Effects of moderately enhanced levels of ozone on the acyl lipid composition of leaves of garden pea (Pisum sativum)

Plants of garden pea ( Pisum sativum L.) were exposed to charcoal-filtered air with or without addition of 65 ± 5 l⁻¹ ozone. Plants were harvested daily for 9 days and lipids were extracted from the second-oldest leaf. Visible injury of this leaf was evident from day 5 on, while the differences in lipids between ozone and control treatments were observed earlier. Ozone caused large decreases in the contents of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG), a slower decrease in the content of phosphatidylcholine (PC), but an increase in the content of phosphatidylethanolamine (PE) per leaf area, compared with exposure to charcoal-filtered air. The content of phosphatidylglycerol (PG) was unaffected by ozone. Compared with charcoal-filtered air, fumigation with ozone resulted in a decrease in the proportion of linolenic acid (18:3) of the total lipid extract, with a concomitant increase in the proportion of linoleic acid (18:2). For individual lipids, ozone caused a similar pattern of decreased 18:3 and increased 18:2 in MGDG, SQDG, PC and PE, while the fatty acid composition of DGDG was unaffected. In PG, ozone decreased the proportions of 18:3 and trans -Δ³-decenoic acid (16:1_trans), balanced by increased proportions of palmitic and oleic acids. The contents of chlorophylls and carotenoids were unaffected by ozone. Our results show that moderately elevated levels of ozone cause significant changes in the polar lipid composition of garden pea leaves and in the level of unsaturation of the lipid acyl groups and, furthermore, that ozone has different effects, which could be direct or indirect, on chloroplast lipids (MGDG, DGDG, SQDG, PG acylated with 16:1_trans) and cytosolic membrane lipids.     

Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants: the origin of the diacylglycerol moiety

The positional distribution of fatty acids in chloroplast polar lipids and phosphatidylcholine from leaves of four plants has been measured in order to determine the origin of the diacylglycerol (DAG) moieties of each lipid. In spinach and tobacco, the DAG of sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG) and digalactosylglycerol (DGDG) were derived partly from the chloroplast and partly from the cytoplasm. The contribution of the chloroplast pathway differed for each lipid, but in both plants the proportion of a lipid derived from that pathway was in the order SQDG greater than MGDG greater than DGDG. In contrast, all the DAG moieties of the three glycolipids of wheat and cucumber were derived from the cytoplasm. The DAG moiety of chloroplast phosphatidylglycerol was synthesized in the chloroplast in all four plants.     

The effect of low oxygen concentration on the acyl-lipid and fatty-acid composition of the C4 plant Amaranthus paniculatus L.

Acyl lipids and their constituent fatty acids were studied in leaves, chloroplasts and bundle-sheath strands of the C₄ plant Amaranthus paniculatus L. grown under normal and 4%-oxygen-containing atmospheres. In all fractions the major lipids were found to be monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovo-syldiacylglycerol and phosphatidylglycerol. Significant quantities of phosphatidylcholine and phosphatidylethanolamine were restricted to leaves and bundle-sheath strands. All lipids, except phosphatidylglycerol where 3-trans-hexadecenoic acid was also present, contained palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. On a chlorophyll basis and compared with whole leaves, the amounts of phosphatidylcholine and phosphatidylethanolamine in bundle-sheath strands were considerably reduced. Three weeks after the change from a normal to a 4% atmospheric O₂ level, the galactolipid content, particularly in the bundlesheath strands, was enhanced. There were no significant differences in the degrees of saturationunsaturation of total acyl lipid for the plants grown in the low oxygen and normal atmospheres, although under 4% O₂ the phosphatidylglycerol contained an increased proportion of 3-trans-hexadecenoic acid at the expense of palmitic acid.Abbreviations DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - SQDG sulphquinovosyldiacylglycerol     

Age-dependent variation in membrane lipid synthesis in leaves of garden pea (Pisum sativum L.).

To study membrane lipid synthesis during the life-span of a dicotyledon leaf, the second oldest leaf of 10-40-d-old plants of garden pea (Pisum sativum L.) was labelled with [1-(14)C]acetate and the distribution of radioactivity between the major membrane lipids was followed for 3 d. In the expanding second oldest leaf of 10-d-old plants, acetate was primarily allocated into phosphatidylcholine (PC) during the first 4 h of labelling. During the following 3 d, labelling of PC decreased and monogalactosyldiacylglycerol (MGDG) became the most radioactive lipid. In the fully expanded second oldest leaf of older plants, acetate was predominantly allocated into phosphatidylglycerol (PG), which remained the major radiolabelled lipid during the 3 d studied. The proportion of radioactivity recovered in MGDG decreased with increasing plant age up to 20 d, suggesting that, in expanded leaves, MGDG is more stable and requires renewal to a lower extent than PG. When the second oldest leaf approached senescence, labelling of MGDG again increased, indicating an increased need for thylakoid repair. The proportion of acetate allocated into phosphatidylethanolamine and free sterols was largest in leaves of 18-26-d-old plants and in the youngest leaves, respectively. Thus, these results demonstrate that the distribution of newly synthesized fatty acids between acyl lipid synthesis in the chloroplast and extraplastidial membranes strongly varies with leaf age, as do the proportion utilized for sterol synthesis. The findings emphasize the importance of defining the developmental stage of the leaf material used when performing studies on leaf lipid metabolism.     

The effects of elevated atmospheric CO2 on lipid metabolism in leaves from mature wheat (Triticum aestivum cv. Hereward) plants

Winter wheat (Triticum aestivum L. cv. Hereward) plants were grown for 35 d either at 350 μ mol mol^–1 CO₂ or at 650 μ mol mol^–1 CO₂. Lipid synthesis was studied in these plants by incubating the 5th leaf on the main stem with [1–¹⁴C]acetate. Increased CO₂ concentrations did not significantly affect the total incorporation of radiolabel into lipids of whole leaf tissue, but altered the distribution for individual lipid classes. Most noticeable amongst acyl lipids was the reduction in labelling of diacylglycerol and a corresponding increase in the proportion of phosphatidylcholine labelling. In the basal regions, there were similar changes and, in addition, phosphatidylglycerol labelling was particularly increased following growth in an enriched CO₂ atmosphere. The stimulation of labelling of the mitochondrial-specific lipid, diphosphatidylglycerol, prompted an examination of the mitochondrial population in wheat plants. Mitochondria were localized in intact wheat sections by immunolabelling for the mitochondrial-specific chaperonin probe. Growth in elevated CO₂ doubled the number of mitochondria compared to growth in ambient CO₂. Fatty acid labelling was also significantly influenced following growth at elevated CO₂ concentrations. Most noticeable were the changes in 16C:18C ratios for the membrane lipids, phosphatidylcholine, phosphatidylglycerol and monogalactosyldiacylglycerol. These data imply a change in the apportioning of newly synthesized fatty acids between the 'eukaryotic' and 'prokaryotic' pathways of metabolism under elevated CO₂.     

Lipid metabolism in green leaves of developing monocotyledons

Lipid synthesis was studied in successive leaf sections from the base to the tip of developing barley (Hordeum vulgare L.), maize (Zea mays L.), rye grass (Lolium perenne L.) and wheat (Triticum aestivum L.) leaves. The endogenous levels of acyl lipids and their constituent fatty acids from the same leaf sections were also analysed. The principle chloroplast acyl lipids showed a relative increase in amount with the age of the leaf section. Their content of -linolenic acid also increased whereas there was little change in the amount of this acid in phosphatidylcholine and phosphatidylethanolamine, which are primarily non-chloroplastic. The content of trans-3-hexadecenoic acid in phosphatidylglycerol increased approximately 20-fold between the youngest (basal) and oldest (distal) leaf sections.The incorporation of [¹⁴C]acetate was always high into monogalactosyldiacylglycerol, phosphatidylcholine and the neutral lipid (mainly pigments) fractions. With increasing age, the neutral lipids were less well labelled. In three of the plant species but not in barley, phosphatidylglycerol was heavily labelled. Monogalactosyldiacylglycerol usually contained the highest amount of radioactivity in the middle leaf sections. Apart from these generalisations, each plant type had its own specific pattern of radiolabelling.     

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.     

Parallel inactivation of alpha 2-adrenergic agonist binding and Ni by alkaline treatment

The isolation of a cytochrome b₆-f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol-plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol-plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.     

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.

     

Etioplast Development in Dark-grown Leaves of Zea mays L

The ultrastructure of etioplasts and the acyl lipid and the fatty acid composition of sequential 2-centimeter sections cut from the base (youngest) to the top (oldest) of nonilluminated 5-day-old etiolated leaves of Zea mays L., and the acyl lipid and fatty acid composition of the etioplasts isolated from them have been investigated. There is a 2.5-fold increase in the size of the plastids from the base to the tip of the leaf, and an increase both in the size of the prolamellar body and in the length of lamellae attached to it. The etioplasts in the bundle sheath and mesophyll cells of the older, but not the younger leaf tissue, are morphologically distinct. The monogalactosyl and digalactosyldiglycerides, phosphatidylcholine, phosphatidylglycerol, and phosphatidylinositol were the only detectable acyl lipids in the isolated etioplast fractions. Together with phosphatidylethanolamine these were also the major acyl lipids in the whole leaf sections. With increasing age of the leaf tissue, increases occurred in two of the major plastid lipids, monogalactosyldiglyceride and phosphatidylglycerol, while the levels of essentially nonplastid lipids remained constant or declined slightly. The monogalactosyldiglyceride to digalactosyldiglyceride ratio increased from 0.4 to 1.1 in the tissue sections of increasing age and from 0.7 to 1.2 in the etioplasts isolated from them. Similarly, the galactolipid to phospholipid ratio increased from 0.8 to 1.4 in the tissue and from 0.5 to 4.5 in the isolated plastids. In the latter, the proportions of phosphatidylglycerol (as a per cent of total phospholipid) increased from 20 to 41% with increasing age of plastids.

Linolenic acid was the major fatty acid in the total lipid of each of the etioplast fractions, but it was only the major fatty acid in the total lipid of the oldest leaf tissue. Its proportion in both total lipid extracts and individual lipids increased with age. The trans Δ³ hexadecenoic acid was absent from all lipids. The protochlorophyllide content of the tissue increased with age. The results are discussed in relation to the use of illuminated etiolated leaves for studying chloroplast development.

     

Involvement of plastoquinone and lipids in electron transport reactions mediated by the cytochrome b6-f complex isolated from spinach

The isolation of a cytochrome b₆-f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol-plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol-plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.     

Spin-label ESR studies of lipid-protein interactions in thylakoid membranes

Lipid-protein interactions in thylakoid membranes, and in the subthylakoid membrane fractions containing either photosystem 1 or photosystem 2, have been studied by using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted membrane lipids interacting directly with the integral membrane proteins. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with the membrane proteins and to determine the selectivity between the different lipid classes for the lipid-protein interaction. The fractions of motionally restricted lipid in the thylakoid membrane are 0.36, 0.39, and 0.53, for the spin-labeled monogalactosyldiacylglycerol, phosphatidylcholine, the phosphatidylglycerol, respectively. Spin-labeled monogalactosyldiacylglycerol exhibits very little preferential interaction over phosphatidylchline, which suggests that part of the role of monogalactosyldiacylglycerol in thylakoid membranes is structural, as is the case for phosphatidylcholine in mammalian membranes. Spin-labeled phosphatidylglycerol shows a preferential interaction over the corresponding monogalactosyldiacylglycerol and phosphatidylcholine analogues, in contrast to the common behavior of this lipid in mammalian systems. This pattern of lipid selectivity is preserved in both the photosystem 1 and photosystem 2 enriched subthylakoid membrane fractions.     

Ultrastructure and lipid composition of etioplasts in developing dark-grown wheat leaves

Changes in the ultrastructure and lipid composition of etioplasts have been evaluated in three regions from the base to the tip of 8-day-old darkgrown wheat leaves and in the upper-2/3 region of etiolated leaves of different ages. In developing darkgrown tissues, the main morphological changes that etioplasts undergo consist of an increase in the amount of thylakoïds which, in the most mature etioplasts, align in parallel arrays. Concomitantly, galactolipids and sulfolipid form an increasing proportion of the total lipids. Trans-3-hexadecenoic acid was not detectable in phosphatidylglycerol (PG) of etioplasts showing appressed thylakoïds isolated from 5-day-old leaves, but was present in significant amounts in etioplasts in the basal part of 8-day-ols leaves in which membrane appression was barely visible. The proportions of this acid increase as etioplasts develop, reaching 25% of the PG fatty acids (1.2% of the total fatty acids) in the most differentiated etioplasts. In wheat etioplasts, it appears that trans-3-hexadecenoic acid may accumulate in considerable amounts in darkgrown tissues and that its accumulation is not directly involved in membrane appression.Abbreviations AP phosphatidic acid - DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PS phosphatidylserine - SL sulfolipid     

Plastid differentiation, acyl lipid, and Fatty Acid changes in developing green maize leaves

Plastid differentiation, acyl lipid, and fatty acid composition have been followed in successive 2-cm sections from the base (youngest tissue) to the tip (oldest tissue) of green Zea mays (maize) leaves grown under a normal diurnal light regime. Although the youngest cells (0-4 cm from the leaf base) had only proplastids with one or two grana, they contained chlorophylls a and b, monogalactosyldiglyceride, digalactosyldiglyceride, sulfolipid, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol. In the more mature sections, the plastids increased in size 5-fold, and differentiation into mesophyll and bundle-shealth chloroplasts had occurred. Concomitantly, the levels of all the lipids increased with the exception of phosphatidylcholine and phosphatidylethanolamine which decreased. With increasing cell maturity, the percentage of linolenic acid increased in all the individual acyl lipids, but palmitic acid remained constant in phosphatidylcholine, phosphatidylethanolamine, and sulfolipid. The Δ^3t-hexadecenoic acid was only detectable in the phosphatidylglycerol of the most mature maize tissue.     

Changes in Lipid and Fatty Acid Metabolism of Vicia faba Mesophyll Protoplasts

The metabolism of the major polar and neutral lipids of Viciafaba protoplasts isolated from ¹⁴CO₂-fed leaves has been examined.The results show large losses in the radioactivity found inphosphatidylcholine and monogalactosyldiacylglycerol while thatof phosphatidylglycerol was stable. This loss was accountedfor by a rapid increase in the ¹⁴C content of the neutral lipids,particularly the triacylglycerols. Analysis of the fatty acidradioactivity in the lipids suggests that protoplast isolationinhibited fatty acid desaturation on phosphatidylcholine andpossibly on other lipids. These results also suggest a roleof phosphatidylcholine in the donation of fatty acids for triacylglycerolsynthesis in mesophyll protoplasts. The results are discussedin terms of the regulation of lipid metabolism and protoplastbiology. (Received April 20, 1984; Accepted August 27, 1984)     

Spin label saturation transfer ESR studies of protein-lipid interactions in Photosystem II-enriched membranes

Saturation transfer ESR has been used to study the dynamic behaviour of lipids in the appressed regions of thylakoid membranes from pea seedlings. Four different phospho- and galacto-lipid spin labels (phosphatidylcholine labelled at the 12 or 14 C-atom positions of the sn-2 chain, phosphatidylglycerol labelled at the 14-position of the sn-2 chain, and monogalactosyldiacylglycerol labelled at the 12-position of the sn-2 chain) were used to probe the lipid environment in photosystem II-enriched membranes prepared by detergent extraction. The ESR spectra show that the majority of the lipid in these preparations is strongly motionally restricted. Values for the effective rotational correlation times of the labelled chains were deduced from the lineheight ratios and integrals of thhe saturation transfer ESR spectra. The effective rotational correlation times were found to be in the 10⁵ range, indicating a very low lipid chain mobility which correlates with the low lipid content of these preparations. Comparison of the effective rotational correlation times deduced from the different diagnostic regions of the spectrum revealed little anisotropy in the chain mobility, indicating that the dominant motional mode was trans-gauche isomerization. The effective rotational correlation times deduced from the spectral integrals were similar to those deduced from the lineheight ratios, consistent with the absence of any appreciable fluid lipid component in these preparations. The results also indicate some selectivity of interaction between the lipid species, with phosphatidylcholine exhibiting appreciably slower motion than either phosphatidylglycerol or monogalactosyldiacylglycerol.     

The effect of cadmium on lipid and fatty acid biosynthesis in tomato leaves

This research aims to examine the effect of cadmium uptake on lipid composition and fatty acid biosynthesis, in young leaves of tomato treated seedlings (Lycopersicon esculentum cv. Ibiza F1). Results in membrane lipids investigations revealed that high cadmium concentrations affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the unsaturated fatty acid content, resulting in a lower degree of fatty acid unsaturation. The level of lipid peroxides was significantly enhanced at high Cd concentrations. Studies of the lipid metabolism using radioactive labelling with [1-¹⁴C]acetate as a major precursor of lipid biosynthesis, showed that levels of radioactivity incorporation in total lipids as well as in all lipid classes were lowered by Cd doses. In total lipid fatty acids, [1-¹⁴C]acetate incorporation was reduced in tri-unsaturated fatty acids (C16:3 and C18:3); While it was enhanced in the palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and linoleic (C18:2) acids. [1-¹⁴C]acetate incorporation into C16:3 and C18:3 of galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] and some phospholipids [phosphatidylcholine (PC) and phosphatidylglycerol (PG)] was inhibited by Cd stress. Our results showed that in tomato plants, cadmium stress provoked an inhibition of polar lipid biosynthesis and reduced fatty acid desaturation process.     

Incorporation of 14C-radioactivity into lipid classes at different maturity of Laminaria japonica Aresch. cultured under natural and partially controlled conditions

A study to assess which environmental or developmental factors predominate in the biosynthesis of lipids of Laminaria japonica Aresch. blades was undertaken by means of ¹⁴C-labelling technique. In experiment 1, kelp blades at different growth stages were collected in different cultural seasons. In experiment 2, kelp blades of different sizes and maturity cultured simultaneously for two months in the same sea area were collected at the same time.The following results were obtained. In experiment 1, the ¹⁴C-incorporation into whole lipids was lowest in juvenile blades collected at the end of autumn and highest in blades of middle size collected in winter. However, the highest counts were incorporated in PC among complex lipid classes from all size classes of blades in both experiments 1 and 2. In experiment 2, ¹⁴C-incorporation patterns of individual lipid classes were characteristically different depending on the sizes of blades even under the same cultural condition. Thus, the biosynthesis of lipids in this kelp seems to be affected essentially by developmental factors.Abbreviation Comp. lip. complex lipid - FA non-esterified fatty acids - Fucost fucosterol - DG diacylglycerol - DGDG digalactosyldiacylglycerol - MG monoacylglycerol - MGDG monogalactosyldiacylglycerol - NL neutral lipids - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidyl inositol - PS phosphatidylserine - SQDG sulphoquinovosyldiacyl glycerol - TG triacylglycerol     

In situ incorporation of Fatty acids into lipids of the outer and inner envelope membranes of pea chloroplasts

When incubated with [1-¹⁴C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, ¹⁴C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg²⁺-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their ¹⁴C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The ¹⁴C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.     

Effects of moderately enhanced levels of ozone on the acyl lipid composition and dynamical properties of plasma membranes isolated from garden pea (Pisum sativum)

Plasma membranes were isolated from leaves of 16-day-old garden pea, Pisum sativum L., that had been grown in the absence or presence of 65 nl l⁻¹ ozone for 4 days prior to membrane isolation. Plasma membranes from ozone-fumigated plants contained significantly more acyl lipids per protein than those from leaves of plants grown in filtered air on a molar/weight ratio. The ratio between the major acyl lipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), also increased due to the ozone fumigation, while the fatty acid unsaturation level was unaltered in total plasma membrane acyl lipids, as well as in PC and PE. The amount of free sterols per protein was unaltered, but the percentage of campesterol increased, concomitant with a decrease in stigmasterol. The dynamical properties of the isolated plasma membranes were assessed using Laurdan fluorescence spectroscopy, which monitors water penetration and mobility at the hydrophilic-hydrophobic interface of the membrane. At 0°C, the molecular mobility was slightly lower in plasma membranes from ozone-fumigated plants than in plasma membranes from control plants, possibly reflecting the increased PE/PC, campesterol/stigmasterol and lipid/protein ratios, and suggesting that ozone-fumigated pea plants may be more susceptible to freezing injuries.