Proteomic analysis of somatic embryogenesis in Medicago truncatula. Explant cultures grown under 6-benzylaminopurine and 1-naphthaleneacetic acid treatments.
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The Medicago truncatula line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than wild-type Jemalong. We have compared proteomes of tissue cultures from leaf explants of these two lines. Both 2HA and Jemalong explants were grown on media containing the auxin 1-naphthaleneacetic acid and the cytokinin 6-benzylaminopurine. Proteins were extracted from the cultures at different time points (2, 5, and 8 weeks), separated by two-dimensional gel electrophoresis, and detected by silver staining. More than 2,000 proteins could be reproducibly resolved and detected on each gel. Statistical analysis showed that 54 protein spots were significantly (P < 0.05) changed in expression (accumulation) during the 8 weeks of culture, and most of these spots were extracted from colloidal Coomassie-stained two-dimensional gel electrophoresis gels and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry analysis. Using a publicly available expressed sequence tag database and the Mascot search engine, we were able to identify 16 differentially expressed proteins. More than 60% of the differentially expressed protein spots had very different patterns of gene expression between 2HA and Jemalong during the 8 weeks of culture. (+info)
Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds.
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1. Peritoneal mast cells from rat were co-incubated in vitro in a platelet aggregometer cuvette with washed rabbit platelets. In response to stimulation with calcium ionophore (A23187; 1-5 microM), the mast cells released a substance which stimulated the platelets to aggregate. These concentrations of ionophore did not stimulate platelet aggregation in the absence of mast cells, nor affect the responsiveness of the platelets to aggregation induced by thrombin or PAF. Release of a PAF-like substance was also observed in response to stimulation of the mast cells with antigen. 2. This pro-aggregatory activity is attributable to the release of PAF by the mast cells, since the activity could be abolished by preincubating the platelets with a specific PAF receptor antagonist (WEB 2086; 10 microM). Furthermore, the platelet-aggregating factor co-migrated with PAF on thin-layer chromatographs and could be abolished by incubation with phospholipase A2 (20 micrograms ml-1) or a specific antibody directed against PAF. 3. The release of PAF by peritoneal mast cells could be inhibited, in a concentration-dependent manner, by PF-5901 (IC50 of 3.9 microM) or Wy-50,295 (IC50 of 1.2 microM), two structurally similar compounds with inhibitory effects on leukotriene synthesis, as well as leukotriene D4 (LTD4) receptor antagonist properties. 4. Inhibition of PAF synthesis was not observed when the mast cells were incubated with a structurally unrelated 5-lipoxygenase inhibitor (A-64077), a structurally dissimilar inhibitor of 5-lipoxygenase activating protein (MK-886) or with a structurally related LTD4 receptor antagonist (MK-571) which lacks inhibitory effects on leukotriene synthesis, each at concentrations of up to 100 microM.5. Neither PF-5901 nor Wy-50,295 (1 or 10 microM) significantly affected histamine release or prostaglandin D2 synthesis by peritoneal mast cells in response to calcium ionophore stimulation.6. These results demonstrate the ability of a class of quinoline-based compounds to inhibit PAF synthesis by peritoneal mast cells. This activity does not appear to be related to effects of these compounds on leukotriene synthesis or LTD4 receptors. The ability of these compounds to inhibit PAF synthesis may contribute to their anti-inflammatory properties. (+info)
NtPDR3, an iron-deficiency inducible ABC transporter in Nicotiana tabacum.
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In plants, the ABC transporter PDR (pleiotropic drug resistance) subfamily is composed of approximately 15 genes, few of which have been analyzed. We have identified NtPDR3, a Nicotiana tabacum PDR gene belonging to a cluster for which no functional data was previously available. NtPDR3 was found to be induced in suspension cells treated with methyl jasmonate, salicylic acid, 1-naphthalene acetic acid, or cembrene, a macrocyclic diterpene. In agreement with the identification of a putative iron deficiency element in the NtPDR3 transcription promoter region, we found that iron deficiency in the culture medium induced NtPDR3 expression, thus suggesting a new function of the PDR transporter family. (+info)
PpEG4 is a peach endo-beta-1,4-glucanase gene whose expression in climacteric peaches does not follow a climacteric pattern.
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In peach (Prunus persica L. Batsch.) the degradation of the pectic compounds of the cell wall is considered to be the principal component responsible for fruit softening. Many genes encoding enzymes acting on the different polymers of the pectic matrix have been shown to be highly expressed during the late phases of softening, with polygalacturonase being the most important. Nevertheless, it is known that softening starts well before the ethylene climacteric rise which occurs concomitant with the maximal expression of the pectolytic enzymes. The cloning and characterization of PpEG4, an endo-beta-1,4-glucanase (EGase) gene preferentially expressed in preclimacteric fruits, are presented here. PpEG4 belongs to the group of EGases containing, at their carboxy-terminus, a peptide similar to the cellulose binding domain of microbial origin. This EGase is also expressed during abscission of both leaves and fruits. The effect of exogenous ethylene treatments on PpEG4 transcription is null in young fruits and negative in preclimacteric ones, while it is positive in abscission zones. Thus, the expression of PpEG4 seems to be more dependent on the type of separation process rather than being influenced by a direct hormone action. The ability of the PpEG4 regulatory sequences to drive transcription in cells undergoing separation events is also maintained in tomato, where about 3 kb of the gene promoter could drive the expression of gusA in preclimacteric fruits and in the fruit abscission zones. (+info)
Somatic embryogenesis in wild relatives of cotton (Gossypium Spp.).
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Wild cotton species can contribute a valuable gene pool for agronomically desirable cultivated tetraploid cultivars. In order to exploit diploid cotton a regeneration system is required to achieve transformation based goals. The present studies aimed at optimizing the conditions for regeneration of local varieties as well as wild species of cotton. Different callus induction media were tested with varying concentrations of hormones in which sucrose was used as nutritional source. Different explants (hypocotyls, cotyledon, root) were used to check the regeneration of both local cotton plants and wild relatives using T & G medium, BAP medium, CIM medium, EMMS medium, and cell suspension medium. Different stages of embryogenicity such as early torpedo stage, late torpedo stage, heart stage, globular stage and cotyledonary stage were observed in wild relatives of cotton. The results of this study pave the way for establishing future transformation methods. (+info)
Organ-specific transcription of putative flavonol synthase genes of grapevine and effects of plant hormones and shading on flavonol biosynthesis in grape berry skins.
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In order to investigate the control mechanism of flavonol biosynthesis of grapevine, we obtained five genomic sequences (FLS1 to FLS5) of putative flavonol synthase genes from Vitis vinifera cv. Cabernet Sauvignon. The mRNA of five FLSs accumulated in flower buds and flowers, while the mRNA of FLS2, FLS4, and FLS5 accumulated in small berry skins and then decreased toward veraison. At the ripening stage, the mRNA of only FLS4 and FLS5 accumulated again. This change in mRNA accumulation did not contradict the flavonol accumulation in the berry skins. Shading of the berries completely inhibited the increase in flavonol content and mRNA accumulation of FLS4, but did not affect the mRNA accumulation of FLS5. The effects of light and plant hormones on flavonol accumulation were different from those on anthocyanin accumulation. Thus flavonol biosynthesis appears to be under a different control system from that of anthocyanin biosynthesis. (+info)
PIN proteins perform a rate-limiting function in cellular auxin efflux.
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Intercellular flow of the phytohormone auxin underpins multiple developmental processes in plants. Plant-specific pin-formed (PIN) proteins and several phosphoglycoprotein (PGP) transporters are crucial factors in auxin transport-related development, yet the molecular function of PINs remains unknown. Here, we show that PINs mediate auxin efflux from mammalian and yeast cells without needing additional plant-specific factors. Conditional gain-of-function alleles and quantitative measurements of auxin accumulation in Arabidopsis and tobacco cultured cells revealed that the action of PINs in auxin efflux is distinct from PGP, rate-limiting, specific to auxins, and sensitive to auxin transport inhibitors. This suggests a direct involvement of PINs in catalyzing cellular auxin efflux. (+info)
Separation of plant hormones from biofertilizer by capillary electrophoresis using a capillary coated dynamically with polycationic polymers.
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A new, simple and rapid capillary electrophoresis (CE) method, using hexadimethrine bromide (HDB) as electroosmotic flow (EOF) modifier, was developed for the identification and quantitative determination of four plant hormones, including gibberellin A3 (GA3), indole-3-acetic acid (IAA), alpha-naphthaleneacetic acid (NAA) and 4-chlorophenoxyacetic acid (4-CA). The optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.005% (w/v) of HDB. The applied voltage was -25 kV and the capillary temperature was kept constant at 25 degrees C. Salicylic acid was used as internal standard for quantification. The calibration dependencies exhibited good linearity within the ratios of the concentrations of standard samples and internal standard and the ratios of the peak areas of samples and internal standard. The correlation coefficients were from 0.9952 to 0.9997. The relative standard deviations of migration times and peak areas were < 1.93 and 6.84%, respectively. The effects of buffer pH, the concentration of HDB and the voltage on the resolution were studied systematically. By this method, the contents of plant hormone in biofertilizer were successfully determined within 7 min, with satisfactory repeatability and recovery. (+info)