Sphingolipids related to apoptosis from the point of view of membrane structure and topology.

Sphingolipids and their metabolites are implicated in signal transduction, but the mechanisms are still poorly understood. In particular, the presumed function of ceramide as a second messenger remains controversial. Here, we emphasize the importance of both ceramide and sphingomyelin for membrane structure. The effects of sphingolipid turnover in the induction and effector phases of apoptosis are explained by their impact on membrane microdomains that are relevant for cell signalling or changes in morphology. The topology of sphingolipid metabolism is important because of their limited transbilayer and inter-membrane movement. For instance, glycosylceramide synthase converts de novo synthesized ceramide to glycosylceramide, but it is neither a general attenuator of ceramide accumulation at the plasma membrane, nor of the apoptotic process. Synthetic alkyl-lysophospholipids modulate membrane-lipid composition and, therefore, apoptosis sensitivity.

The calcium rhythms of different cell types oscillate with different circadian phases.

Transgenic tobacco (Nicotiana plumbaginifolia) seedlings containing the Ca(2+)-sensitive luminescent protein aequorin have been shown to exhibit circadian variations in cytosolic calcium. Concomitant measurements of cytosolic and nuclear calcium show that circadian variations in the cytoplasm are not expressed in the nucleus. To investigate whether all cells of transgenic seedlings contribute equally to circadian variations in cytosolic calcium, different promoters eliciting different expression patterns have been placed upstream of aequorin and used for transformation. The circadian peak occurred at different times in the three transgenic lines constructed. Luminescence imaging of these transgenic lines indicated that aequorin was differentially accumulated among the main tissues and cells of the seedlings and overcoat technology with applied epidermal strips indicated that the surface cell layers contribute the vast majority of luminescent light. We conclude that the Ca(2+) rhythmicities of cells and tissues oscillate with distinct differences in phase, that this might represent different underlying cellular control mechanisms and that these observations have significant implications for our understanding and study of Ca(2+) mediated signal transduction in plant cells.

Elicitation of suspension-cultured tomato cells triggers the formation of phosphatidic acid and diacylglycerol pyrophosphate.

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,N',N",N"'-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.

Distinct calcium signaling pathways regulate calmodulin gene expression in tobacco.

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.

Identification of Endogenous Gibberellins in Petunia Flowers (Induction of Anthocyanin Biosynthetic Gene Expression and the Antagonistic Effect of Abscisic Acid).

The elongation and pigmentation of corollas of Petunia hybrida requires the presence of anthers. The ability of exogenous gibberellic acid (GA3) to substitute for the anthers suggests a role for endogenous GAs. Here we report the identification of endogenous GAs in corollas and in anthers and show that both tissues contain detectable levels of GA1, GA4, and GA9, of which GA4 is the most abundant. These GAs stimulate corolla pigmentation, chalcone synthase (chs) mRNA accumulation, and chs transcription in an in vitro flower bud culture system. Methyl ester derivatives of GA3 and GA4 were not active but did not inhibit the bioactive GAs. Even though it is unknown whether abscisic acid (ABA) is involved in corolla maturation, ABA inhibited pigmentation of intact flowers, overruling the effect of the anthers. In detached flower buds it was shown that ABA prevented activation of the chs promoter by GA3. The synthesis of anthocyanin pigments requires the coordinate expression of at least 15 structural genes. Expression of early biosynthetic genes and of late biosynthetic genes are regulated by different transcriptional activators. GA induces both classes of genes with similar kinetics, indicating that GA acts relatively early in the signaling pathway.

The petunia homologue of the Antirrhinum majus candi and Zea mays A2 flavonoid genes; homology to flavanone 3-hydroxylase and ethylene-forming enzyme.

The synthesis of anthocyanins in higher plants involves many enzymatic steps. Here we describe the isolation and characterization of a cDNA, ant17, which encodes a protein that has 73% amino acid sequence identity with the candi gene product of Antirrhinum majus and 48% with that of the maize a2 gene. This protein may therefore be involved in the synthesis of anthocyanins in the steps after the action of dihydroflavonol 4-reductase. This is consistent with the absence of ant17 expression in the regulatory anthocyanin mutants of petunia an1, an2 and an11. Furthermore, ant17 is predominantly expressed in corollas and anthers and is induced by gibberellic acid.