Photosynthesis of Lipids from CO(2) in Spinacia oleracea

Young expanding spinach leaves exposed to ¹⁴CO₂ under physiological conditions for up to 20 minutes assimilated CO₂ into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the ¹⁴C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO₂in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H¹⁴CO₃ into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor.     

Evidence for the Glutamine Synthetase/Glutamate Synthase Pathway during the Photorespiratory Nitrogen Cycle in Spinach Leaves

Spinach leaf (Spinacia oleracea L.) discs infiltrated with [¹⁵N]glycine were incubated at 25°C in the light and in darkness for 0, 30, 60 and 90 minutes. The kinetics of ¹⁵N-incorporation into glutamine, glutamate, asparagine, aspartate, and serine from [¹⁵N]glycine was determined. At the beginning of the experiment, just after infiltration (0 min incubation) serine, and the amido-N of glutamine and asparagine were the only compounds significantly labeled in both light- and dark-treated leaf discs. Incorporation of ¹⁵N-label into the other amino acids was observed at longer incubation time. The per cent ¹⁵N-enrichment in all amino acids was found to increase with incubation. However, serine and the amido-N of glutamine remained the most highly labeled products in all treatments. The above pattern of ¹⁵N-labeling suggests that glutamine synthetase was involved in the initial refixation of ¹⁵NH₃ derived from [¹⁵N]glycine oxidation in spinach leaf discs.

The ¹⁵N-enrichment of the amino-N of glutamine was found to increase rapidly from 0 to 19% during incubation in the light. There was a comparatively smaller increase (4-9%) in the ¹⁵N-label of the amino-N of glutamine in tissue incubated in darkness. Furthermore the total flux of ¹⁵N-label into each of the amino acids examined was found to be greater in tissue incubated in the light than those in the dark. The above evidence indicates the involvement of the glutamine synthetase/glutamate synthase pathway in the recycling of photorespiratory NH₃ during glycine oxidation in spinach leaves.

     

Accumulation of Maltose during Photosynthesis in Protoplasts Isolated from Spinach Leaves Treated with Mannose

When mannose was included in the enzyme incubation medium during the preparation of protoplasts from leaves of spinach, maltose was an early product of protoplast photosynthesis and, after 12 minutes, accounted for up to 15% of the ¹⁴C incorporated from ¹⁴CO₂. Maltose was not detected in protoplasts prepared in the normal enzyme medium. Rapid separation of cytoplasm and chloroplasts following exposure to ¹⁴CO₂ showed that maltose was present in both fractions. Direct measurements of [¹⁴C]maltose uptake indicated transport across the chloroplast envelope at rates similar to the transport of glucose. The source of maltose and site of its initial formation are discussed.     

Effect of BASF 13-338, a Substituted Pyridazinone, on Lipid Metabolism in Leaf Tissue of Spinach, Pea, Linseed, and Wheat

A substituted pyridazinone (BASF 13-338) inhibited photosynthesis in spinach (Spinacia oleracea, Hybrid 102 Arthur Yates Ltd.) leaf discs and reduced the incorporation of [1-¹⁴C]acetate into trienoic acids of diacylgalactosylglycerol while causing radioactivity to accumulate in diacylgalac-tosylglycerol dienoic acids. Although BASF 13-338 inhibited photosynthesis in isolated spinach chloroplasts, it did not prevent dienoate desaturation. In discs, the labeling of fatty acids was affected by the inhibitor only in diacylgalactosylglycerol. Very little radioactivity was incorporated into trienes of phosphatidylcholine and the proportion of the label recovered in the fatty acids of phosphatidylcholine was not changed by BASF 13-338. The herbicides caused an increase in the proportion of the lipid ¹⁴C incorporated into diacylgalactosylglycerol and a decrease in labeling of phosphatidylcholine, whereas the proportion of ¹⁴C recovered in other lipids remained unchanged. Similar results were obtained with pea (Pisum sativum cv. Victory Freeze), linseed (Linum usitatissimum cv. Punjab), and wheat (Triticum aestivum cv. Karamu). With these species, a greater proportion of the label was incorporated into phosphatidylcholine and less into diacylgalactosylglycerol than with spinach. The data indicate that trienoate synthesis uses diacylgalactosylglycerol as substrate. BASF 13-338 appears to act at that step, and seems to cause in spinach a shift in polyenoate synthesis from the pathway involving microsomal phosphatidylcholine to the pathway operating inside the chloroplast.     

Biosynthesis of ribulose-1,5-bisphosphate carboxylase in spinach leaf protoplasts

Spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts sustain protein-synthesizing activity as measured by the incorporation of [¹⁴C]-leucine into the protein fraction both in the light and in the dark. By the immunoprecipitation of ribulose-1,5-bisphosphate (RuP₂) carboxylase with rabbit antibody raised against the purified spinach enzyme preparation, it was found that approximately 7% of the total radiocarbon incorporated into the protein fraction in the light was in the carboxylase molecules. However, there was no measurable net increase observed in the content of the enzyme protein in the experimental conditions employed. It was found that both chloramphenicol and cycloheximide inhibited the incorporation of [¹⁴C]leucine into RuP₂ carboxylase and its constituent subunits, as measured by the immunoprecipitation of the enzyme molecule and its subunits, A and B.     

Uptake and retention of external solutes from the digest medium during preparation of protoplasts

The extent to which solutes present in the digest medium enter cells and are retained during preparation of protoplasts was investigated. When barley (Hordeum vulgare, L. cv. Clipper) leaf slices were incubated in sorbitol there was considerable uptake of sorbitol into the tissue, which continued for up to 6 h and was dependent on the sorbitol concentration in the external medium. Protoplasts prepared by digesting leaf slices in a medium containing [¹⁴C]sorbitol but isolated and purified in media with unlabelled sorbitol contained significant amounts of [¹⁴C]sorbitol. From measurements of the protoplast volume, the internal sorbitol concentration was calculated to be 100 mM, assuming uniform distribution of the sorbitol throughout the protoplasm. The uptake of sorbitol during digestion and its retention by protoplasts was confirmed by measuring sugars in protoplast extracts by gas sucrose or inositol. Vacuoles prepared from the protoplasts contained 83% of the sorbitol present in protoplasts. It is concluded that considerable uptake of solutes from the external medium occurs during digestion of leaf tissue and that these solutes are retained within the protoplasts during isolation and purification. The solutes appear to be uniformly distributed throughout the subcellular compartments of the protoplast.     

A simple method for estimating intactness of spinach leaf protoplasts by glycolate oxidase assay

A method was developed for the quantitative analysis of intactness of spinach leaf protoplasts using glycolate oxidase activity as an index. Since glycolate does not penetrate into protoplasts at neutral pH, the increase of O₂ consumption by the addition of glycolate to protoplast suspension was due to the glycolate oxidase activity released from damaged protoplasts. The proportion of damaged protoplasts in the whole preparation was calculated from the ratio of released and total glycolate oxidase activity. Freshly prepared spinach leaf protoplasts were found to be 80 to 90% intact as estimated by the method. The effect of osmolarity on the respiratory activities of spinach leaf protoplasts was also examined by applying the same principle.     

CO(2) Fixation in Opuntia Roots

Nonautotrophic CO₂ metabolism in Opuntia echinocarpa roots was studied with techniques of manometry and radiometry. The roots were grown in a one-quarter strength nutrient solution for several days; the distal 2 cm was used for physiological studies. The roots assimilated significant quantities of ¹⁴CO₂ and appeared to show a crassulacean-type acid metabolism with respect to quality and quantity. Most of the ¹⁴C activity was associated with the distal portion of the elongating root indicating correlation with metabolic activity. The ¹⁴CO₂ assimilation was comparable to a crassulacean leaf succulent, but 3 times greater than that found for stem tissue of the same Opuntia species.

The rates of O₂ and CO₂ exchange and estimated CO₂ fixation were 180, 123, and 57 μl/g per hour. A respiratory quotient of 0.66 was found.

The products of ¹⁴CO₂ fixation were similar in most respects to reported experiments with leaf succulents. Equilibration of the predominant malic acid with isocitric, succinic, and fumaric acids was not evident. The latter observation was interpreted as metabolic isolation of the fixation products rather than poor citric acid cycle activity.

A rapid turnover of the fixed ¹⁴CO₂ was measured by following decarboxlyation kinetics and by product analysis after a postincubation period. The first order rate constant for the steady state release was 4.4 × 10⁻³ min⁻¹ with a half-time of 157.5 minutes. Amino acids decayed at a more rapid rate than organic acids.

     

In-situ determination of intracellular concentrations of K+ in barley (Hordeum vulgare L. cv. Kara) using the K+-binding fluorescent dye benzofuran isophthalate

The tetra[acetoxymethyl] ester of the K⁺-binding fluorescent dye benzofuran isophthalate (PBFI-AM) was used to determine changes in intracellular potassium (K⁺) concentrations and to measure net transport of K⁺ in barley (Hordeum vulgare L. cv. Kara) root and leaf protoplasts. When this dye binds to free K⁺ inside the cytoplasm, the fluorescence intensity ratio 340/380 nm increases in direct relation to the K⁺ concentration. Because of a delay in the uptake of dye into the vacuoles, it is possible to determine K⁺ concentrations in the vacuoles and transport of K⁺ from the cytoplasm into the vacuole. The uptake of PBFI-AM in root and leaf protoplasts of barley differed in the absence or presence of external K⁺ and was faster at pH 5.5 than at pH 7.0. The fluorescence intensity of the dye was stable for at least 20 h when the protoplasts were kept at 4°C. In the presence of nigericin, the fluorescence intensity of both cells and protoplasts was linearly related to the external concentration of K⁺ (up to 100 mM).     

In vivo biosynthesis of -linolenic acid in plants

[1-¹⁴C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of $\text{Chlorella}\text{pyrenoidosa}$ and $\text{Candida}\text{bogoriensis}$. In these systems the [¹⁴C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C_1–9 and the C_10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [¹⁴C] was localized in the C_1–9 fragment. While [1-¹⁴C]oleic acid was converted by whole cells of $\text{Chlorella}$ to [1-¹⁴C]linoleic and [1-¹⁴C]linolenic acids, [U-¹⁴C]oleic acid yielded [U-¹⁴C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid.     

Elongation Pathway for alpha-Linolenic Acid Synthesis in Spinach Leaves: A Reexamination

Leaf slices from spinach exhibited considerable variation in their incorporation of [¹⁴C]bicarbonate and [1-¹⁴C]acetate into fatty acids. Reductive ozonolysis studies indicated that all the ¹⁴C-labeled fatty acids were synthesized de novo; no in vivo evidence was found for the previously proposed elongation of hexadecatrienoate to α-linolenate.     

Transport of anions in isolated barley vacuoles : I. Permeability to anions and evidence for a cl-uptake system

The anion contents of young barley leaves and of mesophyll protoplasts from the leaves was compared. Anion loss from the protoplasts during isolation was small. Although only about 60% of the leaf cells were mesophyll cells, phosphate and sulfate contents of the mesophyll cells accounted for almost 90% of the leaf contents. Chloride accumulated in the leaf epidermis. The rapid isolation of vacuoles from mesophyll protoplasts permitted the determination of vacuolar ion concentrations. Sodium and nitrate levels were very low in the cytoplasm, and much higher in the vacuole. When barley plants were grown in the presence of low NaCl levels, chloride concentrations were comparable in cytoplasm and vacuole, and similar observations were made with sulfate. Cytoplasmic phosphate concentrations were close to 30 millimolar and potassium concentrations 100 millimolar. During a 30 minute incubation period at room temperature, anion contents of isolated vacuoles decreased considerably. Efflux of NO₃⁻ was faster than that of Cl⁻. Phosphate and sulfate crossed the tonoplast only slowly. 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid partially inhibited the efflux of nitrate and, to a lesser extent, that of chloride. Decreased efflux was also observed in the presence of MgATP. In remarkable contrast, p-chloromercuribenzene sulfonate and HgCl₂ stimulated the efflux of nitrate and chloride, but not of phosphate. Labeled chloride was taken up by isolated vacuoles. The apparent K_m for chloride uptake at low chloride concentrations was 2.3 millimolar. At elevated chloride concentrations, chloride did not display saturation characteristics but, rather, characteristics of a diffusional process. Uptake was stimulated by ATP.     

The Metabolism of the Germinating Oil Palm (Elaeis guineensis) Seedling : In Vivo Studies

The metabolism of ¹⁴C-labeled fatty acids and triacylglycerols was followed in intact germinating oil palm seedlings as well as in tissue slices. In the germinating seedling, the shoot contained a normal pattern of membrane fatty acids (mainly C₁₆, C_18:1, C_18:2) but the kernel contained about 68% C₁₂ and C₁₄ fatty acids. Haustorium fatty acids were intermediate between the two. [¹⁴C]Acetate was actively metabolized by shoot and haustorium slices but not so actively by the kernel. Approximately 9% to 17% was converted to water-soluble substances, 4% to 6% to CO₂, and 0.5% to 5.9% to lipids. The fatty acids synthesized in the shoot and haustorium were mainly C₁₆, C₁₈, and C_18:1 fatty acids but in the kernel about 18% to 32% of the ¹⁴C-fatty acids were C₁₂ fatty acids.

[¹⁴C]Lauric acid was absorbed and metabolized by haustorium slices and by the haustorium in intact seedlings; it was partly esterified to triacylglycerols and also converted to water-soluble substances and insoluble tissue material. In contrast, tri-[¹⁴C]laurin was absorbed but not metabolized. The haustorium also absorbed other fatty acids but the longer chain (C₁₆ and C₁₈) fatty acids were not esterified or metabolized further. Preincubation of the haustorium with plant hormones or in the presence of kernel tissue did not alter its inactivity towards tri-[¹⁴C]laurin.

When tri-[¹⁴C]laurin or [¹⁴C]lauric acid were injected into the seed or the shoot, there was no movement or radioactivity to other parts of the seedling. When injected into the shoot, but not into the seed, tri-[¹⁴C] laurin was hydrolyzed and partly metabolized to water-soluble substances.

     

Comparison of acetate- and pyruvate-dependent fatty-acid synthesis by spinach chloroplasts

In recent studies using intact chloroplasts of spinach (Spinacia oleracea L.) to investigate the accumulation of acetyl-CoA produced by the activity of either acetyl-CoA synthetase (EC 6.2.1.1) or the pyruvate-dehydrogenase complex, this product was not detectable. These results in combination with new information on the physiological levels of acetate and pyruvate in spinach chloroplasts (H.-J. Treede et al. 1986, Z. Naturforsch. 41 C, 733–740) prompted a reinvestigation of the incorporation of [1-¹⁴C] acetate and [2-¹⁴C] pyruvate into fatty acids at physiological concentrations.The K _m for the incorporation into fatty acids was about 0.1 mM for both metabolites and thus agreed with the values obtained by H.-J. Treede et al. (1986) for acetyl-CoA synthetase and the pyruvate dehydrogenase complex. However, acetate was incorporated with a threefold higher V _max. Saturation for pyruvate incorporation into the fattyacid fraction was achieved only at physiological pyruvate concentrations (<1.0 mM). The diffusion kinetics observed at higher concentrations may be the result of contamination with derivates of the labeled substrate. Competition as well as double-labeling experiments with [³H]acetate and [2-¹⁴C]pyruvate support the notion that, at least in spinach, chloroplastic acetate is the preferred substrate for fatty-acid synthesis when both substrates are supplied concurrently (P.G. Roughan et al., 1979 b, Biochem. J. 184, 565–569).Experiments with spinach leaf discs confirmed the predominance of fatty-acid incorporation from acetate. Radioactivity from [1-¹⁴C]acetate appeared to accumulate in glycerolipids while that from [2-¹⁴C]pyruvate was apparently shifted in favor of the products of prenyl metabolism.Abbreviations Chl chlorophyll - TLC thin-layer chromatography     

Transport and subcellular localization of polyamines in carrot protoplasts and vacuoles

Putrescine and spermidine uptake in carrot (Daucus carota L., cv “Tip top”) protoplasts and isolated vacuoles was studied. Protoplasts and vacuoles accumulated polyamines very quickly, with maximum absorption within 1 to 2 minutes. The insertion of a washing layer containing 100 millimolar unlabeled putrescine or spermidine did not change this pattern, but strongly reduced the uptake of putrescine and spermidine in protoplasts and in vacuoles. The dependence of spermidine uptake on the external concentration was linear up to the highest concentrations tested in protoplasts, while that in vacuoles showed saturation kinetics below 1 millimolar (K_m = 61.8 micromolar) and a linear component from 1 to 50 millimolar. Spermidine uptake in protoplasts increased linearly between pH 5.5 and 7.0, while there was a distinct optimum at pH 7.0 for vacuoles. Preincubation of protoplasts with 1 millimolar Ca²⁺ affected only surface binding but not transport into the cells. Nonpermeant polycations such as La³⁺ and polylysine inhibited spermidine uptake into protoplasts. Compartmentation studies showed that putrescine and spermidine were partly vacuolar in location and that exogenously applied spermidine could be recovered inside the cells. The characteristics of the protoplast and vacuolar uptake system induce us to put forward the hypothesis of a passive influx of polyamines through the plasmalemma and of the presence of a carrier-mediated transport system localized in the tonoplast.     

Effect of Zn deficiency on net photosynthetic rate, 14C partitioning,and oil accumulation in leaves of peppermint

Changes in growth, CO₂ exchange rate, and distribution of photosynthetically fixed ¹⁴CO2 into the primary photosynthetic metabolic pool (sugars, amino acids and organic acids) and essential oil accumulation were determined in leaves (leaf positions 1-6 from apex) of developing peppermint grown in a solution culture at Zn concentrations of 0 and 0.05 g m-3. There was a significant decrease in ¹⁴C incorporation in total, ethanol-soluble and ethanol-insoluble fractions in Zn deficient plants at all leaf positions. 14C incorporated in essential oil and in sugars were significantly higher in leaf pairs 1 to 3 than in leaf pairs 4 to 6. ¹⁴C incorporation into amino acids and organic acids was higher in all leaf pairs in Zn deficient plants. Statistical analysis showed a positive significant association between Zn content of leaf and ¹⁴C incorporation into ethanol-soluble fraction and sugars and a negative correlation with ¹⁴C incorporation into amino acids and organic acids. Hence the content of sugars in leaves significantly influences essential oil accumulation under Zn stress. This revised version was published online in September 2006 with corrections to the Cover Date.     

Manipulating the incorporation of [1-C]acetate into different leaf glycerolipids in several plant species

During short term labeling of expanding leaves of seven plant species with [1-¹⁴C]acetate, 35 to 64% of the label incorporated into lipids was found in phosphatidylcholine and 5 to 24% in phosphatidylglycerol. In pumpkin, sunflower, broad bean, and maize, only 4 to 12% of the label was found in diacylgalactosylglycerol, but in tomato, parsley, and spinach, the proportion was 17 to 31%. The latter group was further distinguished by having diacylgalactosylglycerol containing C16:3.

The proportions of total incorporated [1-¹⁴C]acetate entering the lipids could be manipulated in a predictable manner. Phosphatidylcholine labeling was depressed by treating intact leaves with glycerol or ethylene glycol monomethyl ether or by incubating leaf discs in vitro. An associated increase in phosphatidylglycerol labeling occurred within the first group of plants, whereas an increase in labeling of either diacylgalactosylglycerol, phosphatidylglycerol, or sulfolipid occurred within the second group. Treating intact leaves with glycerol or incubating leaf discs in vitro was shown to elevate cellular concentrations of sn-glycerol 3-phosphate.

These results have been interpreted in terms of the two-pathway hypothesis for glycerolipid biosynthesis, in which it is proposed that phosphatidylcholine is synthesized via a different pathway (eukaryotic) to that for synthesis of phosphatidylglycerol (prokaryotic). Both pathways may contribute toward the synthesis of diacylgalactosylglycerol, with the contribution of each being assessed from the proportion of hexadecatrienoic acid found in the particular plant.

     

Membrane transport of sugars and amino acids in isolated protoplasts

A method has been developed for observing membrane transport in isolated protoplasts. Transport of sugars and amino acids has been studied in protoplasts isolated from the mesophyll of Pisum sativum L. That uptake was not due to passive diffusion through damaged membranes was demonstrated by supplying simultaneously two sugar stereoisomers, the one ³H-labeled and the other ¹⁴C-labeled. The protoplast membranes were sufficiently functional to discriminate strongly between these stereoisomers.

To characterize transport the nonmetabolized glucose analogue 3-O-methyl glucose (MeG) and amino acid analogue α-aminoisobutyric acid (AIB) were employed. When uptake was compared per unit of protein as between leaf strips and protoplasts prepared from the same tissue, it was estimated that the protoplasts had retained approximately 40 to 50% of the uptake ability of the whole cells. Uptake of neither MeG nor AIB by protoplasts was linear with time, but the tendency to flatten was more marked for AIB. Addition of Mg-ATP to buffered medium significantly promoted AIB uptake, an effect not ascribable to either chelation or pH. Transport of both MeG and AIB was markedly pH-dependent, uptake falling with rise in pH.

The stimulatory effect of Mg-ATP and the pH dependence confirm that uptake was not due to a diffusional inward “leak” but involved membrane function.

This work demonstrates the feasibility of using isolated protoplasts for membrane transport studies. The potential advantages of using protoplasts for such studies are pointed out.

     

Utilization of Photosynthetically Fixed 14CO2 into Alkaloids in Relation to Primary Metabolites in Developing Leaves of Catharanthus roseus

Partitioning of current photosynthates towards primary metabolites and its simultaneous incorporation in leaf alkaloids was investigated in developing leaves of medicinally important Catharanthus roseus. Of the total ¹⁴CO₂ assimilated, the leaves at positions 1–6 fixed 8, 22, 25, 19, 13, and 8 %, respectively, and stem 3 %. Leaf fresh mass, chlorophyll content, and CO₂ exchange rate increased up to the third leaf. The total alkaloid content was highest in young actively growing leaves, which declined with age. Total ¹⁴C fixed and its content in ethanol soluble fraction increased up to the third leaf and then declined. The ¹⁴C content in primary metabolites such as sugars and organic acids was also highest in the 3^rd leaf. The utilization of ¹⁴C assimilates into alkaloids was maximum in youngest leaf which declined with leaf age. Hence the capacity to synthesize alkaloids was highest in young growing leaves and metabolites from photosynthetic pathway were most efficiently utilized and incorporated into alkaloid biosynthetic pathway by young growing leaves.     

Transfer of fatty acids and proteins between cytoplasmic membranes and mesosomes of Bacillus subtilis var. niger

A transfer of labelled branched-chain fatty acids or proteins between mesosomes, periplasmic space and protoplasts, is suggested in vivo and demonstrated in vitro.In the sole mesosomal fraction, [¹⁴C]valine or [¹⁴C]isoleucine are, always, more incorporated in fatty acids than in proteins.Mesosomal fatty acids can be transferred to the protoplasts, but protoplasts seems to give essentially amino acids to mesosomes.