Allelic differences at two loci govern different mechanisms of intraflower self-pollination in self-pollinating strains of periwinkle.
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Periwinkle [Catharanthus roseus (L.) G. Don], an ornamental and medicinal plant, is a self-compatible, insect-pollinated plant species in which intraflower self-pollination does not occur because of spatial separation of the stigma and anthers. Recently three self-pollinating strains-MJ, VI, and OR-were identified. Self-pollination in these strains was found to be brought about by continuous increase in gynoecium length from anthesis to self-pollination, in contrast to non-self-pollinating strains, in which the stigma remained below the base of the anthers from anthesis to flower drop. Self-pollination in these strains was found to be controlled by duplicate, recessive genes. Self-pollination in strains MJ and VI was brought about by an increase in gynoecium length resulting from an increase in the length of the ovary, while in the strain OR, the increase in gynoecium length was because of an increase in the length of the style from anthesis to self-pollination. The three strains were intercrossed to determine the relationship between genes governing self-pollination in these strains. The F(1) plants and all plants of the F(2) generation of the cross MJ x VI exhibited self-pollination that was brought about by an increase in the length of the ovary, indicating that the same genes were involved in these two strains. The F(1) plants of crosses OR x MJ and OR x VI, exhibited self-pollination that was brought about by an increase in the length of the ovary, indicating that self-pollination brought about by an increase in the length of the ovary was dominant over self-pollination brought about by an increase in the length of the style. In the F(2) and backcross [(OR x MJ) x OR and (OR x VI) x OR] generations, both self-pollinating and non-self-pollinating plants were observed. The ratio of plants with self-pollination brought about by an increase in the length of the ovary, non-self-pollinating plants, and plants with self-pollination brought about by an increase in the length of the style in the F(2) and backcross generations fit 9:6:1 and 1:2:1 ratios, respectively. All plants of the backcrosses [(OR x MJ) x MJ and (OR x VI) x VI] exhibited self-pollination brought about by an increase in the length of the ovary. The results thus supported the earlier finding that self-pollination in the studied strains was controlled by duplicate, recessive genes and suggested that three alleles at two loci determine the occurrence or nonoccurrence of intraflower self-pollination in periwinkle. (+info)
Sugar import and phytopathogenicity of Spiroplasma citri: glucose and fructose play distinct roles.
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We have shown previously that the glucose PTS (phosphotransferase system) permease enzyme II of Spiroplasma citri is split into two distinct polypeptides, which are encoded by two separate genes, crr and ptsG. A S. citri mutant was obtained by disruption of ptsG through homologous recombination and was proved unable to import glucose. The ptsG mutant (GII3-glc1) was transmitted to periwinkle (Catharanthus roseus) plants through injection to the leaf-hopper vector. In contrast to the previously characterized fructose operon mutant GMT 553, which was found virtually nonpathogenic, the ptsG mutant GII3-glc1 induced severe symptoms similar to those induced by the wild-type strain GII-3. These results, indicating that fructose and glucose utilization were not equally involved in pathogenicity, were consistent with biochemical data showing that, in the presence of both sugars, S. citri used fructose preferentially. Proton nuclear magnetic resonance analyses of carbohydrates in plant extracts revealed the accumulation of soluble sugars, particularly glucose, in plants infected by S. citri GII-3 or GII3-glc1 but not in those infected by GMT 553. From these data, a hypothetical model was proposed to establish the relationship between fructose utilization by the spiroplasmas present in the phloem sieve tubes and glucose accumulation in the leaves of S. citri infected plants. (+info)
Expression, purification, crystallization and preliminary X-ray analysis of strictosidine glucosidase, an enzyme initiating biosynthetic pathways to a unique diversity of indole alkaloid skeletons.
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Strictosidine beta-D-glucosidase, a plant enzyme initiating biosynthetic pathways to about 2000 monoterpenoid indole alkaloids with an extremely large number of various carbon skeletons, has been functionally expressed in Escherichia coli and purified to homogeneity in mg scale. Crystals suitable for X-ray analysis were found by robot-mediated screening. Using the hanging-drop technique, optimum conditions were 0.3 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6 and PEG 4000 (10%) as precipitant buffer. The crystals of strictosidine glucosidase belong to the space group P42(1)2 with unit cell dimensions of a=157.63, c=103.59 A and diffract X-rays to 2.48-A resolution. (+info)
Purification, molecular cloning, and cell-specific gene expression of the alkaloid-accumulation associated protein CrPS in Catharanthus roseus.
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Identification of molecular markers of monoterpenoid indole alkaloid (MIA) accumulation in cell-suspension cultures of Madagascar periwinkle (Catharanthus roseus (L.) G. Don) was performed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the protein patterns from alkaloid-producing and non-producing cells showed the specific occurrence of a 28 kDa polypeptide restricted to cells accumulating MIAs. The polypeptide was purified by preparative two-dimensional gel electrophoresis, digested with trypsin, and microsequenced by the Edman degradation method. Cloning of the corresponding cDNA revealed that the protein which has been named CrPS (Catharanthus roseus Protein S) is a member of the alpha/beta hydrolase superfamily. Time-course expression studies by northern blot analysis confirmed that CrPS gene expression was associated with MIA accumulation in cell suspension cultures. In the whole plant, multicellular compartmentation is required for alkaloid biosynthesis. In situ mRNA hybridization on developing leaves revealed that CrPS mRNA and transcripts encoding the first enzymes of the MIA pathway were co-localized in internal phloem parenchyma cells. The possible implication of the alkaloid-accumulation associated protein CrPS in the signal transduction pathway leading to MIA production is discussed. (+info)
Cytochrome P450 2D6 (CYP2D6) inhibitory constituents of Catharanthus roseus.
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The MeOH-soluble fraction of the water extract of Catharanthus roseus from Indonesia, having shown potent inhibitory activity on the metabolism mediated by CYP2D6, was subjected to activity-guided isolation to yield two triterpenes, ursolic acid (1) and oleanolic acid (2), and three alkaloids, vindoline (3), ajmalicine (4), and serpentine (5). The isolated compounds were tested for their inhibitory activity on the metabolism mediated by CYP3A4 or CYP2D6 using [N-methyl-14C]erythromycin or [O-methyl-14C]dextromethorphan as a substrate, respectively. Ajmalicine (4) and serpentine (5) showed very potent inhibitory activity against CYP2D6 with IC50 values of 0.0023 and 3.51 microM, respectively. All isolated compounds showed weak or no inhibition against CYP3A4. On time-, concentration-, and NADPH-dependent assay, serpentine (5) appear to be the mechanism-based inhibitor for CYP2D6 enzyme in which the inhibition was irreversible and driven by catalytic process. K(I) and k(inact) values for serpentine (5) were 0.148 microM and 0.090 min-1, respectively. On the other hand, ajmalicine (4) showed no time-dependent inhibition or reversible inhibition, and thus appear to be not mechanism-based inhibitor. (+info)
Phytic acid synthesis and vacuolar accumulation in suspension-cultured cells of Catharanthus roseus induced by high concentration of inorganic phosphate and cations.
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We have established a new system for studying phytic acid, myo-inositol hexakisphosphate (InsP(6)) synthesis in suspension-cultured cells of Catharanthus. InsP(6) and other intermediates of myo-inositol (Ins) phosphate metabolism were measured using an ion chromatography method. The detection limit for InsP(6) was less than 50 nM, which was sufficient to analyze Ins phosphates in living cells. Synthesis of Ins phosphates was induced by incubation in high inorganic phosphate medium. InsP(6) was mainly accumulated in vacuoles and was enhanced when cells were grown in high concentration of inorganic phosphates with the cations K(+), Ca(2+), or Zn(2+). However, there was a strong tendency for InsP(6) to accumulate in the vacuole in the presence of Ca(2+) and in nonvacuolar compartments when supplied with Zn(2+), possibly due to precipitation of InsP(6) with Zn(2+) in the cytosol. A vesicle transport inhibitor, brefeldin A, stimulated InsP(6) accumulation. The amounts of both Ins(3)P(1) myo-inositol monophosphate synthase, a key enzyme for InsP(6) synthesis, and Ins(1,4,5)P(3) kinase were unrelated to the level of accumulation of InsP(6). The mechanisms for InsP(6) synthesis and localization into vacuoles in plant cells are discussed. (+info)
Processing methods for differential analysis of LC/MS profile data.
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BACKGROUND: Liquid chromatography coupled to mass spectrometry (LC/MS) has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data. RESULTS: We present a software package MZmine that enables differential LC/MS analysis of metabolomics data. This software is a toolbox containing methods for all data processing stages preceding differential analysis: spectral filtering, peak detection, alignment and normalization. Specifically, we developed and implemented a new recursive peak search algorithm and a secondary peak picking method for improving already aligned results, as well as a normalization tool that uses multiple internal standards. Visualization tools enable comparative viewing of data across multiple samples. Peak lists can be exported into other data analysis programs. The toolbox has already been utilized in a wide range of applications. We demonstrate its utility on an example of metabolic profiling of Catharanthus roseus cell cultures. CONCLUSION: The software is freely available under the GNU General Public License and it can be obtained from the project web page at: http://mzmine.sourceforge.net/. (+info)
Simultaneous determination of vincristine, vinblastine, catharanthine, and vindoline in leaves of catharanthus roseus by high-performance liquid chromatography.
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A simple reversed-phase liquid chromatographic method is developed for the simultaneous quantitation of the anticancerous drugs vincristine, vinblastine, and their precursors catharanthine and vindoline using a Merck Chromolith Performance reversed-phase high-performance liquid chromatography column. A better resolution is obtained in comparison with available particulate-type C18 columns. The column provides good reproducibility and peak symmetry. Chromatography is carried isocratically with a mobile phase of acetonitrile-0.1M phosphate buffer containing 0.5% glacial acetic acid (21:79, v/v; pH 3.5) at a flow rate of 1.2 mL/min and UV detection at 254 nm. Parameters such as linearity, limits of quantitation (LOQ) and detection (LOD), precision, accuracy, recovery, and robustness are studied. The method is selective and linear for alkaloid concentration in the range 0.25 microg-25 microg/mL. The LOQ and LOD are 25, 46, 56, and 32 microg/mL and 8, 14, 18, and 10 microg/mL, respectively. The results of accuracy studies are good. Values for coefficient of variation are 2.50, 1.82, 1.33, and 1.13, respectively. The percent recovery of the alkaloids was found to be 96%, 97%, 98%, and 98%, respectively. Peak purity and homogeneity of these compounds in plant extract is studied using a photodiode-array detector. This simple and rapid method of analysis is applied for the determination of these alkaloids in a large number of leaf extracts of Catharanthus roseus.. (+info)