The physiological role of ceramide formation in response to cell stimulation remains controversial. Here, we emphasize that ceramide is not a priori an apoptotic signalling molecule. Recent work points out that the conversion of sphingomyelin into ceramide can play a membrane structural (physical) role, with consequences for membrane microdomain function, membrane vesiculation, fusion/fission and vesicular trafficking. These processes contribute to cellular signalling. At the Golgi, ceramide takes part in a metabolic flux towards sphingomyelin, diacylglycerol and glycosphingolipids, which drives lipid raft formation and vesicular transport towards the plasma membrane. At the cell surface, receptor clustering in lipid rafts and the formation of endosomes can be facilitated by transient ceramide formation. Also, signalling towards mitochondria may involve glycosphingolipid-containing vesicles. Ceramide may affect the permeability of the mitochondrial outer membrane and the release of cytochrome c. In the effector phase of apoptosis, the breakdown of plasma membrane sphingomyelin to ceramide is a consequence of lipid scrambling, and may regulate apoptotic body formation. Thus ceramide formation serves many different functions at distinct locations in the cell. Given the limited capacity for spontaneous intracellular diffusion or membrane flip-flop of natural ceramide species, the topology and membrane sidedness of ceramide generation are crucial determinants of its impact on cell biology.

Phosphatidic acid (PA) and its phosphorylated derivative diacylglycerol pyrophosphate (DGPP) are lipid molecules that have been implicated in plant cell signaling. In this study we report the rapid but transient accumulation of PA and DGPP in suspension-cultured tomato (Lycopersicon esculentum) cells treated with the general elicitors, N,NЈ,NЉ,Nٞ-tetraacetylchitotetraose, xylanase, and the flagellin-derived peptide flg22. To determine whether PA originated from the activation of phospholipase D or from the phosphorylation of diacylglycerol (DAG) by DAG kinase, a strategy involving differential radiolabeling with [ 32 P]orthophosphate was used. DAG kinase was found to be the dominant producer of PA that was subsequently metabolized to DGPP. A minor but significant role for phospholipase D could only be detected when xylanase was used as elicitor. Since PA formation was correlated with the high turnover of polyphosphoinositides, we hypothesize that elicitor treatment activates phospholipase C to produce DAG, which in turn acts as substrate for DAG kinase. The potential roles of PA and DGPP in plant defense signaling are discussed.

The synthetic alkyl-lysophospholipid (ALP), 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, is an antitumor agent that acts on cell membranes and can induce apoptosis. We investigated how ALP is taken up by cells, how it affects de novo biosynthesis of phosphatidylcholine (PC), and how critical this is to initiate apoptosis. We compared an ALP-sensitive mouse lymphoma Furthermore, ALP was found accumulated in isolated rafts and disruption of rafts also prevented the inhibition of PC synthesis and apoptosis induction in S49 cells. In summary, ALP is internalized by raft-dependent endocytosis to inhibit PC synthesis, which triggers apoptosis.

Exposure of plants to elevated temperatures results in a complexThe responses of plants to HS have received increasing attention in recent years. Elevated temperatures initiate changes in transcription and selective translation of HS mRNA encoding HSPs, thereby enhancing thermotolerance of treated plants (Nover et al., 1989; Nover, 1991;Vierling, 1991; Howarth and Ougham, 1993;Waters et al., 1996). However, the pathways by which HS signals are perceived and transduced to activate gene expression of HSPs and to induced thermotolerance are not understood.In recent years a second-messenger Ca 2ϩ was found to be involved in the perception and regulation of many responses of plants to environmental signals (Gilroy et al., 1993; Poovaiah and Reddy, 1993; Gilroy and Trewavas, 1994; Bush, 1995;Braam et al., 1996;Webb et al., 1996). [Ca 2ϩ ] cyt often shows significant changes in plant cells under the influence of various stress signals such as touch, wind stimulation, cold shock, wounding, and mechanical stimulation (Knight et al., 1991(Knight et al., , 1992(Knight et al., , 1993 Haley et al., 1995;Campbell et al., 1996; Polisensky and Braam, 1996), oxidative stress (Price et al., 1994), salinity (Lynch et al., 1989; Bush, 1996; Okazaki et al., 1996), anoxia (Subbaiah et al., 1994a; Bush, 1996;Sedbrook et al., 1996), and hypoosmotic shock (Takahashi et al., 1997). It has been suggested that a stress-induced change in [Ca 2ϩ ] cyt might be one of the primary transduction mechanisms whereby gene expression and biochemical events are altered to adapt plant cells to environmental stresses (Monroy et al., 1993;Subbaiah et al., 1994aSubbaiah et al., , 1994b Monroy and Dhindsa, 1995;Braam et al., 1996).Several authors have suggested that Ca 2ϩ -mediated second-messenger systems might be involved in the HS responses of animal cells (Lamarche et al., 1985; Calderwood et al., 1988; Landry et al., 1988; Mosser et al., 1990), although other results indicated that Ca 2ϩ was not strictly required for some HSP synthesis (Drummond et al., 1986(Drummond et al., , 1988. In plant cells Klein and Ferguson (1987) observed that the uptake of Ca 2ϩ by suspension-cultured pear cells or protoplasts was significantly enhanced during heat stress. Braam (1992) demonstrated that HS induced a strongly up-regulated expression of calmodulin-related TCH genes in cultured Arabidopsis cells, and external Ca 2ϩ was required for maximal HS induction of these TCH genes. Wu et al. (1992) also indicated that pretreatment of hypocotyl segments and etiolated seedlings of Brassica napus with the Ca 2ϩ ionophore A23187 or the Ca 2ϩ chelator EGTA to modify Ca 2ϩ homeostasis resulted in changes in the synthesis of HSPs. Using the fluorescent dye Indo-1, Biyaseheva et al. (1993) reported that HS induced a 4-fold increase in [Ca 2ϩ ] cyt in pea mesophyll protoplasts, but the further dynamic changes in [Ca 2ϩ ] cyt during HS could not be detected because of limitations in the technique.We recently described a novel technology to measure [Ca 2ϩ ] ...

Cold shock and wind stimuli initiate Ca(2+) transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca(2+) pathways that are spatially distinct, stress-induced nuclear and cytoplasmic Ca(2+) transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca(2+) signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca(2+) dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca(2+) signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm.