Publications - Cell and Metabolic Biology

BackgroundCharacterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.ResultsAgrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2.ConclusionWith this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.

BackgroundPhytohormones are the key metabolites participating in the regulation of multiple functions of plant organism. Among them, jasmonates, as well as abscisic and salicylic acids are responsible for triggering and modulating plant reactions targeted against pathogens and herbivores, as well as resistance to abiotic stress (drought, UV-irradiation and mechanical wounding). These factors induce dramatic changes in phytohormone biosynthesis and transport leading to rapid local and systemic stress responses. Understanding of underlying mechanisms is of principle interest for scientists working in various areas of plant biology. However, highly sensitive, precise and high-throughput methods for quantification of these phytohormones in small samples of plant tissues are still missing.ResultsHere we present an LC-MS/MS method for fast and highly sensitive determination of jasmonates, abscisic and salicylic acids. A single-step sample preparation procedure based on mixed-mode solid phase extraction was efficiently combined with essential improvements in mobile phase composition yielding higher efficiency of chromatographic separation and MS-sensitivity. This strategy resulted in dramatic increase in overall sensitivity, allowing successful determination of phytohormones in small (less than 50 mg of fresh weight) tissue samples. The method was completely validated in terms of analyte recovery, sensitivity, linearity and precision. Additionally, it was cross-validated with a well-established GC-MS-based procedure and its applicability to a variety of plant species and organs was verified.ConclusionThe method can be applied for the analyses of target phytohormones in small tissue samples obtained from any plant species and/or plant part relying on any commercially available (even less sensitive) tandem mass spectrometry instrumentation.

Diadenosine 5′,5′”‐P1,P4‐tetraphosphate (Ap4A) cleaving enzymes are assumed to regulate intracellular levels of Ap4A, a compound known to affect cell proliferation and stress responses. From plants an Ap4A hydrolase was recently purified using tomato cells grown in suspension. It was partially sequenced and a peptide antibody was prepared (Feussner et al., 1996). Using this polyclonal monospecific antibody, an abundant nuclear location of Ap4A hydrolase in 4‐day‐old cells of atomato cell suspension culture is demonstrated here by means of immunocytochemical techniques using FITC (fluorescein‐5‐isothiocyanate) labeled secondary antibodies. The microscopic analysis of the occurrence of Ap4A hydrolase performed for different stages of the cell cycle visualized by parallel DAPI (4,6‐diamidino‐2‐phenylindole) staining revealed that the protein accumulates within nuclei of cells in the interphase, but is absent in the nucleus as well as cytoplasm during all stages of mitosis. This first intracellular localization of an Ap4A degrading enzyme within the nucleus and its pattern of appearance during the cell cycle is discussed in relation to the suggested role of Ap4A in triggering DNA synthesis and cell proliferation.

Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6‐dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX‐95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX‐97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.

The plant growth substance jasmonic acid and its methyl ester (JA‐Me) induce a set of proteins (jasmonate‐induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one‐dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno‐gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP‐23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP‐37 was detected in vacuoles and in the nucleoplasm; JIP‐66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP‐100 was found within the stromal fraction of chloroplasts; JIP‐70 is present in the peroxisome and the nucleus; JIP‐50 and JIP‐6 accumulate in vacuoles. The location of JIP‐66 and JIP‐6 confirms their possible physiological role deduced from molecular analysis of their cDNA.