ABSTRACT
We have recently detected phosphatidylinositol-4-phosphate (PI4P) in the extracellular medium of tomato cell suspensions. Extracellular PI4P was shown to trigger the activation of defence responses induced by the fungal elicitor xylanase. In this study, by applying a differential centrifugation technique, we found that extracellular PI4P is associated with fractions composed of diverse phospholipids and proteins, which were pelleted from the extracellular medium of tomato cell suspensions grown under basal conditions. Using mass spectrometry, we identified the proteins present in these pelleted fractions. Most of these proteins have previously been characterised as having a role in defence responses. Next, we evaluated whether PI4P could also be detected in an entire plant system. For this, apoplastic fluids of tomato plants grown under basal conditions were analysed using a lipid overlay assay. Interestingly, PI4P could be detected in intercellular fluids obtained from tomato leaflets and xylem sap of tomato plants. By employing electrospray ionisation tandem mass spectrometry (ESI-MS/MS), other phospholipids were also found in intercellular fluids of tomato plants. These had a markedly different profile from the phospholipid pattern identified in entire leaflets. Based on these results, the potential role of extracellular phospholipids in plant intercellular communication is discussed.
Subject(s)
Phosphatidylinositol Phosphates/biosynthesis , Plant Proteins/metabolism , Solanum lycopersicum/metabolism , Cells, Cultured , Disease Resistance , Extracellular Fluid/metabolism , Solanum lycopersicum/chemistry , Mass Spectrometry , Phospholipids/metabolism , Plant Leaves/chemistry , Plant Proteins/analysis , Spectrometry, Mass, Electrospray Ionization , Xylem/chemistryABSTRACT
Phospholipase D (PLD, EC 3.1.4.4.) has been implicated in a variety of plant processes, including signalling. In Arabidopsis thaliana a PLD gene family has been described and individual members classified into alpha-, beta- and gamma-classes. Here we describe a second PLD gene family in tomato (Lycopersicon esculentum) that includes three alpha- and two beta-classes. Different expression patterns in plant organs were observed for each PLD. In testing a variety of stress treatments on tomato cell suspensions, PLDbeta1 mRNA was found to rapidly and specifically accumulate in response to the fungal elicitor xylanase. The greatest increase was found 2 h after treatment with 100 microg m1(-1) xylanase (ninefold). In vivo PLD activity increased nearly threefold over a 1.5 h period of treatment. When the elicitor was injected into tomato leaves, PLDbeta1 mRNA accumulation peaked at 2 h (threefold increase), before decreasing to background levels within 72 h. Mutant, non-active xylanase was as effective as the active enzyme in eliciting a response, suggesting that xylanase itself, and not the products resulting from its activity, functioned as an elicitor. When chitotetraose was used as elicitor, no PLDbeta1 mRNA accumulation was observed, thus it is not a general response to elicitation. Together these data show that PLD genes are differentially regulated, reflecting potential differences in cellular function. The possibility that PLDbeta1 is a signalling enzyme is discussed.
Subject(s)
Phospholipase D/genetics , Solanum lycopersicum/enzymology , Amino Acid Sequence , Cells, Cultured , Cloning, Molecular , Cold Temperature , DNA, Complementary , DNA, Plant , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Solanum lycopersicum/genetics , Molecular Sequence Data , Multigene Family , Osmotic Pressure , Plant Leaves/drug effects , Plant Leaves/enzymology , RNA, Messenger/metabolism , Sequence Alignment , Xylan Endo-1,3-beta-Xylosidase , Xylosidases/metabolismABSTRACT
Plants respond to pathogen infection and environmental stress by regulating the coordinate expression of many stress-related genes. In plants, the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced under environmental stress. This work was aimed at investigating whither the expression pattern of cytosolic GAPDH is also modulated upon infection of potato plants (Solanum tuberosum L.) with the late blight fungal agent Phytophthora infestans. Northern blot analysis showed the accumulation of the GAPDH gene transcripts in leaves and stems of inoculated potato plants. When tuber discs were treated with eicosapentaenoic acid (EPA), an elicitor found in P. infestans, GAPDH gene transcripts level increased. The increase was parallel to that of the hydroxymethyl glutharyl coenzyme A reductase (HMGR), an enzyme involved in pathogen defense reactions. Glucans obtained from P. infestans cell wall acts synergistically with EPA on GAPDH and HMGR gene induction. Salicylic acid, an endogenous signal for inducing systemic acquired resistance, was also effective in stimulating the GAPDH transcript accumulation in potato leaves. These experiments suggest that related multi-component factors, which are part of both primary and secondary metabolism, are probably regulated by similar signal transduction pathways when they are induced under biotic or abiotic stress conditions.
