Lipid composition of chloroplasts isolated by aqueous and nonaqueous techniques

Chloroplasts isolated from tobacco leaves in 0.5 M sucrose solution (the 1000 g pellet) contained 83% of the total cellular monogalactosyl diglyceride, 88% of the digalactosyl diglyceride, 76% of the sulfolipid, and 74% of the phosphatidyl glycerol. Phosphatidyl inositol was concentrated in the 15,000 g pellet. Phosphatidyl choline and phosphatidyl ethanolamine were concentrated in the 15,000 g supernatant fraction. Chloroplasts isolated from tobacco leaves by a nonaqueous technique in hexane-carbon tetrachloride show a glycerolipid composition similar to that found in chloroplasts isolated in the aqueous system, even though some lipid, particularly monogalactosyl diglyceride, is extracted by the organic solvent during the process.     

Role of Galactolipids in Spinach Chloroplast Lamellar Membranes: II. Effects of Galactolipid Depletion on Phosphorylation and Electron Flow

A galactolipid lipase from primary bean (Phaseolus vulgaris) leaves has been used to partially deplete spinach chloroplast inner membranes of their galactolipids. Chloroplasts treated with the lipase in the absence of bovine serum albumin lost 91% of their monogalactosyl diglyceride, 83% of their digalactosyl diglyceride, all of their phosphatidyl choline, but none of their sulfolipid. Electron microscopy of this sections revealed that the treated chloroplasts were greatly enlarged and lacked membrane stacking. Linolenic acid had similar effects on the structure of the chloroplasts. Chlorophyll, carotenoids, and coupling factor 1 remained bound to the treated membranes.     

Age dependent changes in phospholipids and galactolipids in primary bean leaves (Phaseolus vulgaris)

Primary leaves of Phaseolus vulgaris show concomitant changes in phospholipid, galactolipid, chlorophyll and fresh weight during leaf development from 3 to 32 days after planting. Phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol show only small changes on a mole per cent lipid phosphate basis during leaf development. The chloroplast lipids, phosphatidyl glycerol, monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) all show marked increases and decreases which are coincident with chloroplast development. The decline in the leaf content of chloroplast polar lipids and chlorophyll become evident upon reaching maximal leaf size. The molar ratio of galactolipids (MGDG/DGDG), reaches a maximum value of 2.3 in expanding leaves, but steadily declines during senescence to a minimum value of 1.5 at abscission. The declining ratio is caused by a preferential loss of MGDG in the senescing leaves.     

Differences in Lipid Composition between Undifferentiated and Mature Maize Chloroplasts

Lipid compositions of undifferentiated maize (Zea mays) chloroplasts, capable of fixing CO₂, were compared with the lipid compositions of mature chloroplasts, which do not fix CO₂, located in both the mesophyll and bundle sheath cells. The major lipids found in all three chloroplast types were the glycolipids, monogalactosyl diglyceride and digalactosyl diglyceride, followed by decreasing amounts of sulfolipid, phosphatidyl glycerol, phosphatidyl choline, phosphatidyl inositol, and diphosphatidyl glycerol. Quantitative differences in lipid components were observed among the chloroplast types. The mesophyll and bundle sheath maize chloroplasts differed in their chlorophyll a/chlorophyll b ratios (2.27 and 4.13 respectively) and their content of glycolipid relative to chlorophyll (51.8% glycolipid to 20.9% chlorophyll and 84.5% glycolipid to 10.1% chlorophyll respectively). A comparison between the lipid compositions of maize mesophyll chloroplasts and mesophyll chloroplasts obtained from spinach, sugar beet, and tobacco showed many similarities.     

Lipids of ripening tomato fruit and its mitochondrial fraction

The lipid composition of tomato fruit and its mitochondrial fraction were examined at various stages of fruit ripeness. Phosphatidyl choline, phosphatidyl ethanolamine, monogalactosyl diglyceride, digalactosyl diglyceride and phosphatidyl inositol were found to be the major lipids of tomato pericarp at all stages of ripeness. Mitochondrial lipids resembled those of the parent tissue except for the absence of monogalactosyl diglyceride and a greater percentage of diphosphatidyl glycerol and phosphatidic acid. Changes in the lipid-protein ratio of mitochondria were noted with ripening.     

Changes in Glycolipid and Phospholipid Compositions in Cotyledons of Germinating Soybeans

This investigation was conducted to observe changes in the compositions of fatty acids, glycolipids (GL) and phospholipids (PL) in cotyledons of soybean seeds which were germinated either in the dark or the light at 28°C for 8 days. The patterns of changes in lipid composition depended on the germinating conditions tested. In general, non-polar lipids were metabolized at a faster rate than polar lipids. Changes in lipid contents in cotyledons were also observed more clearly with the polar lipids than with the non-polar ones, especially in the light-grown seedlings. The major component of lipid, GL in chloroplasts, appeared rapidly at an earlier stage in the cotyledons of light-grown seedlings. During germination of soybean seeds, acyl sterylglucoside in cotyledons decreased rapidly, but monogalactosyl diglyceride and digalactosyl diglyceride (DGD) increased in the light-grown seedlings, whereas sterylglucoside and DGD increased in the dark-grown seedlings.

The major PL present immediately after immersion were phosphatidyl ethanolamine (PE), phosphatidyl choline (PC) and phosphatidyl inositol (PI). During germination under both conditions, light and dark, PE in cotyledons decreased with PC or PI, while phosphatidic acid increased rapidly, and phosphatidyl glycerol and diphosphatidyl glycerol also increased slightly. These changes in glycolipid and phospholipid compositions during germination seem to occur from the formation of photosynthetic tissues and the metabolic interconversion of phospholipids.     

Sequential changes in the lipids of developing proplastids isolated from green maize leaves

Changes in lipid composition were followed as a proplastid develops into a chloroplast. Methods were devised for the isolation of developing proplastids from sections of five different ages from the same 7-day-old maize (Zea mays var. Kelvedon Glory) leaf. Electron micrographs illustrate the homogeneity of the five types of plastid suspension, minimal contamination with other cytoplasmic membranes, and the presence of morphologically intact plastids in the proportions 85% (youngest), 85%, 80%, 70% and 60% (oldest), respectively. Both bundle sheath and mesophyll plastids are well preserved in isolation. Plastid numbers were determined from calibration curves of the chlorophyll content of each type of suspension, and lipid values then expressed as nmoles/10⁶ plastids. Monogalactosyl diglyceride (MGDG), digalactosyl diglyceride (DGDG), sulfoquinovosyl diglyceride, and phosphatidyl glycerol (PG) all increase during plastid development but the rate of increase is different for each lipid. The largest changes are in MGDG (6-fold) and DGDG (4-fold). Phosphatidyl choline shows a continuous decline during plastid development. Phosphatidyl inositol and phosphatidyl ethanolamine were found in all the suspensions in low concentrations (0.4-4.0% of total lipid): calculations showed their presence could not be accounted for by bacterial or mitochondrial contamination. The increase in PG parallels the chlorophyll changes during development and at maturity 1 molecule of PG is present per 3 molecules of chlorophyll. The results are discussed in the context of the molecular structure of the photosynthetic thylakoid membranes.     

Polygalactolipids in Spinach Chloroplast

Two polygalactolipids, designated as components A and B, were isolated from spinach chloroplasts and were also obtained from glycolipid products synthesized with chloroplast enzymes using uridine diphosphate galactose as a galactose donor. These lipids were purified by column and thin layer chromatography. Chemical analysis of component A indicates that the lipid is trigalactosyl diglyceride, whereas component B behaves like tetragalactosyl diglyceride on a thin layer plate. The major fatty acid in trigalactosyl diglyceride was alpha-linolenic acid. Relative amount (molar ratio) of galactolipids in spinach chloroplasts was monogalactosyl diglyceride:digalactosyl diglyceride:trigalactosyl diglyceride:(tetragalactosyl diglyceride) = 60:30:5:1.     

Phosphoglycerides of Trichophyton terrestre and One Phenotype Selected from the Apollo 16 Microbial Ecology Evaluation Device

Total lipid extracted from wild-type Trichophyton terrestre CDC-X285 was found to be 2.0 percent of the dry cell weight. The total lipid contained the following phospholipid components identified by silicic acid-impregnated thin-layer and paper chromatography: phosphatidyl inositol, phosphatidyl choline, phosphatidyl serine, and phosphatidic acid. The total lipid extracted from the phenotype T. terrestre 7048-1 isolated from the Apollo 16 Microbial Ecology Evaluation Device (MEED) was found to vary according to the time at which the phospholipids were extracted. The Trichophyton phenotype was selected from a cuvette housed in the MEED exposed to specific space parameters including ultraviolet light of known wavelengths and energy levels in deep space. The phospholipid components, identified in the phenotype were phosphatidyl ethanolamine and cardiolipin. The major lipid fraction was composed of digalactosyl diglyceride and monogalactosyl diglyceride. An unusual lipid was detected in the phenotype, which appeared to be sterol glycoside.     

Lipids of Chlamydomonas reinhardi under different growth conditions

Photo-, mixo- and heterotrophically grown cultures of Chlamydomonas reinhardi (wild type ss and 2 streptomycin-resistant mutants sr₃ and sr₃₅) have been analyzed for lipids and fatty acids. Ether-soluble lipids, chlorophyll, monogalactosyl diglyceride, digalactosyl diglyceride, sulfolipid, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl glycerol and the relative amounts of fatty acids in total and individual lipids have been determined. The lipid and fatty acid compositions are very similar in the 3 strains and are not affected by the mutations. Fatty acids belong exclusively to the C₁₆ and C₁₈ series, 16:0, 16:4, 18:1, 18:2, 18:3 (6,9,12) and 18:3 (9,12,15) comprising about 90% of the total. 18:3 (6,9,12) is concentrated in phosphatidyl ethanolamine. In streptomycin-bleached sr₃ cells, ether-soluble lipids increase from 7 to 11% of dry weight on greening, mostly due to synthesis of monogalactosyl diglyceride and chlorophyll. Monogalactosyl diglyceride of bleached cells exhibits the same fatty acid pattern before and after greening.     

Total polar lipid biosynthesis during growth of Crotalaria juncea pollen tubes

[1-¹⁴C]-Acetate incorporation into total and polar lipids was studied in the growing pollen tubes of Crotalaria juncea. Ungerminated pollen had phosphatidyl inositol, phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, monogalactosyl diglyceride, digalactosyl diglyceride, sulpholipid and steryl glycosides. In the growing pollen tubes considerable [1-¹⁴C]-acetate incorporation was observed into the individual polar lipids. The exogenous carbon source significantly influenced lipid biosynthesis. Boric acid (20mg/l.) promoted both pollen tube growth and acetate incorporation into phospholipids. In comparison to 5′-adenosine monophosphate, cyclic-3′,5′-adenosine monophosphate (cAMP) promoted tube growth and also enhanced phospho-and glycolipid biosynthesis. The regulation of membrane component biosynthesis by cAMP is suggested.     

Lipid Labeling in Normal and Nitrogen-Deficient Squash (Cucurbita maxima) Leaves

Squash (Cucurbita maxima Duchesne) plants were grown on normal and on nitrogen-deficient nutrients. The degrees of label incorporation into chloroplast lipids as well as non-chloroplast lipids were determined. Nitrogen-deficient tissues contain less chlorophyll, have a decreased chlorophyll a/b ratio, incorporate more label into phosphatidyl choline and phosphatidyl ethanolamine than into the chloroplast lipids such as mono- and digalactosyl diglycerides, have a reduced capacity to incorporate the hexose moieties into the glycolipids but normal capacity to incorporate bases into the phospholipids of non-chloroplast constituents, and have a normal level of total fatty acids even though the level of linolenate is decreased. All of this would suggest that the most evident changes in membrane lipid constituents during nitrogen-deficiency occur as changes in the chloroplast lipid constituents as opposed to the non-chloroplast lipid constituents.     

Lipid composition of cyanidium

The major lipids in Cyanidium caldarium Geitler are monogalactosyl diglyceride, digalactosyl diglyceride, plant sulfolipid, lecithin, phosphatidyl glycerol, phosphatidyl inositol, and phosphatidyl ethanolamine. Fatty acid composition varies appreciably among the lipids, but the major ones are palmitic acid, oleic acid, linoleic acid, and moderate amounts of stearic acid. Trace amounts of other acids in the C₁₄ to C₂₀ range were also present. Moderate amounts of linolenic acid were found in two strains, but not in a third. The proportion of saturated acid is relatively high in all lipids ranging from about a third in monogalactosyl diglyceride to three-fourths in sulfolipid. This may be a result of the high growth temperature. Lipases forming lysosulfolipid, and lysophosphatidyl glycerol are active in ruptured cells; galactolipid is degraded with loss of both acyl residues. Thus the lipid and fatty acid composition of Cyanidium more closely resembles that of green algae than that of the blue-green algae, although there are differences of possible phylogenetic interest.     

Fatty-acid composition of polar lipids in fruit and leaf chloroplasts of “16:3”- and “18:3”-plant species

The fatty-acid composition of polar lipids from fruit and leaf chloroplasts was compared in five Solanaceous and two cucurbit species. The acylated fatty acids in monogalactosyl diglycerides (MGDG) from leaf chloroplasts of all five Solanaceous species included substantial amounts of ^7,10,13-hexadecatrienoic acid (16:3). In contrast, the MGDG from fruit chloroplasts of the Solanaceae contained very little of this plastid-specific polyunsaturate, and instead included a proportionately greater percentage of linoleic acid (18:2). In MGDG from leaf chloroplasts of two cucurbits, -linolenic acid (18:3) constituted 94–95% of the acylated fatty acids. Fruit-chloroplast galactolipids of the cucurbits had a greater abundance of 18:2, and hence a higher 18:2/18:3 ratio, than found in the corresponding leaf lipids. Among the phosphoglycerides, the unusual fatty acid 3-trans-hexadecenoate (trans-16:1) constituted from 15 to 24% of the acylated fatty acids in phosphatidyl glycerol (PG) from leaf chloroplasts (all species). In sharp contrast, trans-16:1 was virtually absent in PG from fruit chloroplasts of both Solanaceous and cucurbit species, and was replaced by a proportionate increase in the content of palmitate (16:0). The observed differences in the polar lipid fatty-acid composition of fruit and leaf chloroplasts are discussed in terms of the relative activity of several intrachloroplastic enzymes involved in lipid synthesis and fatty-acyl desaturation.Abbreviations MGDG monogalactosyldiglyceride - DGDG digalactosyl diglyceride - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PG phosphatidyl glycerol     

Modification of Herbicide Binding to Photosystem II in Two Biotypes of Senecio vulgaris L

The present study compares the binding and inhibitory activity of two photosystem II inhibitors: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron [DCMU]) and 2-chloro-4-(ethylamine)-6-(isopropyl amine)-S-triazene (atrazine). Chloroplasts isolated from naturally occurring triazine-susceptible and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.) showed the following characteristics. (a) Diuron strongly inhibited photosynthetic electron transport from H₂O to 2,6-dichlorophenolindophenol in both biotypes. Strong inhibition by atrazine was observed only with the susceptible chloroplasts. (b) Hill plots of electron transport inhibition data indicate a noncooperative binding of one inhibitor molecule at the site of action for both diuron and atrazine. (c) Susceptible chloroplasts show a strong diuron and atrazine binding (¹⁴C-radiolabel assays) with binding constants (K) of 1.4 × 10⁻⁸ molar and 4 × 10⁻⁸ molar, respectively. In the resistant chloroplasts the diuron binding was slightly decreased (K = 5 × 10⁻⁸ molar), whereas no specific atrazine binding was detected. (d) In susceptible chloroplasts, competitive binding between radioactively labeled diuron and non-labeled atrazine was observed. This competition was absent in the resistant chloroplasts.     

Effect of Growth Temperature on the Lipid Composition of Cyanidium caldarium: II. Glycolipid and Phospholipid Components

Cyanidium caldarium was grown at 20 and 55 C and harvested during exponential growth phase. Lipids were extracted and separated by silicic acid column and thin layer chromatography. The major glycolipids were identified as mono- and digalactosyl diglyceride and sulfolipid. Major phospholipids were identified as phosphatidyl choline and phosphatidyl ethanolamine. The cells grown at 20 C contained significantly larger quantities of these glycolipids and phospholipids than cells grown at 55 C.     

Biosynthesis of galactolipids by enzyme preparations from spinach leaves

The pH optimum for galactolipid synthesis from UDP-galactose by spinach chloroplasts is 7.2 in Tris-HCl or phosphate buffer. The products include sterol glycosides, trigalactosyl diglyceride (tentatively identified), digalactosyl diglyceride, and monogalactosyl diglyceride in increasing order of quantity. The proportion of monogalactosyl diglyceride decreases and that of digalactosyl diglyceride increases as the pH is lowered. The galactolipid synthesis is quite resistant to elevated temperature; maximal incorporation of galactose from UDP-galactose was observed at 45 degrees C. The proportion of monogalactosyl diglyceride was greater at the higher temperatures. As much as 40% of the galactolipid-synthesizing capability of a spinach leaf homogenate is not sedimented by centrifugation for 60 min at 100,000 g. An acetone powder of spinach chloroplasts contains enzymes which catalyze galactolipid synthesis. This preparation is dependent on added diglycerides in order to make galactolipid, whereas the chloroplast preparation is not dependent on added diglycerides. Molecular species of diglycerides were compared as requirements for galactolipid synthesis. The requirement was satisfied best by the diglycerides of highest unsaturation. Methylation of the free hydroxyl of the diglyceride eliminated the effectiveness.     

Investigations on heat resistance of spinach leaves

Exposure of spinach plants to high temperature (35° C) increased the heat resistance of the leaves by about 3° C. This hardening process occurred within 4 to 6 h, whereas dehardening at 20°/15° C required 1 to 2 days. At 5° C dehardening did not take place. Hardening and dehardening occurred in both the dark and the light. The hardiness was tested by exposure of the leaves to heat stress and subsequent measurements of chlorophyll fluorescence induction and light-induced absorbance changes at 535 nm on the leaves and of the photosynthetic electron transport in thylakoids isolated after heat treatment. Heat-induced damage to both heat-hardened and non-hardened leaves seemed to consist primarily in a breakdown of the membrane potential of the thylakoids accompanied by partial inactivation of electron transport through photosystem II. The increase in heat resistance was not due to temperature-induced changes in lipid content and fatty acid composition of the thylakoids, and no conspicuous changes in the polypeptide composition of the membranes were observed. Prolonged heat treatment at 35° C up to 3 days significantly decreased the total lipid content and the degree of unsaturation of the fatty acids of membrane lipids without further increase in the thermostability of the leaves. Intact chloroplasts isolated from heat-hardened leaves retained increased heat resistance. When the stroma of the chloroplasts was removed, the thermostability of the thylakoids was decreased and was comparable to the heat resistance of chloroplast membranes obtained from non-hardened control plants. Compartmentation studies demonstrated that the content of soluble sugars within the chloroplasts and the whole leaf tissue decreased as heat hardiness increased. This indicated that in spinach leaves, sugars play no protective role in heat hardiness. The results suggest that changes in the ultrastructure of thylakoids in connection with a stabilizing effect of soluble non-sugar stroma compounds are responsible for acclimatization of the photosynthetic apparatus to high temperature conditions. Changes in the chemical composition of the chloroplast membranes did not appear to play a role in the acclimatization.Abbreviations DGDG digalactosyl diglyceride - MGDG monogalactosyl diglyceride - PG phosphatidyl glycerol - PGA 3-phosphoglyceric acid Dedicated to Professor Wilhelm Simonis, Würzburg, on the occasion of his 70th birthday     

Chemical compositions and physical states of chloroplast lipids related to atrazine resistance in Conyza canadensis L.

A comparison of the chemical composition and physical states of chloroplast lipids, of atrazine-resistant (R) and sensitive (S) biotypes of Conyza canadensis L. (horseweed), in the rosetta stage showed: (1) the R biotype contains lower amounts of polar lipids in its thylakoids, as expressed on a chlorophyll basis, than the S biotype. (2) The chloroplasts of the R biotype have higher contents of monogalactosyl diacylglycerol (MGDG) and lower contents of digalactosyl diacylglycerol (DGDG) and phosphatidylglycerol (PG), than those of the S biotype. (3) The chloroplast total lipids exhibit a higher degree of unsaturation in the R biotype. This is due to a higher level of linolenic acid, and a lower level of palmitic acid in the glycolipids. The fatty acid compositions of the phospholipids, except that of PG, do not differ significantly. (4) The lipid matrix of the thylakoid membranes of the R biotype is more fluid than that of the S biotype, as measured by the fluorescence polarization technique. The results are discussed in terms of whether these differences are responsible for the herbicide resistance.     

BIOSYNTHESIS OF SMALL MOLECULES IN CHLOROPLASTS OF HIGHER PLANTS

1. Chloroplasts of higher plants contain enzymes which permit them to synthesize many kinds of small molecules in addition to carbohydrates. 2. Either aqueous or non-aqueous techniques may be used to isolate chloroplasts. Aqueous methods permit the isolation of chloroplasts showing high rates of photosynthesis; the organelles can be purified by means of density gradients. Non-aqueously isolated chloroplasts cannot photosynthesize, but show good retention of low-molecular-weight substances and soluble enzymes. 3. Whole cells photoassimilating ¹⁴CO₂ show considerable formation of ¹⁴C-labelled amino acids and lipids, but isolated chloroplasts exhibit very poor synthesis of amino acids and lipids from ¹⁴CO₂. 4. Chloroplasts play an important rôle in reducing nitrate to ammonia. There is controversy about the presence in chloroplasts of nitrate reductase and about the mechanism of the light-dependent reduction of nitrate to nitrite; however, it is generally agreed that non-cyclic electron transport directly supports reduction of nitrite to ammonia via a chloroplastic nitrite reductase. 5. Chloroplasts actively assimilate inorganic nitrogen into amino acids. The assimilation reaction is either the reductive amination of α-ketoglutarate to glutamate or the ATP-dependent conversion of glutamate to glutamine. The enzyme glutamate synthase has recently been found to be present in chloroplasts and may play an important function in nitrogen assimilation. 6. Numerous transaminases (aminotransferases) are present in chloroplasts. 7. The source of α-keto-acid precursors of chloroplastic amino acids is unknown. It remains to be established whether chloroplasts import the required keto acids or whether some of them might be generated via an incomplete tricarboxylic-acid cycle located in the chloroplast. 8. Chloroplasts contain characteristically high levels of mono and digalactosyl diglycerides, sulpholipid and phosphatidyl glycerol. They also have large amounts of polyunsaturated fatty acids. 9. Fatty acids are synthesized by the concerted action of fatty-acid synthetase, elongases and desaturases. Two pathways have been implicated for the formation of α-linolenic acid. 10. The galactosyldiglycerides are synthesized by successive galactosylation of diglyceride. The enzymes responsible are probably located in the chloroplastic envelope. 11. The other major chloroplastic acyl lipids (sulpholipid, phosphatidylglycerol and phosphatidylcholine) have not been, as yet, synthesized de novo by means of isolated chloroplast fractions. However, indirect evidence indicates that the first two are probably formed there. 12. Chlorophyllide synthesis involves the formation of δ-aminolaevulinic acid (δALA) followed by conversion of δALA to protoporphyrin IX, which is then transformed into protochlorophyll. 13. Recent evidence favours the view that δALA synthesis is not mediated by δALA synthetase but by another pathway in which δALA can be derived from α-ketoglutarate or glutamate. It has not been established whether this pathway is localized in plastids. 14. Conversion of δALA to protoporphyrin IX is mediated by soluble enzymes of the plastid stroma. Membrane-bound enzymes mediate the conversion of protoporphyrin to protochlorophyll. 15. Carotenoids are synthesized from acetyl C_oA via geranylgeranyl-pyrophosphate and phytoene intermediates. Evidence has been obtained for both neurosporene and lycopene as precursors of the cyclic carotenoids. 16. The overall pathway of carotenoid formation is subject to photoregulation, particularly during the development of the chloroplast. 17. Carotenes are precursors of xanthophylls, the inserted oxygen being derived from molecular oxygen. 18. Chloroplasts may synthesize or interconvert gibberellin hormones.