Viviparous leaves produced by somatic activation of an inactive cytokinin-synthesizing gene.

Tobacco plants that are somatic mosaics for expression of a cytokinin-synthesizing gene have viviparous leaves. Such a formation of shoots in an abnormal position represents a significant deviation from the usual organization of the plant body where a central axis produces shoots only in the axils of lateral leaf appendages and according to a precise phyllotactic pattern. This report links vivipary to the expression of a gene whose product is involved in the synthesis of the phytohormone cytokinin.

Stainless steel electrospray probe: a dead end for phosphorylated organic compounds?

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.

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.

Diurnal Fluctuations in Ethylene Formation in Chenopodium rubrum.

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.

Dynamics of cytokinins in apical shoot meristems of a day-neutral tobacco during floral transition and flower formation

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.

Antiproliferative effect of plant cytokinin analogues with an inhibitory activity on cyclin-dependent kinases.

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.

Production of auxin and related compounds by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita.

Mass spectrometric analysis revealed the presence of auxin, mainly in conjugated form, in secretions of Heterodera schachtii and Meloidogyne incognita, with or without treatment with DMT or resorcinol. M. incognita showed the highest production rates, though treatment of M. incognita with resorcinol had a negative effect on auxin production. Analysis of auxin precursor molecules in lysates of H. schachtii, M. incognita and Caenorhabditis elegans suggested that auxin is most probably a degradation product of tryptophan and that auxin may be synthesized via several intermediates, including indole-3-acetamide which is an intermediate of a pathway so far only characterized in bacteria. Furthermore, high levels of anthranilate, a degradation product of tryptophan in animals, but possibly also a precursor for auxin were detected.

The ORF8 gene product of Agrobacterium rhizogenes TL-DNA has tryptophan 2-monooxygenase activity.

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.

Cyclic AMP affinity purification and ESI-QTOF MS-MS identification of cytosolic glyceraldehyde 3-phosphate dehydrogenase and two nucleoside diphosphate kinase isoforms from tobacco BY-2 cells.

The soluble protein fraction of tobacco bright yellow 2 cells contained adenosine 3',5'-cyclic monophosphate (cAMP)-binding activity, detected with both a conventional binding assay and a surface plasmon resonance biosensor. A cAMP-agarose-based affinity purification procedure yielded three proteins which were identified by mass spectrometry as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and two nucleoside diphosphate kinases (NDPKs). This is the first report describing an interaction between cAMP and these proteins in higher plants. Our findings are discussed in view of the reported role of the interaction of cAMP with GAPDH and NDPK in animals and yeast. In addition, we provide a rapid method to isolate both proteins from higher plants.

Zeatin is indispensable for the G2-M transition in tobacco BY-2 cells.

The importance of N6-isoprenoid cytokinins in the G2-M transition of Nicotiana tabacum BY-2 cells was investigated. Both cytokinin biosynthesis and entry in mitosis were partially blocked by application at early or late G2 of lovastatin (10 microM), an inhibitor of mevalonic acid synthesis. LC-MS/MS quantification of endogenous cytokinins proved that lovastatin affects cytokinin biosynthesis by inhibiting HMG-CoA reductase. Out of eight different aminopurines and a synthetic auxin tested for their ability to override lovastatin inhibition of mitosis, only zeatin was active. Our data point to a key role for a well-defined cytokinin (here, zeatin) in the G2-M transition of tobacco BY-2 cells.

Levels of endogenous cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY-2 cells.

Correlation between cell cycle progression and endogenous levels of plant hormones was studied in synchronized tobacco BY-2 cell suspension cultures. Sixteen different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) were extracted using solid-phase anion exchange chromatography in combination with immunoaffinity purification, and quantified by mass spectrometry. No significant correlation could be identified for IAA and ABA. In contrast, there were sharp peaks in the levels of specific cytokinins (zeatin- and dihydrozeatin-type) at the end of the S phase and during mitosis. The levels of other cytokinins analyzed, including zeatins N- and O-glucosides, remained low, suggesting that the increased amounts of their corresponding non-glucosylated form resulted from de novo synthesis. These findings suggest that zeatin- and dihydrozeatin-type cytokinins might play a specific regulatory role in the progression of the plant cell cycle. One hypothesis to explain cytokinin action is based on a specific interaction with kinases that regulate cell cycle progression, as has been recently shown for the cytokinin analogue olomoucine.

A low content in zeatin type cytokinins is not restrictive for the occurrence of G1/S transition in tobacco BY-2 cells.

Theories on the importance of cytokinins in G1/S transition control are manifold and contradictory. By establishing a double A(phi-PZ block, maximal synchronization of a BY-2 suspension culture was obtained to investigate the effect of cytokinin depletion on G1/S transition. Lovastatin was used as a specific inhibitor of cytokinin biosynthesis. Flow cytometry showed that the G1/S transition occurred regardless of the cytokinin drop. This observation indicates an extremely low dose requiry for that stage of the cell cycle. It is very likely that precisely the downregulation of zeatin type cytokinins matters for the G1/S transition to occur, since cytokinin addition at early G1 blocked the cycle at G1/S.

Stimulation of indole-3-acetic acid production in Rhizobium by flavonoids.

Flavonoids activate nod gene expression in Rhizobium resulting in the synthesis of Nod signals which trigger organogenesis in the host plant. This paper shows that nod-inducers also stimulate the production of the phytohormone IAA (indole-3-acetic acid).

Effect of indomethacin on cell cycle dependent cyclic AMP fluxes in tobacco BY-2 cells.

The evolution of adenosine 3',5'-cyclic monophosphate (cAMP) levels was investigated in synchronised tobacco BY-2 cells by virtue of a method based on immunoaffinity purification and analysis on electrospray tandem mass spectrometry. A transient peak in cAMP content was observed during the S and G1 phases of the cell cycle. Application of the prostaglandin inhibiting drug indomethacin at early S phase resulted in the loss of the cAMP peak in S phase and inhibited mitotic division. This inhibition of cAMP accumulation suggests the presence of a prostaglandin-dependent adenylyl cyclase activity, analogous to animal cyclases. A potential role for cAMP during the plant cell cycle is postulated.

Indomethacin-induced G1/S phase arrest of the plant cell cycle.

In animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway. To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied. Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S. These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells.

Ecto-nucleotide pyrophosphatase modulates the purinoceptor-mediated signal transduction and is inhibited by purinoceptor antagonists.

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.

Differentiation between isomeric phenylglycidyl ether adducts of 2'-deoxyguanosine and 2'-deoxyguanosine-5'-monophosphate using liquid chromatography/electrospray tandem mass spectrometry.

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.

Apical dominance in Pssu-ipt-transformed tobacco.

In Pssu-ipt-transformed tobacco, apical dominance was released by defoliation of the upper nodes, while the apex remained intact. After defoliation, the concentration of cytokinins (CKs) increased whereas IAA remained constant, evoking an increase in the CK/IAA ratio in the buds. Moreover, defoliation resulted in a tremendous increase in the concentrations of aromatic amines (AAs): tyramine (TYR), phenethylamine (PEA) and an as yet unidentified compound. Although the total aliphatic monoamine and polyamine (PA) concentration remained constant, putrescine (PUT) and spermidine (SPD) concentrations in the axillary buds decreased, whereas the concentration of spermine (SPM) increased. Similar changes in PAs and AAs could be observed in the buds of untransformed SR1 plants after decapitation, whereas defoliation without removal of the apex had no effect. This is the first report on the possible involvement of PAs and AAs in apical dominance.