Labelling in vivo and in vitro of Molecular Species of Lipids from Chloroplast Envelopes and Thylakoids

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Mono-and digalactosyldiacylglycerol (MGDG and DGDG) were isolated from the leaves of sixteen 16:3 plants. In all of these plant species, the sn-2 position of MGDG was more enriched in C1, fatty acids than sn-2 of DGDG. The molar ratios of prokaryotic MGDG to prokaryotic DGDG ranged from 4 to 10. This suggests that 16:3 plants synthesize more prokaryotic MGDG than prokaryotic DGDG. In the 16:3 plant Spinacia oleracea L. (spinach), the formation of prokaryotic galactolipids was studied both in vivo and in vitro. In intact spinach leaves as well as in chloroplasts isolated from these leaves, radioactivity from [1-14C]acetate accumulated 10 times faster in MGDG than in DGDG. After 2 hours of incorporation, most labeled galactolipids from leaves and all labeled galactolipids from isolated chloroplasts were in the prokaryotic configuration. Both in vivo and in vitro, the desaturation of labeled palmitate and oleate to trienoic fatty acids was higher in MGDG than in DGDG. In leaves, palmitate at the sn-2 position was desaturated in MGDG but not in DGDG. In isolated chloroplasts, palmitate at sn-2 similarly was desaturated only in MGDG, but palmitate and oleate at the sn-I position were desaturated in MGDG as well as in DGDG. Apparently, palmitate desaturase reacts with sn-1 palmitate in either galactolipid, but does not react with the sn-2 fatty acid of DGDG. These results demonstrate that isolated spinach chloroplasts can synthesize and desaturate prokaryotic MGDG and DGDG. The finally accumulating molecular species, MGDG(18:3/16:3) and DGDG(18:3/16:0), are made by the chloroplasts in proportions similar to those found in leaves. Biosynthesis of prokaryotic MGDG and DGDG has been measured in vivo by the labeling of leaves from 16:3 plants with ['4C]acetate (4, 26, 29). In de novo-made MGDG, both C16 and C18 fatty acids were labeled and both were gradually desaturated to trienoic fatty acids. Desaturation of newly made DGDG, however, was limited to its labeled C18 fatty acids, suggesting a different action of the desaturases on MGDG and DGDG. Strong evidence that desaturation of MGDG fatty acids, indeed, proceeds as a lipid-linked process has been provided by Sato et al. (25) for the blue-green alga Anabaena variabilis.Following the initial study of galactolipid desaturation in vitro by Roughan et al. (22), it was confirmed recently that isolated spinach chloroplasts convert de novo-made MGDG(18:1/16:0) to ultimately MGDG(18:3/16:3) (1,15). In the present paper, we extend this work and report for the first time the synthesis of prokaryotic DGDG(18:1/16:0) and its subsequent desaturation in isolated chloroplasts. The rates observed were low, but are consistent with the small proportions of prokaryotic DGDG that were found in the leaves of sixteen 16:3 plants. A scheme is proposed for the regulation and interdependence of galactolipid synthesis in 16:3 plants, which tries to explain the typical differences in content and fatty acid composition between the major molecular species of MGDG and DGDG. 3Abbreviat...

Section: Formation and Desaturation Of Prokaryotic Galactolipids In Dsupporting

confidence: 81%

Biosynthesis and Desaturation of Prokaryotic Galactolipids in Leaves and Isolated Chloroplasts from Spinach

Heemskerk¹,

Schmidt²,

Hammer³

et al. 1991

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“…The free fatty acids synthesized by isolated chloroplasts, which consisted of 75 to 80%o oleic acid, were 66% converted to [14Clacyl-CoAs in the presence of exogenous CoA and ATP (20) and have been considered to be the most likely precursors for PC synthesis in vivo (17,19). This high oleic acid content of the free fatty acid fraction and its derivative acyl-CoA is in marked contrast with the fatty acid disposition of l-oleoyl-2-palmitoyl or 1,2-dipalmitoyl found both in the precursor diacylglycerols synthesized in vitro (10,17) and the DGG and PG derived from them in vivo (15,17,23,29 (Table I). Low DGG and SL labeling and high PC labeling was found in pumpkin, sunflower, and broad bean, whereas higher labeling of DGG and SL and lower PC labeling was found in tomato, parsley, and spinach.…”

mentioning

confidence: 79%

Manipulating the Incorporation of [1-¹⁴C]Acetate into Different Leaf Glycerolipids in Several Plant Species

Gardiner¹,

Roughan²,

Slack³

1982

During short term labeling of expanding leaves of seven plant species with 11-"4Clacetate, 35 to 64% of the label incorporated into lipids was found in phosphatidyicholine and 5 to 24% in phosphatidy4gycerol. In pmpk, sunflower, broad bean, and maize, only4to 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 11-'4Clacetate entering the lipids could be manipulated in a predictable manner. Phosphatidylcholine labelng 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 vito 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.

“…Chromoplast membranes from N. pseudonarcissus were prepared by disrupting the organelles with a tight-fitting Potter homogenizer in a medium containing 25 mM Tricine and 5 mM 2-mercaptoethanol (pH 7.6 with KOH). Total chromoplast membranes were then collected by centrifugation at l00,O00gmax for 2 h (4°C).…”

Section: Plastid Membranesmentioning

confidence: 99%

“…In 16:3 plants, the two galactolipids MGDG and DGDG are present in both the prokaryotic and eukaryotic configuration (3,16,20), and thus must be derived partially from plastid and partially from reticular diacylglycerol residues. The galactolipids of 18:3 plants, however, are found only in the eukaryotic configuration.In the membranes of chloroplasts and other plastids, MGDG and DGDG are formed by stepwise galactosylation of diacylglycerol (7,21,25,29). It has been suggested that pro-and eukaryotic galactolipid molecules might be synthesized in plastids by different sets of enzymes, the prokaryotic galactolipids being formed in the inner and eukaryotic lipids in the outer envelope membrane (2, 3,17,18).…”

mentioning

confidence: 99%

“…In the membranes of chloroplasts and other plastids, MGDG and DGDG are formed by stepwise galactosylation of diacylglycerol (7,21,25,29). It has been suggested that pro-and eukaryotic galactolipid molecules might be synthesized in plastids by different sets of enzymes, the prokaryotic galactolipids being formed in the inner and eukaryotic lipids in the outer envelope membrane (2, 3, 17,18).…”

mentioning

confidence: 99%

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Biosynthesis of Digalactosyldiacylglycerol in Plastids from 16:3 and 18:3 Plants

Heemskerk

Storz²,

Schmidt³

et al. 1990

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Intact chloroplasts isolated from leaves of eight species of 16:3 and 18:3 plants and chromoplasts isolated from Narcissus pseudonarcissus L. flowers synthesize galactose-labeled mono-, di-, and trigalactosyldiacylglycerol (MGDG, DGDG, and TGDG) when incubated with UDP- [6-3H]galactose. In all plastids, galactolipid synthesis, and especially synthesis of DGDG and TGDG, is reduced by treatment of the organelles with the nonpenetrating protease thermolysin. Envelope membranes isolated from thermolysin-treated chloroplasts of Spinacia oleracea L. (16:3 plant) and Pisum sativum L. (18:3 plant) or membranes isolated from thermolysin-treated chromoplasts are strongly reduced in galactolipid:galactolipid galactosyltransferase activity, but not with regard to UDP-Gal:diacylglycerol galactosyltransferase. For the intact plastids, this indicates that thermolysin treatment specifically blocks DGDG (and TGDG) synthesis, whereas MGDG synthesis is not affected. Neither in chloroplast nor in chromoplast membranes is DGDG synthesis stimulated by UDP-Gal. DGDG synthesis in S. oleracea chloroplasts is not stimulated by nucleoside 5'-diphospho digalactosides. Therefore, galactolipid:galactolipid galactosyltransferase is so far the only detectable enzyme synthesizing DGDG. These results conclusively suggest that the latter enzyme is located in the outer envelope membrane of different types of plastids and has a general function in DGDG synthesis, both in 16:3 and 18:3 plants.The 16:3 plants differ from 18:3 plants by the occurrence of cis-7,10,1 3-hexadecatrienoic acid in MGDG4 of the chloroplasts (7,16,17,24 doplasmic reticulum. These lipids consequently are called eukaryotic (9,14,17,30). In 16:3 plants, the two galactolipids MGDG and DGDG are present in both the prokaryotic and eukaryotic configuration (3,16,20), and thus must be derived partially from plastid and partially from reticular diacylglycerol residues. The galactolipids of 18:3 plants, however, are found only in the eukaryotic configuration.In the membranes of chloroplasts and other plastids, MGDG and DGDG are formed by stepwise galactosylation of diacylglycerol (7,21,25,29). It has been suggested that pro-and eukaryotic galactolipid molecules might be synthesized in plastids by different sets of enzymes, the prokaryotic galactolipids being formed in the inner and eukaryotic lipids in the outer envelope membrane (2, 3,17,18). A different localization of UDP-Gal:diacylglycerol galactosyltransferase (MGDG synthase) in the outer envelope membrane of pea (a 18:3 plant) chloroplasts (4), but in the inner envelope membrane of spinach (16:3 plant) chloroplasts (2, 12), was considered evidence for such a hypothesis. Moreover, two distinct enzymatic activities have been proposed for the biosynthesis of DGDG. In 1961, Ferrari and Benson (8) suggested a pathway of DGDG synthesis in which MGDG is galactosylated by UDP-Gal. Using extracts from pea leaves, indeed, Siebertz and Heinz (26) succeeded in measuring DGDG formation from the two precursors UDP-Gal and MGDG. Lat...