Antoine Tremolieres

Analysis of molecular species of plant polar lipids by high-performance and gas liquid chromatography

Abstract Total lipid extracts from potato tubers and tobacco leaves are separated into lipid classes by two step HPLC using a silicic column. Elution is first performed for 20 min with a programmed linear gradient of two mixed solvents running from 100% of solution A (isopropanol-hexane, 4:3) to 100% of solution B (isopropanol-hexane-water, 8:6:1.5); the column is then eluted with pure solution B in an isocratic mode for 20 min more. The main polar lipids (MGDG, DGDG, PC, PE, PG) from both plant tissues can be collected and further separated into component molecular species on a simplified HPLC system with a C18 column eluted in an isocratic mode with a polar solvent. Molecular species separations are achieved within 35 min; quantifications are made through GLC analysis of attached fatty acids. Three to five main molecular species are thus clearly identified in each lipid class. In potato tuber, phospholipids (PC, PE) 18:2/18:2 species are predominant. In tobacco leaf, six double bond species (18:3/18:3 and 16:3/18:3) are predominant in galactolipids, whereas PC contains a greater number of molecular species varying by their degree of unsaturation (from 18:3/18:3 to 16:0/18:2). Only certain molecular species of PG contain Δ3-trans-hexadecenoic acid.

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

Restoration of both an oligomeric form of the light-harvesting antenna CP II and a fluorescence state II-state I transition by Δ3-trans-hexadecenoic acid-containing phosphatidylglycerol, in cells of a mutant of Chlamydomonas reinhardtii

Abstract To define the role of the Δ 3 - trans -hexadecenoic acid (16:1- trans ) in the thylakoid membrane, restoration experiments were carried out using a Photosystem II (PS II)-lacking but low-fluorescent mutant, mf 2, of Chlamydomonas reinhardtii . This mutant is unable to synthesize 16:1- trans , lacks an oligomeric form of the main light-harvesting chlorophyll (Chl) a + b antenna, CP II, and shows an impaired regulation of the excitation energy distribution. Whole cells were incubated for 39 h in the presence of liposomes of 16:1- trans -containing phosphatidylglycerol (PG-16:1- trans ), in the light at 25°C. Then lipids and Chl-protein complexes were analyzed and low-temperature fluorescence emission spectra, both in state I and in state II (oxidized and reduced plastoquinone pool), were measured. The results indicated: (1) a relatively important content of 16:1- trans specifically incorporated in the chloroplast phosphatidylglycerol (PG); (2) an appreciable amount of CP II oligomeric form; (3) the occurrence of a clear state II-state I transition, as shown by a ratio of the CP II fluorescence at 682 nm to the photosystem I fluorescence at 712 nm, which was 3.8-times higher in state I than in state II. These restorations were not observed when cells of mf 2 were incubated in the presence of palmitate-containing PG, of oleate-containing phosphatidylcholine or of PG-16:1- trans + cycloheximide. It is concluded that: (1) the oligomeric form of CP II is essential for a good excitation energy transfer towards the PS II region and, consequently, to a good state II-state I transition in the distribution of excitation energy; (2) PG-16:1- trans probably plays an essential role in stabilizing neo-formed CP II oligomers during the assembly of new Chl-protein complexes in the chloroplast.

Oligomeric form of the light-harvesting chlorophyll a + b-protein complex CP II, phosphatidyldiacylglycerol, Δ3-trans-hexadecenoic acid and energy transfer in Chlamydomonas reinhardtii, wild type and mutants

Abstract Analyses of chlorophyll-protein complexes and of lipids were performed with the wild type of Chlamydomonas reinhardtii and three non-photosynthetic mutants: Fl 39, which was a ‘classical’ high-fluorescent Photosystem II (PS II)-lacking mutant, and mf 1 and mf 2, which lacked also functional PS II but were low-fluorescent and showed an abnormally predominant energy transfer from the main light-harvesting antenna towards Photosystem I. An oligomeric form of the chlorophyll a + b -protein complex CP II was clearly isolated from the wild type and the mutant Fl 39 but it was not detected in the mutants mf 1 and mf 2. The three mutants showed total lipid contents close to or greater than that of the wild type. Their phosphatidyldiacylglycerol (PG) contents, on a chlorophyll basis, were higher ( Fl 39) or 1.4- ( mf 1) and 2.0- ( mf 2) times lower than that of the wild type. The fatty acid compositions of the wild type and of the mutant Fl 39 were comparable, showing about equal amounts of a C18 series and a C16 series which included the Δ3- trans -hexadecenoic acid (C16:1- trans ). This C16:1- trans was not detected in the mutants mf 1 and mf 2 which contained the other fatty acids. These results indicate correlations between lack of C16:1- trans -containing PG, lack of an oligomeric form of CP II and an impaired mechanism of the regulation of excitation energy transfer from the main chlorophyll a + b antenna.

Energy coupling, membrane lipids and structure of thylakoids of Lupin plants submitted to water stress

Bioenergetic properties of thylakoids from plants submitted to a water stress stress (watering stopped for 6–15 days) have been measured in two lupin genotypes characterized as resistant or susceptible to drought. This energy coupling was assessed by flow-force relationships relating the phosphorylation rate to the magnitude of the proton gradient % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakabbaaa6daaahjxzL5gapeqa% aiabgs5aenaaxacabaGaeqiVd0galeqabaGaaiOFaaaakmaaBaaale% aacaWGibWaaWbaaWqabeaacqGHRaWkaaaaleqaaaaa!4D55!\[\Delta \mathop \mu \limits^\~ _{H^ + } \]. The fluorescent probe 9-aminoacridine was used to express, as a ΔpH, the whole % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakabbaaa6daaahjxzL5gapeqa% aiabgs5aenaaxacabaGaeqiVd0galeqabaGaaiOFaaaakmaaBaaale% aacaWGibWaaWbaaWqabeaacqGHRaWkaaaaleqaaaaa!4D55!\[\Delta \mathop \mu \limits^\~ _{H^ + } \] by calibrating fluorescence quenching against the phosphate potential ΔGp in ‘state 4’, i.e., when ATP synthesis is strictly balanced by its hydrolysis. This calibration procedure was shown to be unaffected by treatments. At equal energization (iso-ΔpH), ATP synthesis was halved by a medium stress and disappeared for a more severe stress, whereas ΔpH at equal energy input (light) declined only under a severe drought. For an identical ΔpH, PS 1-driven phosphorylation is always more efficient than PS 2, both in control and stressed plants. Thus, uncoupling is not the cause of the phosphorylation decline; moreover, retention of a ‘micro-chemiosmotic’ type of coupling implies that the distribution of photosystems and ATPases is unchanged. Parallel to these functional alterations, the lipid content of thylakoids dramatically dropped. As galactolipids fell strongly, neutral lipids rose slightly. Fatty acids decreased then increased with stress, yet phosphorylation did not recover in the latter case and membrane permeability to protons remained unaffected. Overall, these observations suggest a preserved thylakoid structure and this was indeed observed on electron micrographs, even for a severe stress. Therefore, the membrane integrity is probably preserved more by the protein network than by the lipid matrix and the loss of the phosphorylating activity mainly reflects a loss of ATPases or at least their inactivation, possibly due to their altered lipid environment. Finally, from the bioenergetic point of view, the susceptible genotype was unexpectedly less affected by drought than the resistant.

Restoration of thylakoid appression by Δ3-trans-hexadecenoic acid-containing phosphatidylglycerol in a mutant of Chlamydomonas reinhardtii. Relationships with the regulation of excitation energy distribution

The mf 2 mutant of Chlamydomonas reinhardtii lacks both Photosystem II reaction centers (RC II) and Δ3-trans-hexadecenoic fatty acid (C16:1-trans). Previous studies have shown that, by comparison with a simple mutant lacking only RC II, the additional deficiency in C16:1-trans results in: (1) a very low fluorescence yield; (2) a predominant and persistent energy transfer from the main light-harvesting antenna towards Photosystem I, which cannot be reversed by state II-state I transition; (3) absence of the oligomeric form of the chlorophyll a + b-protein complex CP II. These defects can be reversed by incorporating this fatty acid in vivo into the photosynthetic membranes of the mf 2 mutant with C16:1-trans-containing phosphatidylglycerol (PG-C16:1-trans) liposomes. In this paper, subsequent ultrastructural analyses show that: (1) by comparison with the simple RC-II-lacking mutant, the additional C16:1-trans deficiency in the mf 2 mutant results in extensive thylakoid destacking; (2) supplementation of the mf 2 mutant with PG-C16:1-trans liposomes during growth induces a significant re-appression of thylakoid membranes; (3) such restacking was not observed in the presence of cycloheximide or using PG-C16:0 (PG-palmitate) liposomes. A strong correlation therefore appears between the presence of C16:1-trans in the thylakoid membranes of C. reinhardtii and the ability of these membranes to stack, to present the oligomeric organization of CP II and to restore state II-state I transitions in excitation energy distribution between the two photosystems. The possible involvement of PG-C16:1-trans in the synthesis and/or in the stabilization of the CP II apoproteins during the biogenesis of thylakoids is considered.

Correlation between the endogenous circadian rhythmicity in growth rate and fluctuations in oleic acid content in expanding stems of Chenopodium rubrum L.

Chenopodium rubrum L. plants exhibit an endogenous circadian rhythm in their instantaneous stem extension rate in continuous light (A. Lecharny and E. Wagner, 1984, Physiol. Plant. 60, 447–453). Stem extension rate and fatty-acid composition of two stem parts were measured in plants kept in continuous light for 90 h following a 12-h dark period. Fluctuations in the relative size of the oleic acid pool were evidenced in the stem tissues. The peaks (minima and maxima) of the oleic acid content occurred at the same times after the end of the 12-h dark periods as the peaks of the stem extension rate. This rhythmic behaviour ceased when growth was completed. No significant rhythmic changes were observed in any other fatty acid pools. Lipids in which the oleate content is rhythmically modified were exclusively phosphatidylcholine and phospha-tidylethanolamine. Thus, there was a specific correlation between the relative amount of oleic acid in phosphatidylcholine and phosphatidylethanolamine and the rate of instantaneous growth in the same tissue. The rhythmic variations in the oleic acid may be linked to the endomembrane flow in relation to the rate of growth.

Chlorophyll a′ and pheophytin a, as determined by HPLC, in photosynthesis mutants and double mutants of Chlamydomonas reinhardtii

Abstract HPLC analysis of chlorophyll (Chl) a ′ and pheophytin (Pheo) a content was run for wild type Chlamydomonas reinhardtii and seven mutants with impaired photosynthetic functions. Four of them had no or little Photosystem II (PS II) activity. Two had no Photosystem I (PS I) activity and no P-700. In the seventh mutant, both photosystems were present but the main light-harvesting antenna (the chlorophyll-protein complex corresponding to PS II: CP II) was totally lacking because of the absence of Chl b . In mutants impaired in PS II activity, Pheo a is either lacking or found in only low amounts in direct relation with the loss in PS II activity. These results are in agreement with the well-known role of Pheo a as a primary electron acceptor in PS II. In mutants impaired in PS II activity the Chl a ′ level is normal. In mutants impaired in PS I activity (P-700 less mutants), only half of the normal content of Chl a ′ is present. So Chl a ′ is associated exclusively with PS I as demonstrated before by Watanabe (Watanabe, T., Kobayashi, M., Hongu, A., Nakazato, M., Hiyama, T. and Murata, N. (1985) FEBS Lett. 191, 252–256). But only half the Chl a ′ content could be P-700. This last result contradicts Watanabes conclusion that the whole Chl a ′ constitutes P-700. On the other hand, there is no relation between the presence of Chl a ′ and the second Chl a -protein complex of PS I: CP 0a. The possible function of Chl a ′ related to PS I activity is discussed.

Three chloroplast membrane models corresponding to different photosynthetic potentialities in the same plant

The biochemical characteristics and photosynthetic activities of chloroplast membranes of the two regions of the green tomato fruit were compared to those of the leaves which were taken as controls. Membranes may have qualitatively (and even quantitatively) identical components, such as lipids and chlorophylls, and yet have different photosynthetic abilities. Three models then are proposed.

Accumulation of C20 and C22 unsaturated fatty acids in triacylglycerols from developing seeds of Limnanthes alba

Abstract In Limnanthes alba seeds, an accumulation of phosphatidic acid (PA) occurred during seed development until the day 15 after pollination (DAP). Between the 15 and the 20 DAP, a rapid synthesis of triacylglycerols (TG) took place with a simultaneous decrease of PA. During the same period, very long-chain fatty acids (VLCFA), viz. 20: 1 Δ 5 , 22:1 Δ 13 and 22:2Δ 5,13 were synthesized and rapidly channelled into TG. These results suggest the involvement of glycerol-3-phosphate in TG biosynthesis. Moreover, TG synthesis appears to be a time-compartmentalized event; the first accumulation of PA having short-chain acyl moieties then, from the 15 DAP, synthesis of VLCFA which are esterified in TG originating from the PA accumulated during the first period.