Pascale Redig

Analysis of Cytokinin Metabolism in ipt Transgenic Tobacco by Liquid Chromatography-Tandem Mass Spectrometry.

The endogenous levels of the major, naturally occurring cytokinins in Pisum sativum ribulose-1,5-bisphosphate carboxylase small subunit promoter-isopentenyl transferase gene (Pssu-ipt)-transformed tobacco (Nicotiana tabacum L.) callus were quantified using electrospray-liquid chromatography-tandem mass spectrometry during a 6-week subcultivation period. An ipt gene was expressed under control of a tetracycline-inducible promoter for a more detailed study of cytokinin accumulation and metabolism. Activation of the ipt in both expression systems resulted in the production of mainly zeatin-type cytokinins. No accumulation of isopentenyladenine or isopentenyladenosine was observed. In Pssu-ipt-transformed calli, as well as in the tetracycline-inducible ipt leaves, metabolic inactivation occurred through O-glucoside conjugation. No significant elevation of cytokinin N-glucosides levels was observed. Side-chain reduction to dihydrozeatin-type cytokinins was observed in both systems. The levels of the endogenous cytokinins varied in time and were subject to homeostatic regulatory mechanisms. Feeding experiments of ipt transgenic callus with [3H]isopentenyladenine and [3H]isopentenyladenosine mainly led to labeled adenine-like compounds, which are degradation products from cytokininoxidase activity. Incorporation of radioactivity in zeatin riboside was observed, although to a much lesser extent.

Gibberellins play a role in the interaction between imidazole fungicides and cytokinins in Araceae

Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA3 inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA3, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.

Uptake and biochemical analysis of 2,4-D in cultured zygotic embryos of Zea mays L.

Summary In order to determine the influence of 2,4-D on the regeneration of zygotic embryos of maize, the uptake and metabolism of 2,4-D by immature embryos were compared in an embryogenic inbred line (A188) and a non-embryogenic inbred line (A632), cultured on induction medium with 2mg/L 2,4-D that was partly 14 C-labelled. Uptake of 2,4-D under exhaustive conditions, i.e. without subculture, was analyzed from the onset of culture until 14 days of culture. During the so called shock response period, i.e. the first 24 h of culture, uptake of 2,4-D was observed in both lines, with a higher uptake in the embryos of A632. The availability of 2,4-D in the medium became a limiting factor for uptake from 3 days of culture onwards. Then the concentration of 2,4-D per gram fresh weight was up to 125 times higher than the concentration in the induction medium for both inbred lines. Differences in uptake between the two lines were observed until 5 days of culture. In this period a lower concentration of 2,4-D per gram fresh mass was found in A188. After 5 days of culture, however, the concentrations of 2,4-D per gram fresh mass were comparable in both lines. Therefore, the difference in embryogenicity is likely not caused by differences in uptake of 2,4-D. Supplementing TIBA to the induction medium under exhaustive conditions caused a drop in the uptake of 2,4-D in both inbred lines, but TIBA neither inhibited the uptake completely nor prevented the induction of callus. As killed embryos only showed a residual uptake, not influenced by TIBA, it is concluded that immature embryos of the two inbred lines do accumulate the greater part of the 2,4-D in an active manner. The 14 C labelled 2,4-D that had accumulated in cultured embryos was analyzed biochemically. Up to 70% of the radioactive 2,4-D accumulated in A188 embryos was present as free 2,4-D after 24 h of culture, and 37% in A632 embryos. Conjugation of 2,4-D to sugars and amino acids started after 16 h of culture. Metabolization of 2,4-D was observed from 2 h onwards and increased with ongoing culture. As compared with A188, A632 embryos showed a higher metabolization rate of 2,4-D. It might well be that the higher levels of free 2,4-D in A188, together with the higher metabolization of 2,4-D in A632, do cause differences in the influence of 2,4-D on the 2 inbred lines, i.e. the different response on tissue culture. Thus, it is concluded that the developmental differences found in cultured immature embryos of embryogenic and non-embryogenic inbred lines might be caused by differences in levels of free 2,4-D and metabolization of 2,4-D. This could be regulated by genetic differences between the two inbred lines; however, other genetic factors might determine the sensitivity of the embryos to 2,4-D as well.

Sterols and polyamines in **ipt**-transformed tobacco plants

Abstract Free sterol and free polyamine contents were determined in the apex and the leaves of control and Pssu-ipt transformed tobacco ( Nicotiana tabacum L. cv. Petit Havana SR1). The older leaves of ipt -transformed plants contained a much higher putrescine (put) content than those of control SR1 plants, whereas no significant differences for spermidine (spd) or spermine (spm) were found between control and ipt plants. Putrescine content corresponded well with endogenous cytokinin (free-bases) content and with ornithine- and ornithine-decarboxylose (ODC and ADC) activities. Plants transformed with ipt were characterized by a higher sterol content in the leaves and by a delay in the increase in the stigmasterol/sitosterol ratio that occurs from the upper to the lower leaves.

Chicken egg yolk antibodies for cytokinin analysis

The production, isolation, and purification of specific chicken immunoglobulins (Igs) against three main groups of naturally occurring cytokinins are reported. The specific Igs directed against, respectively, zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine are extracted from the egg yolk and used in radioimmunoassays that allow the quantification in parallel of pmol of the cytokinins in plant extracts. As little as 50 fmol of zeatin riboside, 20 fmol of isopentenyladenosine, and 40 fmol of dihydrozeatin riboside can be detected. The levels of cytokinins measured in the radio-immunoassay correlate well with physicochemical analysis methods such as high performance liquid chromatography (HPLC) with UV spectrum detection and HPLC-coupled mass spectrometric detection. Cross-reactivity studies indicate that the assay is not affected by most of the structurally related compounds. The respective antibody preparations recognized zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine and the corresponding free bases. The results obtained when analyzing crude plant extracts are expressed as zeatin riboside equivalents, dihydrozeatin riboside equivalents, and isopentenyladenosine equivalents.