The complete general secretory pathway in gram-negative bacteria

Phosphatidic acid (PtdOH) is emerging as an important signalling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using 32P-labelled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid 32P-PtdOH increase, peaking within 2 and 5 min, respectively. In principle, PtdOH can be generated through three different pathways, i.e. i) via de novo phospholipid biosynthesis (through acylation of lyso-PtdOH), ii) via phospholipase D hydrolysis of structural phospholipids or iii) via phosphorylation of diacylglycerol (DAG) by DAG kinase (DGK). Using a differential 32P-labelling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid 32P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of 32P-PtdInsP, correlating in time, temperature dependency and magnitude with the increase in 32P-PtdOH, suggested that a PtdInsP-hydrolyzing PLC generated the DAG in this reaction. Testing T-DNA insertion lines available for the seven DGK genes, revealed no clear changes in 32P-PtdOH responses, suggesting functional redundancy. Similarly, known cold-stress mutants were analyzed to investigate whether the PtdOH response acted downstream of the respective gene products. The hos1, los1 and fry1 mutants were found to exhibit normal PtdOH responses. Slight changes were found for ice1, snow1, and the overexpression line Super-ICE1, however, this was not cold-specific and likely due to pleiotropic effects. A tentative model illustrating direct cold effects on phospholipid metabolism is presented.

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Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase

The signal peptide.

Phospholipids play multiple roles in cells by establishing the permeability barrier for cells and cell organelles, by providing the matrix for the assembly and function of a wide variety of catalytic processes, by acting as donors in the synthesis of macromolecules, and by actively influencing the functional properties of membrane-associated processes. The function, at the molecular level, of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin in specific cellular processes is reviewed, with a focus on the results of combined molecular genetic and biochemical studies in Escherichia coli. These results are compared with primarily biochemical data supporting similar functions for these phospholipids in eukaryotic organisms. The wide range of processes in which specific involvement of phospholipids has been documented explains the need for diversity in phospholipid structure and why there are so many membrane lipids.

MOLECULAR BASIS FOR MEMBRANE PHOSPHOLIPID DIVERSITY: Why Are There So Many Lipids?

Plants produce a diverse array of secondary metabolites, many of which have antifungal activity. Some of these compounds are constitutive, existing in healthy plants in their biologically active forms. Others, such as cyanogenic glycosides and glucosinolates, occur as inactive precursors and are activated in response to tissue damage or pathogen attack. This activation often involves plant enzymes, which are released as a result of breakdown in cell integrity. Compounds belonging to the latter category are still regarded as constitutive because they are immediately derived from preexisting constituents (Mansfield, 1983). VanEtten et al. (i994) have proposed the term “phytoanticipin” to distinguish these preformed antimicrobial compounds from phytoalexins, which are synthesized from remote precursors in response to pathogen attack, probably as a result of de novo synthesis of enzymes. In recent years, studies of plant disease resistance mechanisms have tended to focus on phytoalexin biosynthesis and other active responses triggered after pathogen attack (Hammond-Kosack and Jones, 1996, in this issue). In contrast, preformed inhibitory compounds have received relatively little attention, despite the fact that these plant antibiotics are likely to represent one of the first chemical barriers to potential pathogens. The distribution of preformed inhibitors within plants is often tissue specific (e.g., Price et ai., 1987; Poulton, 1988; Davis, 1991; Fenwick et ai., 1992; Bennett and Wallsgrove, 1994), and r there is a tendency for these compounds to be concentrated in the outer cell layers of plant organs, suggesting that they may indeed act as deterrents to pathogens and pests. Some diffusible preformed inhibitors, such as catechol and protocatechuic acid (which are found in onion scales), may influence fungal growth at the plant surface. In general, however, preformed antifungal compounds are commonly sequestered in vacuoles or organelles in healthy plants. Therefore, the concentrations that are encountered by an invading fungus will depend on the extent to which that fungus causes tissue damage. Biotrophs may avoid the release of preformed inhibitors by minimizing damage to the host, whereas necrotrophs are likely to cause substantial release of these compounds. The nature and leve1 of preformed inhibitors to which a potential pathogen is exposed will also vary, depending on factors such as host genotype, age, and environmental conditions (Price et ai., 1987; Davis, 1991). There have been numerous attempts to associate natural variation in levels of preformed inhibitors in plants with resistance to particular pathogens, but often these attempts have failed to reveal any positive correlation. However, whereas preformed inhibitors may be effective against a broad spectrum of potential pathogens, successful pathogens are likely to be able to circumvent the effects of these antibiotics either by avoiding them altogether or by tolerating or detoxifying them (Schonbeck and Schlosser, 1976; Fry and Myers, 1981; Bennett and Wallsgrove, 1994; VanEtten et al., 1995; Osbourn, 1996). The isolation of plant mutants defective in the biosynthesis of preformed inhibitors would allow a direct genetic test of the importance of these compounds in plant defense. However, in most cases this approach is technically difficult because of the problems associated with screening for lossof the compounds. In the absence of plant mutants, a complementary approach involving the study of fungal mechanisms of resistance to preformed inhibitors, and of the contribution of this resistance to fungal pathogenicity to the relevant host plants, offers another route toward investigating the importance of these inhibitors in plant defense. A large number of constitutive plant compounds have been reported to have antifungal activity. Well-known examples include phenols and phenolic glycosides, unsaturated lactones, sulphur compounds, saponins, cyanogenic glycosides, and glucosinolates (reviewed in Ingham, 1973; Schonbeck and Schlosser, 1976; Fry and Myers, 1981; Mansfield, 1983; Ku6, 1992; Bennett and Wallsgrove, 1994; Grayer and Harborne, 1994; Osbourn, 1996). More recently, 5-alkylated resorcinols and dienes have been associated with disease resistance, in this case, resistance of subtropical fruits to infection by Collefotrichum gloeosporioides (Prusky and Keen, 1993). However, only a few classes of preformed inhibitor have been studied in detail to determine their possible roles in plant defense against fungal pathogens. This review focuses on three of these classes-saponins, cyanogenic glycosides, and glucosinolates-and summarizes our current knowledge of the role of these preformed inhibitors in determining the outcome of encounters between plants and phytopathogenic fungi.

Preformed Antimicrobial Compounds and Plant Defense against Fungal Attack.

We provide direct evidence for phospholipase D (PLD) signaling in plants by showing that this enzyme is stimulated by the G protein activators mastoparan, ethanol, and cholera toxin. An in vivo assay for PLD activity in plant cells was developed based on the use of a "reporter alcohol" rather than water as a transphosphatidylation substrate. The product was a phosphatidyl alcohol, which, in contrast to the normal product phosphatidic acid, is a specific measure of PLD activity. When 32P-labeled cells were treated with 0.1% n-butanol, 32P-phosphatidyl butanol (32P-PtdBut) was formed in a time-dependent manner. In cells treated with any of the three G protein activators, the production of 32P-PtdBut was increased in a dose-dependent manner. The G protein involved was pertussis toxin insensitive. Ethanol could activate PLD but was itself consumed by PLD as transphosphatidylation substrate. In contrast, secondary alcohols (e.g., sec-butyl alcohol) activated PLD but did not function as substrate, whereas tertiary alcohols did neither. Although most of the experiments were performed with the green alga Chlamydomonas eugametos, the relevance for higher plants was demonstrated by showing that PLD in carnation petals could also be activated by mastoparan. The results indicate that PLD activation must be considered as a potential signal transduction mechanism in plants, just as in animals.

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G Protein Activation Stimulates Phospholipase D Signaling in Plants.

Newly synthesized proteins to be exported out of the cytoplasm of bacterial cells have to pass across the inner membrane. In Gram-negative bacteria ATP, a membrane potential, the products of the sec genes and leader peptidases (enzymes which cleave the N-terminal signal peptides of the precursor proteins) are required. The mechanism of translocation, however, remains elusive. Important additional roles for membrane lipids have been repeatedly suggested both on theoretical grounds and on the basis of experiments with model systems but no direct evidence had been obtained. We demonstrate here, using mutants of Escherichia coli defective in the synthesis of the major anionic membrane phospholipids, that phosphatidylglycerol is involved in the translocation of newly synthesized outer-membrane proteins across the inner membrane.
AD	- 	Department of Biochemistry, University of Utrecht, The Netherlands.

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Phosphatidylglycerol is involved in protein translocation across Escherichia coli inner membranes.

Molecular basis of glycoalkaloid induced membrane disruption.

Identification of Diacylglycerol Pyrophosphate as a Novel Metabolic Product of Phosphatidic Acid during G-protein Activation in Plants *

Optimal posttranslational translocation of the precursor of PhoE protein across Escherichia coli membrane vesicles requires both ATP and the protonmotive force

T. de Vrije

Papers

G Protein Activation Stimulates Phospholipase D Signaling in Plants.

Phosphatidylglycerol is involved in protein translocation across Escherichia coli inner membranes.

Molecular basis of glycoalkaloid induced membrane disruption.

Identification of Diacylglycerol Pyrophosphate as a Novel Metabolic Product of Phosphatidic Acid during G-protein Activation in Plants *

Optimal posttranslational translocation of the precursor of PhoE protein across Escherichia coli membrane vesicles requires both ATP and the protonmotive force