Jean-Paul Dubacq

Unsaturated fatty acids in maturing seeds of sunflower and rape: Regulation by temperature and light intensity

Abstract In rape seed, low temperatures and low light intensities increase the level of linoleic acid at the expense of oleic acid biosynthesis without change o

Study of the supramolecular organization of light-harvesting chlorophyll protein (LHCP): Conversion of the oligomeric form into the monomeric one by phospholipase A2 and reconstitution with liposomes

The photosynthetic membranes are composed mainly of lipids and proteins arranged in lipo-protein complexes. Among these complexes, the light-harvesting chlorophyll protein (LHCP) represents 40-50% of the total chlorophyll found in the membrane. Consequently, it is supposed to play a fundamental role in the structure of thylakoids, e.g., in grana stacking (reviews [l-4]). Due to its heterogeneous composition including pigments, lipids and polypeptides, it cannot be established with certainty which of its constituents plays the key role in the stacking process. sidered as a passive support. In [7] a more functional role of lipids was proposed and it was suggested that specific arrangements of components necessary for the functioning of the light reactions of photosynthesis can be regulated to a large extent by the lipid content of the membrane [7].

Lipid composition of chlorophyl-protein complexes: Specific enrichment in trans-hexadecenoic acid of an oligomeric form of light-harvesting chlorophyll aa/b protein

Thylakoid membranes of higher plants and green algae can be resolved by SDS-polyacrylamide gel electrophoresis into at least 2 different chlorophyll-protein complexes and -30 polypeptides. The 2 chlorophy~-protein complexes correspond to the P700 chI a--protein complex (CPI) assumed to originate from the reaction centre of photosystem I and to the lightharvesting chl a&protein complex named LHCP or CP II [ 1 ] which is considered to represent the antenna chlorophyll of photosystem II. It is now well known that using milder conditions of solubilization, additional &l-proteins can be obtained. They correspond to a new chla-protein complex generally termed CPa, which may represent the photosystem II reaction centre complex 12-41, and to several oligomeric forms of CP 1 and LHCP f3-131. Although these different &l-protein complexes are characterized by some of their physiological, biochemical and physical properties (reviews [ 1,14--16]), little is known about their lipid composition other than pigments. The &-protein complexes were firstly considered to contain only traces of lipids [ 171. Two recent analyse? -’ -d the lipid composition of sub-

Protein-mediated transfer of phosphatidylcholine from liposomes to spinach chloroplast envelope membranes

Abstract We have demonstrated that an active transfer of phosphatidylcholine from liposomes towards spinach chloroplast envelope was catalyzed by a phospholipid-transfer protein purified from spinach leaves. The transfer is actually a complex process. During the first 10 min of the incubation, the exchange of phosphatidylcholine between liposomes and isolated envelope vesicles was predominant, as shown by the equilibration of phosphatidylcholine specific activity to the same level in both the liposomes and the envelope vesicles. Further incubation led to a 35% increase of the phosphatidylcholine content of envelope membranes, thus corresponding to a net transfer of phosphatidylcholine from liposomes towards envelope vesicles. After incubation of intact chloroplasts and liposomes in the presence of purified phospholipid-transfer protein, most of the radioactive phosphatidylcholine transferred to intact chloroplasts was recovered with the envelope membrane fraction. In addition, a mild phospholipase C treatment of intact chloroplasts after phosphatidylcholine transfer has demonstrated that all the radioactive phosphatidylcholine remained in the cytosolic leaflet of the outer envelope membrane and was not redistributed towards internal chloroplast membranes. Such a result, which mimics the in vivo situation, suggests that the phospholipid-transfer protein might be partly responsible (together with the apparent lack of transmembrane lipid diffusion) for the different lipid composition of the outer envelope membrane (when compared with the other plastid membranes) and for the asymmetrical distribution of phosphatidylcholine within this membrane.

Evidence for a Transient Association of New Proteins with the Spirulina maxima Phycobilisome in Relation to Light Intensity.

Environmental parameters are known to affect phycobilisomes. Variations of their structure and relative composition in phycobiliproteins have been observed. We studied the effect of irradiance variations on the phycobilisome structure in the cyanobacterium Spirulina maxima and discovered the appearance of new polypeptides associated with the phycobilisomes under an increased light intensity. In high light, the six rods of phycocyanin associated with the central core of allophycocyanin contained only one to two phycocyanin hexamers instead of the two to three they contained in low light. The concomitant disappearance of a 33-kD linker polypeptide was observed. Moreover, in high light three polypeptides of 29, 30, and 47 kD, clearly unrelated to linkers, were found to be associated with the phycobilisome fraction: protein labeling showed that a specific association of these polypeptides was induced by high light. One polypeptide, at least, would play the role of a chaperone protein. Not only the synthesis of these proteins, which appeared slightly increased in high light, but also their association with phycobilisome structure are light intensity dependent.

Purification of acyl-CoA: glycerol-3-phosphate acyltransferase from pea leaves

Abstract The enzyme (EC 2.3.1.15) catalyzing lysophosphatidic acid synthesis has been purified to homogeneity from pea leaves. The different steps of purification were gel filtration, anion exchange chromatography and fast protein liquid chromatography (FPLC) on a mono Q column. The purified enzyme exhibited a single band after polyacrylamide gel electrophoresis with sodium dodecyl sulfate corresponding to a molecular mass of 40.5 kDa. The enzyme was selective towards oleoyl-CoA and oleoyl-ACP, and had an apparent Km of 11 μM with oleoyl-CoA and 33 μM with glycerol 3-phosphate. The activity was sensitive to NaCl and oleic acid.

Oleyl-coenzyme a metabolization by sub-cellular fractions from growing pea leaves

Abstract This work argues for the desaturation of oleyl-CoA in young Pea leaves to be tightly linked to the ‘microsomal fractions’. The weak desaturase activity found in plastid fractions from young leaves is always associated with a NADH-cyt. c reductase and an oleyl-CoA phosphatidylethanolamine acylase. During the development of the leaf the desaturase activity reaches its maximum about 8 days after the germination and light enhances markedly this activity.

Localization and some properties of a Mg2+-dependent ATPase in the inner membrane of pea chloroplast envelopes

Abstract Pea chloroplasts have been found to display an envelope-bound ATPase activity. The enzyme is located exclusively in the inner envelope membrane. The ATPase activity is stimulated 2.5 times at low concentrations of CaCl2 (about 0.3 mM) and 10–12 times at much higher concentrations of MgCl2 (4–10 mM). In the presence of both cations, the activity is further enhanced by calmodulin. The apparent K m - value for Mg-ATP is 0.14 mM. Under optimal conditions (4 mM ATP, 4 mM MgCl2, pH 7.8 and 37°C) the specific activity ranged from 100 to 290 nmol Pi · min−1 · mg−1 protein. The enzyme is sensitive to ADP, Na3 VO4, LaCl3, oligomycin, ammonium molybdate, N- ethylmaleimide , N- bromosuccinimide and cyanuric chloride. However, NaN3, ouabain, dicyclohexylcarbodiimide and phlorizin do not inhibit the ATPase activity. Similarities and differences between chloroplast envelope ATPase from pea and spinach are discussed.

In vitro cooperation between plastids and microsomes in the biosynthesis of leaf lipids

It has been suggested recently that the chloroplast is the major site for de novo synthesis of saturated and monounsaturated fatty acids in plants [l-6]. According to [7,8] plastids are indeed the only sites for the de novo synthesis of acyl groups requiring an obligatory acyl carrier protein. It is well shown by [9-131 that CDP choline and CDP eth~olamine transferases, acyltransferases and desaturases are firmly bound to the endoplasmic reticulum. At the same time, galactosylation and assembly of monogalactosyldiacylglycerol (MGDG) is tightly linked to the ~~oroplast envelope [ 141. Taken together these results suggest that in the plant cells a cooperation between plastids and endoplasmic reticulum membranes is required for the final total synthesis of the membrane lipids. Here we argue for such a cooperation in an in vitro reconstituted system: intact ~~oroplasts plus microsomal fractions from spinach leaves.

Time course of a dedifferentiation process during isolation and culture of protoplasts: Comparison between protoplasts from young and mature regions of the mung bean hypocotyl

Abstract Protoplasts were isolated from successive sections along the mung bean hypocotyl. High yields were obtained with mature tissues. The fatty acid composition of the protoplasts was similar to that found in the original cells which indicates that no major structural alteration of membranes occurs during protoplast isolation. In contrast, all the cells regenerated from the protoplasts exhibited a modified lipid composition and isoperoxidase profiles when compared to hypocotyl cells.