Given the close relationship between cytokinins (CKs), photosynthesis and nitrogen metabolism, this study assessed the effect of arsenic (As) contamination on these metabolic components in the As-hyperaccumulators Pteris cretica L. var. Albo-lineata (Pc-A) and var. Parkerii (Pc-P) as well as the As-non-hyperaccumulator Pteris straminea Mett. ex Baker (Ps). The ferns were cultivated in a pot experiment for 23 weeks in soil spiked with As at the levels 20 and 100 mg·kg-1. For the purpose of this study, the CKs were placed into five functionally different groups according to their structure and physiological roles: bioactive forms (bCKs; CK free bases); inactive or weakly active forms (dCKs; CK N-glucosides); transport forms (tCKs; CK ribosides); storage forms (sCKs; O-glucosides); and primary products of CK biosynthesis (ppbCKs; CK nucleotides). An important finding was higher CKs total content, accumulation of sCKs and reduction of dCKs in As-hyperaccumulators in contrast to non-hyperaccumulator ferns. A significant depletion of C resources was confirmed in ferns, especially Ps, which was determined by measuring the photosynthetic rate and chlorophyll fluorescence. A fluorescence decrease signified a reduction in the C/N ratio, inducing an increase of bioactive CKs forms in Pc-P and Ps. The impact of As on N utilization was significant in As-hyperaccumulators. The glutamic acid/glutamine ratio, an indicator of primary N assimilation, diminished in all ferns with increased As level in the soil. In conclusion, the results indicate a large phenotypic diversity of Pteris species to As and suggest that the CKs composition and the glutamic acid/glutamine ratio can be used as a tool to diagnose As stress in plants.

• MeSH
• Amino Acids metabolism   MeSH
• Arsenic toxicity   MeSH
• Biomass MeSH
• Cytokinins metabolism   MeSH
• Nitrogen metabolism   MeSH
• Photosynthesis drug effects   MeSH
• Soil Pollutants toxicity   MeSH
• Plant Leaves drug effects   growth & development   metabolism   MeSH
• Pteris drug effects   growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Arsenic MeSH
• Cytokinins MeSH
• Nitrogen MeSH
• Soil Pollutants MeSH

Plant hormones are master regulators of plant growth and development. Better knowledge of their spatial signaling and homeostasis (transport and metabolism) on the lowest structural levels (cellular and subcellular) is therefore crucial to a better understanding of developmental processes in plants. Recent progress in phytohormone analysis at the cellular and subcellular levels has greatly improved the effectiveness of isolation protocols and the sensitivity of analytical methods. This review is mainly focused on homeostasis of two plant hormone groups, auxins and cytokinins. It will summarize and discuss their tissue- and cell-type specific distributions at the cellular and subcellular levels.

• Keywords
• auxin, cellular level, cytokinin, phytohormone metabolism, phytohormone transport, subcellular level,
• MeSH
• Biological Transport MeSH
• Cytokinins metabolism   MeSH
• Plant Physiological Phenomena * MeSH
• Homeostasis * MeSH
• Intracellular Space metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Metabolic Networks and Pathways MeSH
• Organelles metabolism   MeSH
• Plant Growth Regulators metabolism   MeSH
• Plant Cells metabolism   MeSH
• Plant Development * MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Cytokinins MeSH
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH

Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize β-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of β-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.

• Keywords
• enzyme kinetics, fluorescence, glucose, glycoside hydrolases, lab automation, β-glucosidase,
• MeSH
• Automation MeSH
• beta-Glucosidase chemistry   MeSH
• Catalysis MeSH
• Kinetics MeSH
• Zea mays enzymology   MeSH
• Plant Proteins chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta-Glucosidase MeSH
• Plant Proteins MeSH

Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

• MeSH
• Enzyme Activation MeSH
• beta-Glucosidase genetics   metabolism   MeSH
• Databases, Protein MeSH
• Gene Library MeSH
• Hydrolysis MeSH
• Codon MeSH
• Zea mays genetics   metabolism   MeSH
• Mutagenesis MeSH
• Protein Engineering * methods   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Substrate Specificity MeSH
• Computational Biology MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta-Glucosidase MeSH
• Codon MeSH
• Plant Proteins MeSH

In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels.

• Keywords
• Arabidopsis thaliana, cytokinin, cytokinin oxidase/dehydrogenase, isopentenyl transferase, metabolome, proteome.,
• MeSH
• Alkyl and Aryl Transferases metabolism   MeSH
• Arabidopsis drug effects   genetics   metabolism   MeSH
• Chromatography, Liquid MeSH
• Cytokinins pharmacology   MeSH
• Dexamethasone pharmacology   MeSH
• Plants, Genetically Modified MeSH
• Mass Spectrometry MeSH
• Hordeum drug effects   metabolism   MeSH
• Metabolome drug effects   genetics   MeSH
• Metabolomics MeSH
• Arabidopsis Proteins metabolism   MeSH
• Proteome metabolism   MeSH
• Proteomics MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Seedlings drug effects   genetics   MeSH
• Up-Regulation drug effects   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• adenylate isopentenyltransferase MeSH Browser
• Alkyl and Aryl Transferases MeSH
• Cytokinins MeSH
• Dexamethasone MeSH
• Arabidopsis Proteins MeSH
• Proteome MeSH

BACKGROUND AND AIMS: Cytokinins are positive regulators of shoot development. However, it has previously been demonstrated that efficient activation of the cytokinin biosynthesis gene ipt can cause necrotic lesions and wilting in tobacco leaves. Some plant pathogens reportedly use their ability to produce cytokinins in disease development. In response to pathogen attacks, plants can trigger a hypersensitive response that rapidly kills cells near the infection site, depriving the pathogen of nutrients and preventing its spread. In this study, a diverse set of processes that link ipt activation to necrotic lesion formation were investigated in order to evaluate the potential of cytokinins as signals and/or mediators in plant defence against pathogens. METHODS: The binary pOp-ipt/LhGR system for dexamethasone-inducible ipt expression was used to increase endogenous cytokinin levels in transgenic tobacco. Changes in the levels of cytokinins and the stress hormones salicylic, jasmonic and abscisic acid following ipt activation were determined by ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). Trends in hydrogen peroxide content and lipid peroxidation were monitored using the potassium iodide and malondialdehyde assays. The subcellular distribution of hydrogen peroxide was investigated using 3,3'-diaminobenzidine staining. The dynamics of transcripts related to photosynthesis and pathogen response were analysed by reverse transcription followed by quantitative PCR. The effects of cytokinins on photosynthesis were deciphered by analysing changes in chlorophyll fluorescence and leaf gas exchange. KEY RESULTS: Plants can produce sufficiently high levels of cytokinins to trigger fast cell death without any intervening chlorosis - a hallmark of the hypersensitive response. The results suggest that chloroplastic hydrogen peroxide orchestrates the molecular responses underpinning the hypersensitive-like response, including the inhibition of photosynthesis, elevated levels of stress hormones, oxidative membrane damage and stomatal closure. CONCLUSIONS: Necrotic lesion formation triggered by ipt activation closely resembles the hypersensitive response. Cytokinins may thus act as signals and/or mediators in plant defence against pathogen attack.

• Keywords
• Cytokinin, Nicotiana tabacum, abscisic acid, hydrogen peroxide, hypersensitive response, jasmonic acid, lipid peroxidation, non-photochemical quenching, pathogenesis-related proteins, photosynthesis, salicylic acid, stomatal conductance,
• MeSH
• Alkyl and Aryl Transferases genetics   MeSH
• Cell Death MeSH
• Chlorophyll metabolism   MeSH
• Chloroplasts genetics   metabolism   MeSH
• Cytokinins genetics   metabolism   MeSH
• Dexamethasone pharmacology   MeSH
• Photosynthesis genetics   MeSH
• Plants, Genetically Modified MeSH
• Host-Pathogen Interactions * MeSH
• Plant Leaves cytology   genetics   physiology   MeSH
• Necrosis genetics   MeSH
• Oxidative Stress genetics   MeSH
• Hydrogen Peroxide metabolism   MeSH
• Lipid Peroxidation MeSH
• Plant Stomata physiology   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Plant Growth Regulators genetics   metabolism   MeSH
• Nicotiana genetics   microbiology   physiology   MeSH
• Gene Silencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• adenylate isopentenyltransferase MeSH Browser
• Alkyl and Aryl Transferases MeSH
• Chlorophyll MeSH
• Cytokinins MeSH
• Dexamethasone MeSH
• Hydrogen Peroxide MeSH
• Plant Growth Regulators MeSH

Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants.

• MeSH
• Electrophoresis, Gel, Two-Dimensional MeSH
• Arabidopsis chemistry   genetics   metabolism   MeSH
• Cytokinins metabolism   MeSH
• Phosphoproteins chemistry   genetics   metabolism   MeSH
• Arabidopsis Proteins chemistry   genetics   metabolism   MeSH
• Proteome chemistry   genetics   metabolism   MeSH
• Proteomics MeSH
• Gene Expression Regulation, Plant MeSH
• Signal Transduction MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokinins MeSH
• Phosphoproteins MeSH
• Arabidopsis Proteins MeSH
• Proteome MeSH