[Show abstract][Hide abstract] ABSTRACT: The three-dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1, 091 NOE-derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (ϕ, ψ, χ1) derived from NOEs and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C-terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413–422) constitutes a new architecture for α-class proteins. 1H NMR and fluorescence studies show that this protein forms well-defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, Kd, of 1.9 ± 0.6 × 10−6 M and >10−3 M were obtained with lyso-C16 and -C12, respectively. A structure model for a lipid—protein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipid—protein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189–199).
[Show abstract][Hide abstract] ABSTRACT: In plants, a temperature downshift represents a major stress that will lead to the induction or repression of many genes. Therefore, the cold signal has to be perceived and transmitted to the nucleus. In response to a cold exposure, we have shown that the phospholipase D (PLD) and the phospholipase C (PLC)/diacylglycerol kinase pathways are simultaneously activated. The role of these pathways in the cold response has been investigated by analyzing the transcriptome of cold-treated Arabidopsis (Arabidopsis thaliana) suspension cells in the presence of U73122 or ethanol, inhibitors of the PLC/diacylglycerol kinase pathway and of the phosphatidic acid produced by PLD, respectively. This approach showed that the expression of many genes was modified by the cold response in the presence of such agents. The cold responses of most of the genes were repressed, thus correlating with the inhibitory effect of U73122 or ethanol. We were thus able to identify 58 genes that were regulated by temperature downshift via PLC activity and 87 genes regulated by temperature downshift via PLD-produced phosphatidic acid. Interestingly, each inhibitor appeared to affect different cold response genes. These results support the idea that both the PLC and PLD pathways are upstream of two different signaling pathways that lead to the activation of the cold response. The connection of these pathways with the CBF pathway, currently the most understood genetic system playing a role in cold acclimation, is discussed.
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositol (PtdIns) synthase 1 from the plant Arabidopsis thaliana has been expressed in Escherichia coli in order to study the synthetic capacities of the enzyme. Analysis of the total fatty acid content of the bacteria shows that PtdIns synthase activity does not have a profound effect on the proportions of the different fatty acids produced, even if the presence of an extra acidic phospholipid leads to a global reduction of the lipid content. A closer analysis carried out on individual phospholipids reveals a global fatty acid composition almost unchanged in the two major bacterial lipids phosphatidylethanolamine (PtdEtn) and phosphatidylglycerol (PtdGro). Phosphatidylinositol has a very unusual composition that shows the ability of the plant enzyme to use CDP-diacylglycerol molecular species absent from plants. We identified the various PtdIns molecular species. They represent a pool of the major molecular species of PtdEtn and PtdGro. These results, together with the determination of the apparent affinity constants of AtPIS1 for myo-inositol and CDP-diacylglycerol, allow us to discuss some of the constraints of PtdIns synthesis in plants in terms of specificity, which will depend on the subcellular localization of the protein.
[Show abstract][Hide abstract] ABSTRACT: Two types of phospholipid degrading enzyme, phospholipase D (PLD; EC 188.8.131.52) and phosphatidyl- inositol-specific phospholipase C (PIP(2)-PLC; PI-PLC 184.108.40.206) were studied during the development of seeds and plants of Brassica napus. PLD exhibits two types of activity; polyphosphoinositide-requiring (PIP(2)-dependent PLD) and polyphosphoinositide-independent requiring millimolar concentrations of calcium (PLDalpha). Significantly different patterns of activity profiles were found for soluble and membrane-associated forms of all three enzymes within both processes. Membrane-associated PIP(2)-dependent PLD activity shows the opposite trend when compared to PLDalpha, while the highest PI-PLC activity appears in the same stages of development of seeds and plants as for PLDalpha. In subcellular fractions of hypocotyls of young plants, phospholipases were localized predominantly on plasma membranes. The biochemical characteristics (Ca(2+), pH) of all three enzymes associated with plasma membrane vesicles, isolated by partitioning in an aqueous dextran- polyethylene glycol two-phase system, are also described. Direct interaction of PLDalpha with G-proteins under in vitro conditions was not confirmed.
Journal of Experimental Botany 03/2003; 54(383):691-8. · 5.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The signaling events generated by a cold exposure are poorly known in plants. We were interested in checking the possible activation of enzymes of the phosphoinositide signaling pathway in response to a temperature drop. In Arabidopsis suspension cells labeled with (33)PO(4)(3-), a cold treatment induces a rapid increase of phosphatidic acid (PtdOH) content. This production was due to the simultaneous activation of phospholipase C (through diacylglycerol kinase activity) and phospholipase D, as monitored by the production of inositol triphosphate and of transphosphatidylation product, respectively. Moreover, inhibitors of the phosphoinositide pathway and of diacylglycerol kinase reduced PtdOH production. Enzyme activation occurred immediately after cells were transferred to low temperature. The respective contribution of both kind of phospholipases in cold-induced production of PtdOH could be estimated. We created conditions where phospholipids were labeled with (33)PO(4)(3-), but with ATP being nonradioactive. In such conditions, the apparition of radioactive PtdOH reflected PLD activity. Thus, we demonstrated that during a cold stress, phospholipase D activity accounted for 20% of PtdOH production. The analysis of composition in fatty acids of cold-produced PtdOH compared with that of different phospholipids confirmed that cold-induced PtdOH more likely derived mainly from phosphoinositides. The addition of chemical reagents modifying calcium availability inhibited the formation of PtdOH, showing that the cold-induced activation of phospholipase pathways is dependent on a calcium entry.
[Show abstract][Hide abstract] ABSTRACT: Rape (Brassica napus L. var. Bienvenue) is a 16:3 plant which contains predominantly prokaryotic species of monogalactosyldiacylglycerol i.e. sn-1 C18, sn-2 C16 (C18/C16 MGDG). Rape plants were exposed to a restricted water supply for 12 days. Under drought conditions, considerable changes in lipid metabolism were observed. Drought stress provoked a decline in leaf polar lipids, which is mainly due to a decrease in MGDG content. Determination of molecular species in phosphatidylcholine (PC) and MGDG indicated that the prokaryotic molecular species of MGDG (C18/C16) decreased after drought stress while the eukaryotic molecular species (C18/C18) remained stable. Drought stress had different effects on two key enzymes of PC and MGDG synthesis. The in vitro activity of MGDG synthase (EC. 220.127.116.11) was reduced in drought stressed plants whereas cholinephosphotransferase (EC. 18.104.22.168) activity was not affected. Altogether these results suggest that the prokaryotic pathway leading to MGDG synthesis was strongly affected by drought stress while the eukaryotic pathway was not. It was also observed that the molecular species of leaf PC became more saturated in drought stressed plants. This could be due to a specific decrease in oleate desaturase activity.
Physiologia Plantarum 07/2002; 115(2):221-227. DOI:10.1034/j.1399-3054.2002.1150207.x · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to study some of its enzymatic properties, phosphatidylinositol synthase 1 (AtPIS1) from the plant Arabidopsis thaliana was expressed in Escherichia coli, a host naturally devoid of phosphatidylinositol (PtdIns). In the context of the bacterial membrane and in addition to de novo synthesis, the plant enzyme is capable of catalysing the exchange of the inositol polar head for another inositol. Our data clearly show that the CDP-diacylglycerol-independent exchange reaction can occur using endogenous PtdIns molecular species or PtdIns molecular species from soybean added exogenously. Exchange has been observed in the absence of cytidine monophosphate (CMP), but is greatly enhanced in the presence of 4 microm CMP. Our data also show that AtPIS1 catalyses the removal of the polar head in the presence of much higher concentrations of CMP, in a manner that suggests a reverse of synthesis. All of the PtdIns metabolizing activities require free manganese ions. EDTA, in the presence of low Mn2+ concentrations, also has an enhancing effect.
European Journal of Biochemistry 06/2002; 269(9):2347-52. DOI:10.1046/j.1432-1033.2002.02893.x · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A cDNA (Vupat1) encoding a predicted 43 kDa protein was isolated from drought-stressed cowpea (Vigna unguiculata) leaves. It has homology with patatin, a potato tuber storage protein with lipolytic acyl hydrolase activity. The recombinant protein VUPAT1 expressed in the baculovirus system displays preferentially galactolipid acyl hydrolase activity. Phospholipids are very slowly hydrolyzed and apparently triacylglycerols are not deacylated. Vupat1 promoter contains putative drought-inducible sequences. Northern blots showed that gene expression is stimulated by drought stress and is more pronounced in a drought-sensitive cultivar than in a drought-tolerant one. An involvement in drought-induced galactolipid degradation is proposed for VUPAT1.
[Show abstract][Hide abstract] ABSTRACT: This paper reports the cloning of a cDNA (Vupat1) expressed in Vigna unguiculata leaves coding for a protein with 48% sequence homology to patatin, the major protein from potato tuber which has lipolytic acylhydrolase activity. Two cultivars differing in drought tolerance were examined in Northern-blot analyses. Expression of Vupat1 is stimulated by drought stress, especially in the drought-sensitive cultivar. Vupat1 was expressed in the baculovirus system as a fusion protein secreted in the culture medium. The recombinant protein displays lipolytic activity towards monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerols.
Biochemical Society Transactions 01/2001; 28(6):779-81. DOI:10.1042/BST0280779 · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lipid transfer proteins (LTPs) are small, basic and abundant proteins in higher plants. They are capable of binding fatty acids and of transferring phospholipids between membranes in vitro. LTPs from this family contain a signal peptide and are secreted in the cell wall. Their biological function is presently unknown. LTPs have been suggested to participate to cutin assembly and to the defense of the plants against pathogens. A genetic approach should prove useful to provide clues on their in vivo functions. Here, the characterization of the LTP gene family in Arabidopsis thaliana is described. At least 15 genes were identified, their map position determined and the expression pattern characterized for six of them. All the sequences exhibit the typical features of plant LTPs. The molecular weight is close to 9 kDa, the isoelectric point is near 9 (except for three acidic LTPs), and typical amino acid residues such as cysteines are conserved. Genomic DNA blotting hybridization experiments performed using ltp1 to ltp6 as probes indicate that ltps form distinct 1–3 gene subfamilies which do not cross hybridize. Expression studies indicate that all the genes tested are expressed in flowers and siliques, but not in roots. Ltp1, ltp5 and ltp2 are expressed significantly in leaves, while ltp6 is detected only in 2–4-week-old leaves. In addition, ltp4 and ltp3 are strongly upregulated by abscisic acid (ABA). Tandem repeats can be noted concerning ltp1 and ltp2 on chromosome 2, ltp3 and ltp4 on chromosome 5 and ltp5 and ltp12 on chromosome 3. While ltp7, ltp8 and ltp9 map at the same position on chromosome 2, the other genes are dispersed throughout the genome. The characterization of the Arabidopsis ltp gene family will permit to initiate a genetic approach for determining the in vivo function(s) of these proteins.
[Show abstract][Hide abstract] ABSTRACT: The biosynthesis of polyphosphoinositides was studied in cultured tobacco cells from Wisconsin 38 (W 38) and Bright yellow 2 (BY 2) lines. Following changes in the culture conditions the labelling with [14C] inositol or [33P] orthophosphate of the phosphoinositides and other phospholipids was largely affected. The labelling depended on the growth stage and was higher in the actively growing BY 2 cells than in W 38 cells. Addition of hormones such as 2,4-D or abscisic acid (ABA) resulted in transient changes in the labelling of the PI cycle metabolites whereas a cold stress preferentially stimulated PA and PIP labelling. In such cells, the limited change of PIP2 compared with the high level of labelled PIP and/or PA suggested a direct participation of these latter compounds to the signalling process in tobacco cells.
[Show abstract][Hide abstract] ABSTRACT: Like other eukaryotic cells, plant cells contain proteins able to bind or to transfer lipids. Since they are able to facilitate movements of various phospholipids between membranes and are also capable of binding fatty acids or acyl-CoAs, they have been termed lipid-transfer proteins (LTP). LTPs are basic proteins containing 90 to 95 residues (molecular mass 9 kDa), eight of them being cysteines found in conserved locations. These proteins have been used to manipulate in vitro the lipid composition of isolated membranes either from plant or mammalian sources. In addition to purified LTPs, recombinant LTPs produced by genes expressed in microorganisms can be used for this purpose. Several genes coding for these proteins have been characterized in various plants with different patterns of expression. However, it remains to be investigated whether these recombinant proteins behave functionally as LTPs. The use of purified or recombinant LTPs is promising for the study of the effect of lipid composition on membrane functional properties.
[Show abstract][Hide abstract] ABSTRACT: Lipid-transfer proteins are small, basic proteins, and have been purified from various plant sources. They are able to transfer lipids between membranes in vitro and, on the basis of this, were initially thought to participate in the intracellular flux of lipids during membrane synthesis. However, the finding that these proteins are located in the cell wall and can be secreted has led to the suggestion that they are not required for intracellular lipid transport. Instead, they may be involved in cutin biosynthesis, surface wax formation, pathogen-defence reactions, or the adaptation of plants to environmental changes.
[Show abstract][Hide abstract] ABSTRACT: Lipid-transfer proteins (LTP) are basic, 9-kDa proteins present in high amounts (as much as 4% of the total soluble proteinss) in higher plants. LTPs can enhance the in vitro transfer of phospholipids between membranes and can bind acyl chains. On the basis of these properties, LTPs were thought to participate in membrane biogenesis and regulation of the intracellular fatty acid pools. However, the isolation of several cDNAs and genes revealed the presence of a signal peptide indicating that LTPs could enter the secretory pathway. They were found to be secreted and located in the cell wall. Thus, novel roles were suggested for plant LTPs: participation in cutin formation, embryogenesis, defense reactions against phytopathogens, symbiosis, and the adaptation of plants to various environmental conditions. The validity of these suggestions needs to be determined, in the hope that they will elucidate the role of this puzzling family of plant proteins.
[Show abstract][Hide abstract] ABSTRACT: Detergent-solubilized cytochrome b5 from potato tuber microsomes was purified in its native form to aspecific content of 25.8 nmol per mg of protein, by using a combination of conventional and high performance (FPLC) chromatography. The highly purified cytochrome bs has an apparent molecular mass of 17 kDa on SDS-PAGE.
[Show abstract][Hide abstract] ABSTRACT: A non-specific lipid transfer protein isolated from coleoptiles of maize (Zea mays L.) was used to study the transfer of [3H]-labelled phosphatidylinositol in reconstituted systems isolated from seedlings of soybean (Glycine max [L.] Merrill). Inclusion of lipid transfer protein in the assay stimulated transfer of phosphatidylinositol from liposomes or from a fraction enriched in endoplasmic reticulum to plasma membrane vesicles in solution or immobilized on strips of nitrocellulose. The rate of transfer increased with increasing concentration of lipid transfer protein and with increasing concentration of acceptor plasma membrane present in the assay. During post-transfer incubation of strips with ATP and without lipid transfer proteins, a portion of the phosphatidylinositol transferred to the plasma membrane vesicles was further metabolized into lysophosphatidylinositol in the absence of Mg2+, and, in the presence of Mg2+ATP, to lysophosphatidylinositol, phosphatidylinositolmonophosphate and, to a small extent, phosphatidylinositolbisphosphate. This result demonstrates that the transferred lipid had been incorporated in the plasma membrane to function as substrate for lipid-metabolizing enzymes. Taken together, our results are in agreement with the hypothesis that lipid transfer proteins are involved in the intracellular phosphatidylinositol dynamics in plant cells.
[Show abstract][Hide abstract] ABSTRACT: The approaches of molecular biogenesis have been increasingly used these recent years in order to explore and to control the lipid metabolism of plants. This is linked to the important roles played by the lipid constituents in plant cells, especially at the level of membranous processes, and, also, to the biotechnological use of storage lipids. The approaches of molecular biology are used either after purification of the enzymes corresponding to steps of lipid biosynthesis—fatty acid synthase, desaturases, acyltransferases—and isolation of the corresponding genes or by complementation of mutants disturbed in one of these genes. The genes or cDNAs, coding for several key enzymes of lipid biosynthesis, recently characterized, have been used to manipulate the fatty acid composition of storage lipids, especially in rapeseed and Arabidopsis, in order to obtain oils rich in saturated fatty acids or shorter acyl chains. Fascinating perspectives related to lipid manipulation, either to improve the resistance of plants against environmental stresses or to develop their bio- technological interest, are thus opened for the next future years.
Acta botanica Gallica: bulletin de la Société botanique de France 01/1993; 140(7):735-754. DOI:10.1080/12538078.1993.10515673 · 0.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A small basic 10 kD abundant protein in barley seeds can convey up to 7% of the phosphatidylcholine in liposomes to potato
mitochondria whereas cholesteryloleate is not transported. The demonstration of this activity combined with a somewhat more
than 50% homology of its primary structure to that of other plant phospholipid transfer proteins are the bases for our naming
it a barley lipid transfer protein (LTP).
Carlsberg Research Communications 03/1989; 54(2):81-84. DOI:10.1007/BF02907587
[Show abstract][Hide abstract] 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.