SUMMARY The presence of different types of cytokinins was analysed in exudates and lysates of stage-2 juveniles of Heterodera schachtii and Meloidogyne incognita and in mixed stages of Caenorhabditis elegans. For all species, cytokinins were detected in lysates and exudates in which benzyladenine and zeatin-type cytokinins were the most prominent forms. The production of cytokinins by Meloidogyne was much higher than by Heterodera, and the detected levels were in a range which interfered with the physiological activities of the host plant. The presence of 5-methoxy-N,N-dimethyltryptamine hydrogen oxalate did not affect hormone production by H. schachtii, whereas resorcinol slightly stimulated hormone production by M. incognita. The exuded cytokinins may play a role in feeding site induction, more particularly in cell cycle activation and in establishing the feeding site as a nutrient sink.
This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.
Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid. Indoleacetic acid (IAA), 4-chloro-IAA (4-Cl-IAA), and 5-Cl-IAA were metabolized to different extents by strains 61A24 and 110. Metabolites were isolated and analyzed by high-performance liquid chromatography and conventional mass spectrometry (MS) methods, including MS-mass spectroscopy, UV spectroscopy, and high-performance liquid chromatography-MS. The identified products indicate a novel metabolic pathway in which IAA is metabolized via dioxindole-3-acetic acid, dioxindole, isatin, and 2-aminophenyl glyoxylic acid (isatinic acid) to anthranilic acid, which is further metabolized. Degradation of 4-Cl-IAA apparently stops at the 4-Cl-dioxindole step in contrast to 5-Cl-IAA which is metabolized to 5-Cl-anthranilic acid.Bradyrhizobium japonicum reduces atmospheric nitrogen to ammonia when the bacterium is located in the root nodules of soybean (Glycine max) plants. The formation of root nodules results from a complex series of interactions between the bacterium and the plant and requires the growth and differentiation of both partners. Indoleacetic acid (IAA) has long been assumed to play a role in one or more aspects of nodule growth and development (11), although no specific role for IAA in nodule formation or development has been suggested.Bradyrhizobia have the enzymatic capacity to produce IAA in culture (7,8), and it has been demonstrated that nodules induced by an IAA-overproducing B. japonicum strain contain much greater amounts of IAA than nodules induced by the parental strain (5, 6), thus indicating that the bacterium can affect nodule IAA levels.In addition to being capable of IAA synthesis, some strains of B. japonicum are able to catabolize IAA. It was hypothesized that the first enzyme catalyzes an oxygen-consuming opening of the indole structure. A degradation pathway with anthranilic acid as the eventual degradation product was proposed (2). In this paper we report the analysis of the degradation products from IAA, 4-chloro-IAA (4-Cl-IAA), and 5-Cl-IAA, determined by conventional mass spectrometry (MS) and by high-performance liquid chromatography (HPLC)-thermospray MS. Using these methods, we have identified the intermediates in IAA catabolism in order to compare the degradation products with those of the degradation pathway postulated for B. japonicum (2). MATERIALS AND METHODSStrains and media. B. japonicum strain 110 (4), a derivative of USDA110, and strain 61A24 (14), showing different kinetics of IAA degradation, were used in this study. The strains were grown in yeast broth medium (1) on a rotary shaker with gentle shaking (100 rpm) at 28ЊC until turbidity reached an optical density at 450 nm of 0.35 to 0.45 (3 to 5 days).Preparing cultures for in vivo experiments. IAA was added to a final concentration of 0.2 mM to a stationary liquid culture and incubated overnight to induce IAA-catabolizing enzymes. The following day, the culture was centrifuged at 20,000 ϫ g for 10 min at 4ЊC. The pellet was w...
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