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Pea (Pisum sativum L., cv. Scinado) seedlings were exposed to low doses of ultraviolet-B (UV-B; 4.4 and 13.3 kJ m−2 d−1) or UV-C (0.1 and 0.3 kJ m−2 d−1) radiation for 14 d. Aminocyclopropane carboxylic acid (ACC), indoleacetic acid (IAA) and abscisic acid (ABA) contents were quantified by gas chromatography coupled to mass spectrometry (GC-MS). The accumulation of ACC upon irradiation was dose-dependent. ABA content was reduced and IAA content increased upon UV-C treatment whereas the UV-B doses used did not cause significant changes in ABA and IAA contents.

A study of the interaction of phosphorylated organic compounds with the stainless components of a liquid chromatography-electrospray ionisation-mass spectrometry system (LC-ESI-MS) was carried out to disclose a (forgotten?) likely pitfall in the LC-ESI-MS analysis of phosphorylated compounds. The retention behaviour of some representative compounds of different important classes of phosphorylated biomolecules such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC-ESI-MS setup were able to retain and trap phosphorylated compounds when these compounds were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25-50% ammonium hydroxide, ca. pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realise adsorption on a stainless surface. Furthermore, the number of phosphate moieties seemed to enhance the adsorption properties of the molecules and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was found to be an important factor in the course of these adsorption phenomena.

The role of phytohormones in genetic tumor formation on radish crop-roots was investigated using the collection of inbred Raphanus sativus lines as a model system. The genetic analysis showed that the trait > was recessive and monogenic in some crossings. The spectrum of main phytohormones in tumor and non-tumor radish lines has shown that at the initiation of tumor formation (30 days old plants) the amounts of main cytokinins in the lower part of plants from the tumor line were dramatically increased. The transformation of the non-tumor line by the ipt gene of Agrobacterium tumefaciens resulted in tumor formation in plants of the T1 progeny. We propose that increasing the cytokinin/auxin ratio may lead to tumor formation on radish crop roots.

The expression of the mitotic cyclin Arath; CYCB1;1 and of the cyclin-dependent kinase Arath; CDC2a was located by beta-glucuronidase histochemical detection and in situ hybridization, and was quantified by 4-methylumbelliferyl beta- D-glucuronide assay in tobacco stem tissues during both in vivo differentiation and in vitro dedifferentiation. The changes in localization of endogenous cytokinins were also determined during both processes using quantitative analysis and in situ immunocytochemistry. The CDC2a promoter was expressed continuously during stem development, with particularly high expression in the shoot apical meristem and in the internal and external primary phloem. CYCB1 expression was not restricted to the dividing cells; its expression in the shoot apical meristem was particularly high in the leaf-forming peripheral cells but the gene was also expressed throughout development in the internal and external phloem in which the rate of cell division was reduced or zero. Following in vitro culture, the internal phloem cells appeared to be particularly competent to re-enter the cell cycle within a short lag phase while the pith tissue reactivated later. In culture, cells that resumed division were found to accumulate cytokinins. The high competency of primary phloem to dedifferentiate was associated with its capacity to express CDC2a and CYCB genes and the presence of high cytokinin levels, providing some insights into the determinants of competency for resuming cell division.

In this study, analogues of olomoucine, a previously described plant cytokinin analogue with cyclin-dependent kinase (CDK) inhibitory activity, were investigated for effect on CDK1 and CDK2 and for effect on cell proliferation. Eight new compounds exhibit stronger inhibitory activity on CDK1 and CDK2 and on cell proliferation than olomoucine. Some active compounds showed low inhibition of proliferation of normal myeloid growth. Improvement of inhibitory activity of known compounds with a C6-benzylamino group was brought about by substitution with one hydroxyl. Also, new C2 substituents associated with inhibitory activity on CDK and on cell proliferation are described. There was a significant correlation between effect on CDK and antiproliferative effect on the KG1 and Molt3 cell lines and on primary human lymphocytes, strongly suggesting that at least part of the antiproliferative effect of cytokinin analogues was due to inhibition of CDK activity. Cytokinin analogues induced apoptosis in a time- and concentration-dependent manner and changes in cell cycle distribution. The antiproliferative and pro-apoptotic effects of plant cytokinin analogues suggest that they are a new class of cytostatic agents and that they may find an application in the chemotherapy of cancer.

Adventitious shoot regeneration was obtained from callus produced from main vegetative apices of pear of in vitrogrown shoots of Italian cultivars Spadona and Precoce di Fiorano and wild pear genotypes ISF54 and ISF61. The highest morphogenetic response was obtained on a medium containing 8.8 μM 6-benzyladenine, 1.0 μM α-naphthaleneacetic acid and 250 mg l−1cefotaxime. The explants were maintained for 30 days in darkness and then transferred to an auxin-free medium and to the light. Histological studies revealed that the new vegetative buds originated from callus that completely altered the morphology of the explant tissues by the 30th day of culture. The in situ localisation of cytokinins, performed using antibodies with marked specificity against zeatin (Z) and isopentenyladenine, revealed an accumulation of Z in the cambiform cells of the leaf primordia and in the shell zone of the new forming buds showing a primary role of this cytokinin in cell differentiation of in vitro pear organogenesis.

Our previous results demonstrated that endogenous cytokinins are involved in the shooty potential of tumors initiated on Eucalyptus globulus plantlets inoculated with Agrobacterium tumefaciens strain 82.139 [A. Azmi et al. (1997a) Plant Sci 127: 81-90]. In order to investigate whether or not these hormones are distributed homogeneously in the tumors prior to the onset of bud regeneration, decapitated hypocotyls were inoculated with the strain C58pMP90/T139 GUS-INT harboring the wild transferred DNA (T-DNA) of strain 82.139 tagged with the beta-glucuronidase (gus)-reporter gene. In situ immunolocalization of zeatin, dihydrozeatin and isopentenyladenine was performed in the developing tumors and combined with the histo-enzymological beta-glucuronidase assay. It was found that the expression of the T-DNA was restricted to only some small areas located deeply in the tumors. These sites were also provided with a high cytokinin signal while the untransformed parts of the tumors displayed a weaker signal, except in the early differentiating tracheary elements. The regenerated buds were untransformed and originated from superficial parts of the tumors provided with a moderate signal for cytokinins. The method of colocalization of both cytokinins and gus expression developed here might be helpful for further studies concerning the role of these hormones in controlling gene expression at cell and tissue levels.

The control of cell division by growth regulators is critical to proper plant development. The isolation of single-gene mutants altered in the response to plant hormones should permit the identification of essential genes controlling the growth and development of plants. We have isolated mutants pasticcino belonging to 3 complementation groups (pas1, pas2, pas3) in the progeny of independent ethyl methane sulfonate and T-DNA mutagenized Arabidopsis thaliana plants. The screen was performed in the presence or absence of cytokinin. The mutants isolated were those that showed a significant hypertrophy of their apical parts when grown on cytokinin-containing medium. The pas mutants have altered embryo, leaf and root development. They display uncoordinated cell divisions which are enhanced by cytokinin. Physiological and biochemical analyses show that cytokinins are probably involved in pas phenotypes. The PAS genes have been mapped respectively to chromosomes 3, 5 and 1 and represent new plant genes involved in the control of cell division and plant development.

The biosynthetic origin of cytokinins (CKs) in plants has been debated ever since the recognition of CKs as plant hormones. Although several possible biosynthetic pathways have been suggested, none of the data published to date are conclusive. The enzymes involved in CK biosynthesis have not yet been purified and characterized in detail, nor have plant genes encoding CK biosynthetic enzymes been identified.

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.

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.

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.

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.

The sensitivity to ABA or GA4+7 was measured in bulblets of Lilium speciosum regenerated on scale explants in vitro at temperatures (15, 20 and 25°C) that allowed development of various levels of dormancy. ABA content at the time of bulblets sprouting did not correlate with dormancy development. Sensitivity to ABA or GA4+7 was defined as the difference in sprouting of excised bulblets on medium with or without the above hormones. The sensitivity to ABA decreased with temperature of regeneration and age of bulblets. In bulblets on ABA-containing medium, the ABA content was always higher. Quite inexpectedly, on GA4+7 containing medium, the ABA levels were lower that those on control medium. It was concluded that the sensitivity to ABA plays a key role in dormancy development. Studies on factors other than ABA or GA should be carried out further to clarify the dormancy phenomena.

Direct enzyme immunoassays (ELISA) for determination of zeatin- and isopentenyladenine-cytokinins were developed. The assays are sensitive and measuring ranges extend from 0.5 to 75 pmol for zeatinriboside and from 0.5 to 50 pmol for isopentenyladenosine. The accuracy and specificity of ELISA were checked by LC-MS. Direct enzyme immunoassay (ELISA) for indole-3-acetic acid determination was developed. The method was verified by HPLC with fluorometric detection.

The physiological state of dormancy is one of the important stages in the plant life cycle. It is accepted that ABA and GAs are among the ruling regularities of the process. Here we report for the first time of endogenous ABA levels determination in Lilium bulblets, regenerated at various conditions. An indirect ELISA was applied using monoclonal antibody validated by LC-MS. The mother bulbls tissues were with low ABA levels—a possible consiquence of the stock storing conditions. No correlation was found between development, maintainence and release of dormancy and the ABA contents of the explants as well as of the regenerated at various temperature conditions, bulblets. Dormancy of Lilium bulblets regenerated in vitro depends not only on their endogenous ABA levels butq in great extent on their ABA—and GA—sensitivity.

Localization of target cells for Agrobacterium-mediated transformation in the carrot root disc model has been achieved after inoculation with a disarmed A. tumefaciens strain harbouring a GUS-intron construct. The first GUS positive cells could be detected on both sides of the discs 48 h after inoculation. The transformed cells were always more numerous on the apical side, mainly localized in the intrafascicular cambium and in the immature phloem strands. The kinetics of free endogenous IAA levels on both sides after wounding have been determined, indicating that rapid IAA accumulation on the apical side was not simply due to polar migration from the basal side. Attempts to optimize transformation efficiency were made by pretreating the discs with various concentrations of acetosyringone (AS) and/or naphthalene acetic acid (NAA). Surprisingly, while 25 muM AS applied to bacteria prior to the inoculations was ineffective, the same AS concentration applied as a pretreatment to the discs strongly increased the number of transformed cells in the target tissues and decreased the lag time for the appearance of the first GUS positive cells. NAA pretreatment on the basal side enhanced the AS effect. AS pretreatment was found both to advance the reentry of competent cells with a potential for cell division into the S phase of the cell cycle and to stimulate bacterial attachment to the cell walls. The relationship between transformation efficiency and DNA synthesis in the host cells is discussed. AS treatment of plant tissues prior to inoculation is proposed as a means of increasing the transformation rates.

Diurnal variations in endogenous IAA levels inFatsia japonica leaves, maintaining constant other external factors such as temperature and relative humidity, were studied. Plants were cultivated in a growth chamber (20 °C, 75 % RH, 16 h photoperiod, 400 µmol m-2 s-1 PAR). IAA analyses were carried out by analytical IP-HPLC with on-line spectrofluorimetry. Rhythmic variation of endogenous IAA levels was found. At the onset of the light period the IAA concentration dropped very rapidly from 1070 pmol g-1 (fr.m.) to 144 pmol g-1 (fr.m.). This concentration was nearly constant throughout the entire light period. During the subsequent dark period the IAA levels increased again to about 1000 pmol g-1 (fr.m.) at the end of the dark phase. These results were not confirmed in open field conditions where many other external factors probably influence the endogenous IAA content.

Summary In the present paper we correlate endogenous IAA and IAM levels with morphological and histological changes of decapitated Pisum sativum epicotyls after inoculation with pA-303,d16Agrobacterium derived strains. Callus and subsequent root formation in the infected region are correlated with endogenous IAA accumulation as a result of expression of the TR aux genes, concomitant with IAM accumulation. Irrespective of the bacterial strain analyzed IAA synthesis by the bacteria themselves was found to be constitutive and therefore not related to root proliferation. Inoculation with strains harbouring the aux genes (TR or TR + TL) induced callus formation, root initiation and histological changes such as tracheid differentiation and development of cribrovascular nodules surrounded by cambial-like layers. The presence of TL largely abolished the geotropic growth of the neoformed roots. Inoculations with strains harbouring only TL DNA or the Eco15 fragment covering orf10 to 12 lead to limited histological effects and root initiation. Morphology and growth pattern of root clones obtained from inoculations with the wild type A4 pA-303,d16oculations with strains containing either TL or TR alone differed greatly and could be related to varying endogenous IAA levels. High endogenous IAA and IAM levels were found in root clones obtained after inoculation with bacterial strains carrying only TL DNA.

The resumption of development upon light activation of Artemia embryos is accompanied by an immediate drop in cyclic adenosine monophosphate (cAMP) concentration. The concentration of cyclic guanosine monophosphate (cGMP) remains unaltered, however. A similar effect is observed when the cysts are treated with H2O2, with which we demonstrated that its action mimics light activation. The demonstrated decrease in cAMP is the earliest observed intracellular event after light induction. Possible regulatory roles of cyclic nucleotides in light-induced resumption of development are discussed.

A rapid and reliable method based on immuno-affinity chromatography and preparative high pressure liquid chromatography was used for extraction and purification of cAMP from the two trematode species Gastrothylax crumenifer and Explanatum explanatum. The purified cAMP was quantified by electrospray tandem mass spectrometry in an amount of 44 and 83 picomole/g fresh weight of Gastrothylax crumenifer and Explanatum explanatum respectively.

Transgenic tobacco lines expressing Arath-CYCD2 or Arath-CYCD3 genes under a cauliflower mosaic virus 35S promoter are modified in the timing of their development, but not in the phenotype of their vegetative organs. They display an increased rate of leaf initiation, which is shown to be associated with distinct changes in the structural organization of their shoot apical meristem (SAM). Constitutive expression of Arath-CYCD2 leads to a progressive modification of the SAM structural organization with predominant periclinal divisions in the L3 layer and to the loss of the classical cytophysiological zonation, the central zone being reduced to the central cells of the L1 and L2 layers. These changes reveal a particular sensitivity of the corpus cells (L3) to Arath-CYCD2 over-expression and suggest a role for CYCD2 in controlling the planes of cell division in these cells. The SAM structural modifications in the Arath-CYCD3 over-expressing lines are less drastic; only an increased cell number together with a reduced cell size, particularly in the L1 layer, characterizes the peripheral zones. This could be related to the shortening of the G1-phase duration that renders cell growth incomplete between successive mitoses. Cell proliferation continues beyond the SAM in the developing internodes and confers a delayed senescence to Arath-CYCD3 over-expressing juvenile tissues. In addition, the ploidy levels of mature stem tissues in both types of transgenic lines are unaffected, suggesting that the studied G1 to S cell-cycle genes have no effect on the extent of endoreduplication in tobacco stem tissues.

The open reading frame 8 (ORF8) is located on the TL-DNA of the phytopathogenic soil bacterium Agrobacterium rhizogenes strain A4. The predicted ORF8 protein has a particular structure and is possibly a natural fusion protein. The N-terminal domain shows homology to the A. rhizogenes rolB protein and may modulate the auxin responsiveness of host cells. The C terminus has up to 38% homology to tryptophan 2-monooxygenases (t2m). We show that ORF8 overexpressing plants contain a fivefold higher concentration of indole-3-acetamide (IAM) than untransformed plants. Protein extracts from seedlings and Escherichia coli overexpressing ORF8 show significantly higher turnover rates of tryptophan to IAM than negative controls. We conclude that the ORF8 gene product has tryptophan 2-monooxygenase activity.

1. The effect of ecto-nucleotide pyrophosphatase (ecto-NPPase; EC 3.6.1. 9) on the ATP- and ADP-mediated receptor activation was studied in rat C6 glioma cells. The P2-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC(50)=12+/-3 microM) of the latter enzyme. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS), 5'-phosphoadenosine 3'-phosphate (PAP) and suramin were less potent inhibitors with an IC(50) of 22+/-4, 36+/-7 and 72+/-11 microM respectively. 2. P1-purinoceptor antagonists CGS 15943, cyclo-pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto-NPPase. 3. ATP- and ADP-mediated P2Y(1)-like receptor activation inhibited the (-)-isoproterenol-induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y-antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto-NPPase. 4. We conclude that ecto-NPPase has a modulator effect on purinoceptor-mediated signalling in C6 glioma cell cultures.

The reaction between phenylglycidyl ether and 2'-deoxyguanosine or 2'-deoxyguanosine-5'-monophosphate yields a variety of different nucleoside and nucleotide adducts. The corresponding mixtures were analyzed by liquid chromatography/electrospray tandem mass spectrometry and the product ion spectra of the different isomers are discussed using the correct cone voltage and collision energy. The latter were selected by looking at the energy-resolved spectra. From these data the formation of N7 and N2 isomers was proposed. However, detailed NMR analysis of the latter proved this isomer to be the N(1)-alkylated adduct. The reaction between phenylglycidyl ether and 2'-deoxyguanosine or 2'-deoxyguanosine-5'-monophosphate yields a variety of different nucleoside and nucleotide adducts. The corresponding mixtures were analyzed by liquid chromatography/electrospray tandem mass spectrometry and the product ion spectra of the different isomers are discussed using the correct cone voltage and collision energy. The latter were selected by looking at the energy-resolved spectra. From these data the formation of N7 and N2 isomers was proposed. However, detailed NMR analysis of the latter proved this isomer to be the N(1)-alkylated adduct. 2'-Deoxyguanosine-5'-monophosphate was alkylated by phenylglycidyl ether at the 5'-phosphate position. If the nucleotide base moiety was alkylated, the corresponding tandem mass spectrometric data strongly suggested alkylation of N7. In the case of bis-PGE-dGMP adduct, evidence was found for simultaneous 5'-phosphate and N7-alkylation.2'-Deoxyguanosine-5'-monophosphate was alkylated by phenylglycidyl ether at the 5'-phosphate position. If the nucleotide base moiety was alkylated, the corresponding tandem mass spectrometric data strongly suggested alkylation of N7. In the case of bis-PGE-dGMP adduct, evidence was found for simultaneous 5'-phosphate and N7-alkylation.

The Arabidopsis thaliana (L,) Heynh, 1-aminocyclopropane-1-carboxylate synthase gene I (AT-ACS1) was found to he developmentally regulated with pronounced activity in immature tissues. The effect of different hormones on this expression pattern was studied by using a transgenic Line carrying the beta-glucuronidase (GUS) gene under control of the AT-ACS1 promoter. In addition, the level of mRNA for AT-ACS1, was assayed in different ethylene and auxin-insensitive backgrounds, as well as in an auxin homeostasis mutant bg using competitive reverse transcriptase polymerase chain reaction (RT-PCR), The results support the existence of a hormonal cross-talk involving auxin, cytokinin, and ethylene in the regulation of AT-ACS1. These hormonal controls act in a developmental and tissue-dependent manner.

Fundamental to expected responses when excised plant tissues are placed in vitro is the question to which extent expiant factors, especially the endogenous hormonal situation, are influencing the receptivity to external stimuli (Cassells et al., 1982). In addition, the main consideration of the recent approaches dealing with the regulation of gene expression by hormones has been the effect of exogenous compounds providing no information on the patterns of distribution of endogenous hormones. This is particularly true for cytokinins which are known to trigger a variety of developmental events both in whole plants and in in vitro cultures (Kaminek, 1992; Brzobohaty et al, 1994). Analytical methods to quantify cytokinins have been strongly improved during the recent past years, however, the various approaches to localize cytokinins in situ have long been questionable due to their diffusible property. Pioneer works in the area of cytokinin immunocytochemistry (Zavala, Brandon, 1983; Eberle et al., 1987; Sossountsov et al., 1988) stressed the role of aldehyde fixation in the linkage between the cytokinin bases isopentenyladenin (iP), zeatin (Z) and dihydrozeatin (DHZ) and BSA and also emphasized the use of antibodies obtained by immunization with cytokinin ribosides coupled to BSA according to the periodate oxidation method (Erlanger, Beiser, 1964). Following these works, we have developed a new immunocytochemical method focusing on the localization of cytokinin bases which were evidenced to be the active forms (Laloue, Pethe, 1982; Vesely et al., 1994).

ethylene to trigger the process when IAA levels are low. Ethylene production was studied during the develop- ment and senescence of seeds and pericarp tissues Key words: Oilseed rape, pod shatter, ethylene metabolism, of oilseed rape (Brassica napus L.) pods (siliquae). In aminoethoxyvinylglycine (AVG), parthenocarpy. the course of the rise to a pre-senescence climacteric, little change in 1-aminocyclopropane-1-carboxylic acid Introduction (ACC) was recorded in the seeds, indicating a rapid conversion to ethylene. In contrast, very small Seed loss from fully mature pods (pod shatter) is a amounts of ethylene were produced by the pod wall problem with monocultures of dicotyledonous crop (PW) tissues, which included the dehiscence zone (DZ), species with an extended flowering period and many- while levels of free and conjugated ACC in the PW seeded, dry fruits, such as Brassica spp., soybean (Glycine increased consistently. As climacteric ethylene pro- max L.), and sesame (Sesame indicum L.). Pod shattering duction by the seeds declined, biosynthesis of ethy- is a particular problem in oilseed rape (Brassica napus L.) lene by the PW increased. Effects of reducing ethylene because of a marked tendency of fully mature pods to production by various means were examined in relation open. Average annual losses of about 20%, represent a to cell separation in the dehiscence zone. Amino- production eYciency that is far less than in any other ethoxyvinylglycine (AVG) applied during the pre- major arable crop. There is little variation in the suscepti- senescence climacteric reduced ACC levels and bility of current cultivars to pod shatter (Downey and ethylene production by the seeds, but did not affect Robbelen, 1989), and conventional breeding has so far subsequent values in the PW. The production of been unable to produce new cultivars which are more b-1,4-glucanase and the separation of the cells of the resistant to opening. DZ were delayed for 3-4 d by AVG, but the force The oilseed rape pod (silique or siliqua) consists of two required to open fully mature pods was unaltered. In carpels whose walls are separated by a replum containing parthenocarpic (seedless) pods, ethylene was pro- the main vascular tissue of the pod. A layer of 2-3 duced during senescence. Cell separation in the DZ parenchymatous cells between the carpel edges and the took place as in seeded pods, although it was also replum forms the separation or dehiscence zone (DZ ). delayed by 3-4 d. The results are related to changes Separation of the cells in the DZ takes place about 7 in indole-3-acetic acid (IAA) levels in oilseed rape pods weeks after anthesis, just before moisture loss is complete. which decline in PW and DZ tissues during senes- The pods open as a result of external force supplied by cence. It is concluded that separation in the cells of contact with other pods, racemes or harvesting machinery, which severs the vascular connections. the dehiscence zone requires only small amounts of

In this work, the coupling of liquid nanochromatography to NanoFlow electrospray mass spectrometry was evaluated for the detection of DNA adducts. The NanoFlow ES LC/MS system was compared with the capillary and conventional ES LC/MS system by analyzing an in vitro reaction mixture resulting from the interaction of 2'-deoxyguanosine 5'-monophosphate with bisphenol A diglycidyl ether and by injecting 2'-deoxyadenosine. By using NanoFlow ES LC/MS, the mass sensitivity could be improved by a factor of 3300. Three different injection methods used in liquid nanochromatography, i.e., split, large-volume, and column-switching injections were compared in terms of sensitivity. Furthermore, NanoFlow ES LC/MS was used to detect 2'-deoxynucleotide adducts isolated from an in vitro mixture of calf thymus DNA and bisphenol A diglycidyl ether. Different 2'-deoxynucleotide adducts could be identified by monitoring typical product ions, diagnostic for the adducts.

In our studies of the possible involvement of ethylene in photoperiodic flower induction in Chenopodium sp. we have shown that ethylene inhibits flower induction in short-day (SD) C. rubrum when applied (in the form of Ethrel) before or during the first half of the inductive darkness [1]. To achieve this inhibition, 100 - 300 μM Ethrel solution had to be used, after application of which ethylene formation achieved values of 260 - 480 nl g-1FW h-1 (unpublished results). Moreover, ethylene was shown to mediate auxin (IAA) - induced inhibition of flowering in both SD C. rubrum and long-day (LD) C. murale [2]. In this case, ethylene production was not by far as high as after Ethrel application; 500 μM IAA induced ethylene formation of 110 nl.g-1FW h-1 at the maximum. If applied 1–2 days after the end of inductive treatment, both Ethrel and IAA at 100-300 μM stimulated flowering.

Concentrations of both free and conjugated indole-3-acetic acid (IAA) were studied during development of pod wall, dehiscence zone and seeds of Brassica napus pods. A decrease in auxin content prior to moisture loss in the pods was observed specifically in the dehiscence zone, which was correlated with a tissue specific increase in β-1,4-glucanase activity. Furthermore, treatment of the pods with the auxin mimic 2-methyl-4-chlorophenoxyacetic acid resulted in a delay of 10 d of β-1,4-glucanase activity and concomitant cell separation in the dehiscence zone. This indicates that the activity of hydrolytic enzymes involved in cell separation in the dehiscence zone is regulated by auxin activity. Comparison of parthenocarpic pods with seeded pods pointed to the seeds as the source of IAA. Levels in the dehiscence zone of these pods were low over the entire sampling period, while cell separation in the dehiscence zone was delayed by about 4 d. These results indicate that a low level of auxins in the dehiscence zone is necessary for dehiscence to take place, but other factors may also be important.

Ethylene formation was studied in 5- to 6-d-old Chenopodium rubrum seedlings under the following light regimes: continuous light (CL), continuous darkness (CD), and alternating light/darkness (12 h of each). No significant regular oscillations in ethylene formation were found in either the CL or CD groups. In the light/dark regime, pronounced diurnal fluctuations in ethylene formation were observed. Activity of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase was transiently increased on transfer from light to dark and vice versa. In CL, ACC oxidase activity did not change significantly, whereas in CD, it decreased continuously after the initial increase. The in vivo levels of ACC and N-malonyl-ACC (MACC) were constant for the first few hours of darkness, then decreased dramatically, but increased again in the light. In constant darkness, the level of ACC displayed endogenous rhythm, with minimum values at h 12 and 44, and a maximum value at h 32 to 36. The level of MACC in both shoots and roots decreased in the CD group until h 12, and then remained constant until h 30 before decreasing continuously. We conclude that the photoperiodic regime affects both ACC and MACC levels, as well as the conversion of ACC to ethylene. Correlation of the described changes in ethylene formation to photoperiodic flower induction is discussed.

Bud regeneration from seedling hypocotyls and stem cuttings arising from micropropagated plantlets of Eucalyptus globulus was achieved using inoculation with the wild Agrobacterium tumefaciens strain 82.139 which induced shooty tumors. The adventitious buds regenerated on the shooty tumors were untransformed as revealed by the use of a C58pMP90/T139GUS-INT strain harbouring the intron inactivated uidA (gus) reporter gene. Subsequent rooting and acclimatization were achieved for some of the regenerants. Flow cytometric analyses of nuclear DNA contents indicated that both the tumors and the regenerants were diploid. Endogenous hormone quantification was performed in shooty and non shooty tumors induced by strains 82.139 and C58, respectively. The shooty potential of the strain 82.139 was associated with high Z and ZR levels in the tumors while auxin levels were similar in shooty and non shooty tumors. The efficiency of the method could be useful for both regeneration and genetic transformation from organs of cloned E. globulus.

Water stress induced an increase in endogenous concentrations of ABA in Lavandula stoechas L. plants to 13100 pmol ABA g−1 FW, which may contribute to the maintenance of water relations between the second and the third day of water stress treatment. After the third day, a sharp decrease in ABA levels was observed to 2630 pmol ABA g−1 FW, together with a decrease in water content and water potential and a loss of plant response to water stress. Water deficit did not induce an increase in endogenous ABA concentration, which remained at 514 pmol ABA g−1 FW in Rosmarinus officinalis L., which is more sclerophyllous than L. stoechas. Nevertheless, the relative water content of Rosmarinus officinalis L. after seven days of water stress decreased more than 40% and ψ reached values of −3.2 MPa. R. officinalis showed lower levels of ABA, but significantly higher levels of IAA and ZR than L. stoechas (4 times and 6 times respectively in well watered-plants). The increase in ABA levels is not a common mechanism in these two Mediterranean shrubs which survive under water stress conditions.

We have isolated seven allelic recessive Arabidopsis mutants, designated superroot (sur1-1 to sur1-7), displaying several abnormalities reminiscent of auxin effects. These characteristics include small and epinastic cotyledons, an elongated hypocotyl in which the connection between the stele and cortical and epidermal cells disintegrates, the development of excess adventitious and lateral roots, a reduced number of leaves, and the absence of an inflorescence. When germinated in the dark, sur1 mutants did not develop the apical hook characteristic of etiolated seedlings. We were able to phenocopy the Sur1- phenotype by supplying auxin to wild-type seedlings, to propagate sur1 explants on phytohormone-deficient medium, and to regenerate shoots from these explants by the addition of cytokinins alone to the culture medium. Analysis by gas chromatography coupled to mass spectrometry indicated increased levels of both free and conjugated indole-3-acetic acid. sur1 was crossed to the mutant axr2 and the altered-auxin response mutant ctr1. The phenotype of both double mutants was additive. The sur1 gene was mapped on chromosome 2 at 0.5 centimorgans from the gene encoding phytochrome B.

Ultrastructural alterations in epidermal and mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in PEG-treated plants of Fatsia japonica Decne & Plank. Under stress induced by PEG vesicles containing fibrous material and electron-dense bodies associated with plasma membranes were observed. Cytochemical examination indicated that electron-dense bodies corresponded to lipids and the fibrous material of the vesicles were polysaccharides. Chloroplasts, mitochondria, nuclei and Golgi apparatus also showed modifications. A strong relationship was found between increasing PEG-induced water stress, increasing endogenous ABA and ultrastructural changes. In relation with leaf ontogeny and ABA concentration a higher ABA level was observed in younger than in older leaves. The differences in the endogenous concentrations of indole-3-acetic acid are unclear, except after 7 days of PEG-treatment. The increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.

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. Udgivelsesdato: 1995-Oct

Ultrastructural alterations in mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in water-stressed field-grown plants of Fatsia japonica. Under water deficit cellular membranes were modified and an increase in vesicles was observed. The main damage to the chloroplasts included thylakoid swelling and disruption of the chloroplast envelope. Concomitant variations in abscisic acid and indole-3-acetic acid were observed. Despite the expected increased in endogenous ABA concentration in relation to water stress, after the highest concentration of ABA, observed at predawn in severely stressed plants (psi(predawn) = -0.681 MPa), there was a sharp decline from 2768 pmol g fw(-1) to 145 pmol g fw(-1); thus in severely stressed plants ABA levels were not related to changes in bulk leaf ABA contents. Water stress did not influence the concentrations of indole-3-acetic acid, although the increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.

Summary Transgenic plants obtained from roots transformed with the T-DNA of Agrobacterium rhizogenes display a modified phenotype. We analyzed endogenous indole-3-acetic acid, abscisic acid and cytokinin concentrations in apices, leaves and stems at juvenile and mature stages of normal, pRi transformed and rol A tobacco plants. We have observed alterations in the hormonal levels in the stem apices of transformed plants. In transformed plants with a moderately altered (T) phenotype, we observed an accumulation of cytokinins at a very young age, which was not observed in transformed plants with an extremely altered phenotype (T′ or rol A). All transformed plants showed reduced endogenous IAA levels in the stem apical region and consequently an attenuated basipetal auxin gradient. At the same time, the transient ABA accumulation observed in the apices of untransformed plants during the vegetative developmental stage was absent for all transformed plants. The detailed kinetic hormonal analyses presented in this paper emphasize the relevance of varying hormone levels in the shoot apical region on the developmental pattern of both transgenic and normal plants.

The adducts formed between 2′-deoxyadenosine (dAdo), 2′-deoxycytidine (dCyd) and 2′-deoxyuridine (dUrd) and phenyl glycidyl ether (PGE) were analysed by HPLC and LC—thermospray (TSP)-MS. Good results were obtained on a 10 RP Select B column (12.5 cm × 4 mm I.D.) using 0.1 M NH4OAcCH3OH at a flow-rate of 0.8 ml/min. The mass spectra of the 2′-deoxynucleoside—PGE adducts, obtained under LC-TSP-MS conditions were all characterized by the presence of the protonated molecule [MH]+ and [BH + H]+ ions. The PGE-dCyd adduct underwent hydrolytic deamination to the corresponding PGE-dUrd adduct. There was an indication that this process of hydrolytic deamination also took place in the TSP interface. Localization of the alkylation site was possible in the PGE-dUrd adduct by the presence of an RDA rearrangement leading to a fragment ion at m/z 194. Preliminary sensitivity studies on PGE-dUrd showed a detection limit of 500 pg (signal-to-noise ratio = 2) in multiple ion monitoring at m/z 263 and 379.

Plant development is greatly influenced by several plant growth regulators including two major classes, cytokinins and auxins. Most experimental systems studied to date with respect to hormonal effects on plant development make use of exogenously applied hormones, and consequently contain uncontrolled variables related to uptake and transport of these compounds. Plant genetic engineering allows us to circumvent this problem by constructing plants transgenic for genes able to modify phytohormone content and activity.

The promoter activity of a 2.2-kb DNA fragment from the 5' flanking sequence of the tap1 gene of snapdragon has been characterised in tobacco with the β-glucuronidase reporter gene. The tap1 promoter conferred to the reporter gene an expression that was limited to the early stages of anther development. Expression of the rolB gene of Agrobacterium rhizogenes under the control of the tap1 promoter impaired the development of tobacco flowers. Tobacco plants transgenic for the tap1-rolB chimeric gene showed altered anthers and a reduction in whole flower growth. Such growth alterations were correlated with an increase in free IAA content and a decrease in gibberellin activity present in anthers. Since the rolB gene codes for an indole β-glucosidase (Estruch et al. 1991), we interpret the phenotypic alterations to be a consequence of the increased content and activity of auxin in anthers. The perturbation of anther development would in turn affect gibberellin production and content, which is reflected in a reduced elongation of flowers.