and 1.80 mmol/L ALA showed significant increases in the photosynthetic rate at 50 and 100 mmol/L NaCl, while that of 0.18 mmol/L ALA did not show any changes at 50 mmol/L NaCl and a gradual decrease at 100 mmol/L NaCl. In contrast, the rate with 0 mmol/L ALA showed reduction at both concentrations of NaCl. The increase of hydrogen peroxide content by treatment with 0.60 and 1.80 mmol/L ALA were more controlled than that of 0 mmol/L ALA under both NaCl conditions. These ALA-treated spinach leaves also exhibited a lower oxidized/reduced ascorbate acid ratio and a higher reduced/oxidized glutathione ratio than the 0 mmol/L-treated spinach leaves when grown at both NaCl conditions. With regard to the antioxidant enzyme activities in the leaves, ascorbate peroxidase, catalase, and glutathione reductase activities were enhanced remarkably, most notably at day 3, by treatment with 0.60 and 1.80 mmol/L ALA under both NaCl conditions in comparison to that of 0 and 0.18 mmol/L ALA. These data indicate that the protection against oxidative damage by higher levels of antioxidants and enzyme activities, and by a more active ascorbate-glutathione cycle related to the increase of the photosynthesis rate, could be involved in the increased salt tolerance observed in spinach by treatment with 0.60 to 1.80 mmol/L ALA with NaCl.     

Physiological rates of starch breakdown in isolated intact spinach chloroplasts

Starch breakdown with rates above 10 μatom carbon per mg chlorophyll per hour has been monitored in spinach chloroplasts and compares favorably with the rates in whole leaves. Intact starch-loaded chloroplasts were prepared from protoplasts to avoid rupture during mechanical homogenization and rapid centrifugation. Particular attention was paid to the identification of all the products of starch degradation and to measuring the actual rates of their accumulation. The products of starch breakdown included triose phosphate, 3-phosphoglycerate, CO₂, glucose, and some maltose. Comparison of the rates of metabolism of added glucose and of the conversion of starch to phosphorylated intermediates showed that starch phosphorolysis was the major pathway leading to phosphorylated endproducts. From the results, the relative contribution of phosphorolysis and hydrolysis to starch breakdown and the contribution of glycolysis and the oxidative pentose phosphate cycle can be estimated. Phosphate has a large influence on the metabolism of the chloroplast in the dark.     

Site of synthesis of geranylgeraniol derivatives in intact spinach chloroplasts

Chloroplasts isolated from fully developed spinach leaves and incubated in the presence of isopentenyl pyrophosphate were able to synthesize rapidly geranylgeranyl chlorophyll a and geranylgeraniol.The biosynthesis of the geranylgeraniol derivatives from isopentenyl pyrophosphate is a compartimentalized process. The membrane fractions (thylakoid and envelope membranes) were essentially unable to synthesize geranylgeraniol, geranylgeranyl pyrophosphate and geranylgeranyl chlorophyll a. When stromal and thylakoid fractions were combined the capacity to synthesize geranylgeranyl chlorophyll a and geranylgeraniol was restored. When stromal and envelope membrane fractions were combined the capacity to synthesize geranylgeranyl pyrophosphate and geranylgeraniol was restored. The products of the reaction were discharged inside the lipid phase of the membranes.     

The amino acid sequence of plastocyanin from spinach. (Spinacia oleracea L.).

The amino acid sequence of spinach (Spinacia oleracea L.) plastocyanin was determined. It consists of a single polypeptide chain of 99 residues and has a sequence molecular weight of 10415. The sequence was determined by using a Beckman 890C automatic sequencer and by the dansyl--phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. Overlap through the two methionine residues was not shown. Sedimentation equilibrium in the ultracentrifuge gave a molecular weight for spinach plastocyanin of about 9000, in contrast with the value of 21000 reported previously by Katoh et al. (1962).     

Purification and properties of glutathione synthetase from spinach (Spinacia oleracea) leaves

Glutathione synthetase (γ-l-glutamyl-l-cysteine:glycine ligase [ADP-forming], EC 6.3.2.3) was partially-purified (100-fold) from spinach (Spinacia oleracea) leaves and its properties determined. At least part of the enzyme activity is localized in chloroplasts. The properties of the enzyme suggest that GSH synthesis would be facilitated at the pH and Mg²⁺ concentration in the stroma of illuminated chloroplasts, but glutathione synthetase does not appear to be ‘light-activated’ in isolated type A chloroplasts.     

Photosynthetic oxygen reduction in isolated intact chloroplasts and cells in spinach

The time course of light-induced O(2) exchange by isolated intact chloroplasts and cells from spinach was determined under various conditions using isotopically labeled O(2) and a mass spectrometer. In dark-adapted chloroplasts and cells supplemented with saturating amounts of bicarbonate, O(2) evolution began immediately upon illumination. However, this initial rate of O(2) evolution was counterbalanced by a simultaneous increase in the rate of O(2) uptake, so that little net O(2) was evolved or consumed during the first approximately 1 minute of illumination. After this induction (lag) phase, the rate of O(2) evolution increased 3- to 4-fold while the rate of O(2) uptake diminished to a very low level. Inhibition of the Calvin cycle, e.g. with dl-glyceraldehyde or iodoacetamide, had negligible effects on the initial rate of O(2) evolution or O(2) uptake; both rates were sutained for several minutes, and about balanced so that no net O(2) was produced. Uncouplers had an effect similar to that observed with Calvin cycle inhibitors, except that rates of O(2) evolution and photoreduction were stimulated 40 to 50%.These results suggest that higher plant phostosynthetic preparations which retain the ability to reduce CO(2) also have a significant capacity to photoreduce O(2). With near-saturating light and sufficient CO(2), O(2) reduction appears to take place primarily via a direct interaction between O(2) and reduced electron transport carriers, and occurs principally when CO(2)-fixation reactions are suboptimal, e.g. during induction or in the presence of Calvin cycle inhibitors. The inherent maximum endogenous rate of O(2) reduction is approximately 25 to 50% of the maximum rate of noncyclic electron transport coupled to CO(2) fixation. Although the photoreduction of O(2) is coupled to ion transport and/or phosphorylation, this process does not appear to supply significant amounts of ATP directly during steady-state CO(2) fixation in strong light.