Bax-induced cell death in yeast depends on mitochondrial lipid oxidation.

The oxidant function of pro-apoptotic protein Bax was investigated through heterologous expression in yeast. Direct measurements of fatty acid content show that Bax-expression induces oxidation of mitochondrial lipids. This effect is prevented by the coexpression of Bcl-xL. The oxidation actually could be followed on isolated mitochondria as respiration-induced peroxidation of polyunsaturated cis-parinaric acid and on whole cells as the increase in the amount of thiobarbituric acid-reactive products. Treatments that increase the unsaturation ratio of lipids, making them more sensitive to oxidation, increase kinetics of Bax-induced death. Conversely, inhibitors of lipid oxidation and treatments that decrease the unsaturation ratio of fatty acids decrease kinetics of Bax-induced death. Taken together, these results show that Bax-induced mitochondrial lipid oxidation is relevant to Bax-induced cell death. Conversely, lipid oxidation is poorly related to the massive Bax-induced superoxide and hydrogen peroxide accumulation, which occurs at the same time, as chemical or enzymatic scavenging of ROS does not prevent lipid oxidation nor has any effects on kinetics of Bax-induced cell death. Whatever the origin of mitochondrial lipid oxidation, these data show that it represents a major step in the cascade of events leading to Bax-induced cell death. These results are discussed in the light of the role of lipid oxidation both in mammalian apoptosis and in other forms of cell death in other organisms.     

Expression of Rapeseed Microsomal Lysophosphatidic Acid Acyltransferase Isozymes Enhances Seed Oil Content in Arabidopsis

In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.With increasing environmental challenges and concerns, there is renewed interest in deriving plant-based sustainable alternatives for petroleum products, including carburants, lubricants, and industrial feed stocks. Modifying oilseed crops to produce oils of uniform composition containing fatty acids varying in chain length or possessing reactive functional groups is a primary objective (Jaworski and Cahoon, 2003), as is that of increasing the yield of seed oil (Lardizabal et al., 2008; Zheng et al., 2008). Early success in modifying seed oils to produce the more common fatty acids has been tempered by limited success in the production of high levels of unusual fatty acids (UFAs) in cultivated oilseeds (Thelen and Ohlrogge, 2002; Drexler et al., 2003). Such studies have led to the conclusion that in order to achieve levels of UFAs similar to those present in the oil of native species, enzymatic activities additional to fatty acid modification are necessary to optimize the synthesis (Mekhedov et al., 2001), stability (Eccleston and Ohlrogge, 1998), and channeling (Bafor et al., 1990) of the desired fatty acid into triacylglycerol (TAG).The synthesis of glycerolipids occurs in the cytoplasm using de novo-synthesized fatty acids exported from the plastid as acyl-CoA thioesters. The fatty acyl groups are incorporated into membrane and storage lipids by the sequential esterification of glycerol-3-phosphate by the action of glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) at sn-1 to form lysophosphatidic acid followed by lysophosphatidic acid acyltransferase (LPAAT; EC 2.3.1.51) at sn-2 to form phosphatidic acid (PA; Somerville et al., 2000). Dephosphorylation of PA results in the formation of diacylglycerol (DAG), which in developing seeds may be directed into the production of TAG by acyl-CoA-independent reactions or by diacylglycerol acyltransferase (DAGAT; EC 2.3.1.20; Roscoe, 2005). The substrate preferences for acyl-thioesters and the selectivities for the acceptor molecules displayed by the microsomal acyltransferases play a crucial role in establishing the acyl composition of lipids (Frentzen, 1998). The TAG synthesized in most oilseeds of agronomic importance contains fatty acids that are the same as those present in cytoplasmic membrane lipids. In contrast, the seeds of species that synthesize TAGs with exotic fatty acid compositions possess microsomal acyltransferases that facilitate the incorporation of UFAs into storage lipids because of their broad GPAT and/or their selective DAGAT specificities (Wiberg et al., 1994; Frentzen, 1998). Furthermore, oilseeds characterized by TAGs that contain UFAs at sn-2 possess additional seed-specific microsomal LPAATs (Brown et al., 1995; Hanke et al., 1995; Knutzon et al., 1995) that exhibit a wide variation in substrate preference and that serve to ensure the channeling of UFAs to this position, thereby segregating incompatible fatty acids away from membrane lipids.Cloning of cDNAs from cultivated and exotic plants and the availability of entirely sequenced genomes from plant and algal species have revealed that a minimum of two classes of genes encoding microsomal LPAATs exist (Frentzen, 1998) within a larger, LPAAT-like gene family containing acyltransferases as yet functionally uncharacterized but distinct from GPATs (Roscoe, 2005). The class A microsomal LPAATs defined by Frentzen (1998) possess substrate preferences for C18:1-CoA typical of enzymes involved in membrane lipid synthesis and are ubiquitously expressed in the plant. In contrast, individual members of the class B LPAATs display preferences for distinct, unusual saturated or unsaturated acyl groups and are normally expressed in storage organs. Although class B LPAATs have been exploited to alter the stereochemical composition of rapeseed (Brassica napus) oil to permit the incorporation of modified fatty acids at sn-2 (Lassner et al., 1995; Knutzon et al., 1999), a significant increase in the total amount of UFAs was not accomplished by the expression of the class B LPAATs alone. In contrast, the transformation of rapeseed and Arabidopsis (Arabidopsis thaliana) with a yeast gene encoding a variant LPAAT, SLC1-1, capable of accepting very long chain fatty acyl (VLCFA)-CoA substrates resulted in an increase in the total VLCFAs and, unexpectedly, in total oil content (Zou et al. 1997).In our efforts to modify the fatty acid composition of oil in rapeseed, in particular to increase the content of VLCFAs, we have addressed the question of optimizing the environment for the correct functioning of LPAATs encoded by transgenes. The above studies using the various LPAAT transgenes indicate that channeling of UFAs into sn-2 of oilseed species remains problematic. The ability to obtain oils with uniform composition strongly depends on the occupancy of sn-2 by UFAs, yet the level of occupancy of sn-2 by fatty acids corresponding to the selectivity of the introduced LPAAT is variable and relatively modest. Occupancy of sn-2 is determined in part by the ability of the LPAAT encoded by the transgene to compete with the endogenous enzyme, a function of the acyl-CoA substrates available to the enzymes and the relative efficiencies of the enzymes to compete for the donor and acceptor substrates. We argued that there is latitude for the reduction of competing activities using an antisense strategy, and although microsomal LPAATs have been cloned from rapeseed, there are no reports of the characterization of the enzyme. Our objectives in this work were to identify and evaluate the potential contribution of LPAAT isozymes to TAG biosynthesis in rapeseed, thereby discerning targets for optimizing efforts to modify oils for industrial purposes. In this study, we catalogue a previously undescribed complexity in microsomal LPAAT diversity and identify a LPAAT isozyme likely to play an important role in TAG synthesis in rapeseed. In contrast to diverged LPAATs of plant origin, we demonstrate a positive effect of the overexpression of microsomal LPAATs on oil content and seed weight.     

Taxonomy of gymnospermae: multivariate analyses of leaf fatty acid composition

The fatty acid composition of photosynthetic tissues from 137 species of gymnosperms belonging to 14 families was determined by gas chromatography. Statistical analysis clearly discriminated four groups. Ginkgoaceae, Cycadaceae, Stangeriaceae, Zamiaceae, Sciadopityaceae, Podocarpaceae, Cephalotaxaceae, Taxaceae, Ephedraceae and Welwitschiaceae are in the first group, while Cupressaceae and Araucariaceae are mainly in the second one. The third and the fourth groups composed of Pinaceae species are characterized by the genera Larix, and Abies and Cedrus, respectively. Principal component and discriminant analyses and divisive hierarchical clustering analysis of the 43 Pinaceae species were also performed. A clear-cut separation of the genera Abies, Larix, and Cedrus from the other Pinaceae was evidenced. In addition, a mass analysis of the two main chloroplastic lipids from 14 gymnosperms was performed. The results point to a great originality in gymnosperms since in several species and contrary to the angiosperms, the amount of digalactosyldiacylglycerol exceeds that of monogalactosyldiacylglycerol.     

Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane

A large body of evidence from the past decade supports the existence of functional microdomains in membranes of animal and yeast cells, which play important roles in protein sorting, signal transduction, or infection by pathogens. They are based on the dynamic clustering of sphingolipids and cholesterol or ergosterol and are characterized by their insolubility, at low temperature, in nonionic detergents. Here we show that similar microdomains also exist in plant plasma membrane isolated from both tobacco leaves and BY2 cells. Tobacco lipid rafts were found to be greatly enriched in a sphingolipid, identified as glycosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methylcholesterol, and cholesterol. Phospho- and glycoglycerolipids of the plasma membrane were largely excluded from lipid rafts. Membrane proteins were separated by one- and two-dimensional gel electrophoresis and identified by tandem mass spectrometry or use of specific antibody. The data clearly indicate that tobacco microdomains are able to recruit a specific set of the plasma membrane proteins and exclude others. We demonstrate the recruitment of the NADPH oxidase after elicitation by cryptogein and the presence of the small G protein NtRac5, a negative regulator of NADPH oxidase, in lipid rafts.     

Role of cardiolipin on tBid and tBid/Bax synergistic effects on yeast mitochondria

The apoptotic effector Bid regulates cell death at the level of mitochondria. Under its native state, Bid is a soluble cytosolic protein that undergoes proteolysis and yields a 15 kDa-activated form tBid (truncated Bid). tBid translocates to mitochondria and participates in cytochrome c efflux by a still unclear mechanism, some of them at least mediated by Bax. Using mitochondria isolated from wild-type and cardiolipin (CL)-synthase-less yeast strains, we observed that tBid perturbs mitochondrial bioenergetics by inhibiting state-3 respiration and ATP synthesis and that this effect was strictly dependent on the presence of CL. In a second set of experiments, heterologous coexpression of tBid and Bax in wild-type and CL-less yeast strains showed that (i) tBid binding and the subsequent alteration of mitochondrial bioenergetics increased Bax-induced cytochrome c release and (ii) the absence of CL favors Bax effects independently of the presence of t-Bid. These data support recent views suggesting a dual function of CL in mitochondria-dependent apoptosis.