ABSTRACT
Under unfavorable environmental conditions, the stress phytohormone ABA inhibits the developmental transition from an embryo in a dry seed into a young seedling. We developed a genetic screen to isolate Arabidopsis thaliana mutants whose early seedling development is resistant to ABA. Here, we report the identification of a recessive mutation in AUXIN RESISTANT1 (AUX1), encoding a cellular auxin influx carrier. Although auxin is a major morphogenesis hormone in plants, little is known about ABA-auxin interactions during early seedling growth. We show that aux1 and pin2 mutants are insensitive to ABA-dependent repression of embryonic axis (hypocotyl and radicle) elongation. Genetic and physiological experiments show that this involves auxin transport to the embryonic axis elongation zone, where ABA enhances the activity of an auxin-responsive promoter. We propose that ABA represses embryonic axis elongation by potentiating auxin signaling in its elongation zone. This involves repression of the AUXIN INDUCIBLE (Aux/IAA) gene AXR2/IAA7, encoding a key component of ABA- and auxin-dependent responses during postgerminative growth.
Subject(s)
Abscisic Acid/pharmacology , Arabidopsis/drug effects , Arabidopsis/metabolism , Germination/physiology , Indoleacetic Acids/metabolism , Plant Growth Regulators/metabolism , Arabidopsis/embryology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/physiology , Blotting, Northern , Blotting, Western , Chromatography, High Pressure Liquid , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Germination/drug effects , Germination/genetics , Microscopy, Fluorescence , Plants, Genetically Modified/drug effects , Plants, Genetically Modified/embryology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Seedlings/drug effects , Seedlings/embryology , Seedlings/genetics , Seedlings/metabolism , Seeds/drug effects , Seeds/embryology , Seeds/genetics , Seeds/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
We have developed a simple, high-throughput batch immunoextraction (IAE) micropurification procedure for extracting a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) from plant tissues in solutions that are compatible with ultra-performance liquid chromatography (UPLC), thereby facilitating sensitive subsequent analysis. The UPLC system was coupled to a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray interface (ESI). Small (mg) amounts of tissues were purified by solid-phase extraction (SPE) followed by an immunoaffinity clean-up step and two fast chromatographic separations of most cytokinin metabolites (bases, ribosides, and 9-glucosides in the first, O-glucosides and nucleotides in the second). Using UPLC, the runs were up to 4-fold faster than in standard cytokinin analyses, and both retention times and injection volumes were less variable (RSDs, 0.15-0.3% and 1.0-5.5%, respectively). In multiple reaction monitoring (MRM) mode, the detection limit for most of the cytokinins analyzed was close to 1 fmol (5-25 fmol for O-glucosides and nucleotides) and the linear range spanned at least five orders of magnitude. The extraction and purification method was optimized using poplar (Populusxcanadensis Moench, cv Robusta) leaf samples, and the analytical accuracy was further validated using IAE-purified 10-day-old Arabidopsis thaliana plants spiked with 1 and 10 pmol of cytokinin derivatives. This approach can be used for rapid, sensitive qualitative and/or quantitative analysis of more than 50 natural cytokinins in minute amounts of plant tissues with high performance, robustness, and accuracy.
Subject(s)
Chromatography, High Pressure Liquid/methods , Cytokinins/chemistry , Plants/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Cytokinins/immunology , Cytokinins/isolation & purification , Molecular Structure , Plant Extracts/chemistry , Plant Extracts/immunology , Time FactorsABSTRACT
Cytokinin activity of forty-eight 6-benzyladenosine derivatives at both the receptor and cellular levels as well as their anticancer properties were compared in various in vitro assays. The compounds were prepared by the condensation of 6-chloropurine riboside with corresponding substituted benzylamines and characterized by standard collection of physico-chemical methods. The majority of synthesized derivatives exhibited high activity in all three of the cytokinin bioassays used (tobacco callus, wheat leaf senescence and Amaranthus bioassay). The highest activities were observed in the senescence bioassay. For several of the compounds tested, significant differences in activity were found between the bioassays used, indicating that diverse recognition systems may operate. This suggests that it may be possible to modulate particular cytokinin-dependent processes with specific compounds. In contrast to their high activity in bioassays, the tested compounds were recognized with only very low sensitivity in both Arabidopsis thaliana AHK3 and AHK4 receptor assays. The prepared derivatives were also investigated for their antiproliferative properties on cancer and normal cell lines. Several of them showed very strong cytotoxic activity against various cancer cell lines. On the other hand, they were not cytotoxic for normal murine fibroblast (NIH/3T3) cell line. This anticancer activity of cytokinin ribosides may be important, given that several of them occur as endogenous compounds in different organisms.
Subject(s)
Adenosine/analogs & derivatives , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cytokinins/chemistry , Cytokinins/pharmacology , Adenosine/chemistry , Adenosine/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Arabidopsis Proteins , Biological Assay , Cells, Cultured , Cytokinins/chemical synthesis , Fibroblasts/drug effects , Histidine Kinase , Mice , NIH 3T3 Cells , Plant Cells , Plants/drug effects , Protein Kinases , Receptors, Cell SurfaceABSTRACT
A range of benzylaminopurines naturally occur in plants and exhibit high biological activity. Others have been synthesized, such as 6-(2-hydroxy-3-methoxybenzylamino)purine riboside (2OH3MeOBAPR), which has shown interesting anti-cancer activity under in vitro conditions. In order to study the biological activity of this interesting compound in more detail, a rapid and highly efficient method for its purification from complex samples (e.g. blood and plant extracts) is needed. Therefore, we prepared monoclonal antibodies against 2OH3MeOBAPR. The antibody had undetectable cross-reactivity with all natural isoprenoid cytokinins, but relatively high cross-reactivity with aromatic cytokinins as well as some synthetic di- and tri-substituted 6-benzylaminopurines and the corresponding ribosides. The antibody also showed strong responses and specificity in enzyme-linked immunoassays (ELISAs). In addition, it was used to prepare, for the first time, an immunoaffinity sorbent with high specificity and capacity for aromatic cytokinins. A batch immunoextraction method was then developed and optimized for the purification of 2OH3MeOBAPR from murine blood samples. The high efficacy and simplicity of this method (in off-line combination with HPLC-MS) for the isolation of target analytes from biological material is demonstrated in this study.
