The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

• Keywords
• auxin, auxin metabolism, flow cytometry, nucleus, subcellular fractionation,
• MeSH
• Arabidopsis drug effects   metabolism   ultrastructure   MeSH
• Cell Nucleus drug effects   metabolism   ultrastructure   MeSH
• Cell Culture Techniques MeSH
• Centrifugation methods   MeSH
• Cell Fractionation instrumentation   methods   MeSH
• Mass Spectrometry MeSH
• Homeostasis physiology   MeSH
• Indoles metabolism   pharmacology   MeSH
• Indoleacetic Acids metabolism   MeSH
• Protoplasts chemistry   MeSH
• Flow Cytometry MeSH
• Plant Growth Regulators metabolism   MeSH
• Plant Cells drug effects   metabolism   ultrastructure   MeSH
• Nicotiana drug effects   metabolism   ultrastructure   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• indole MeSH Browser
• Indoles MeSH
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH

Plant hormones regulate numerous developmental and physiological processes. Abiotic stresses considerably affect production and distribution of phytohormones as the stress signal triggers. The homeostasis of plant hormones is controlled by their de novo synthesis and catabolism. The aim of this work was to analyse the contents of total and individual groups of endogenous cytokinins (CKs) as well as indole-3-acetic acid (IAA) in AtCKX overexpressing centaury plants grown in vitro on graded NaCl concentrations (0, 50, 100, 150, 200 mM). The levels of endogenous stress hormones including abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were also detected. The elevated contents of total CKs were found in all analysed centaury shoots. Furthermore, increased amounts of all five CK groups, as well as enhanced total CKs were revealed on graded NaCl concentrations in non-transformed and AtCKX roots. All analysed AtCKX centaury lines exhibited decreased amounts of endogenous IAA in shoots and roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all AtCKX lines. In shoots and roots of both non-transformed and AtCKX transgenic centaury plants, salinity was associated with an increase of ABA and JA and a decrease of SA content.

• MeSH
• Centaurium growth & development   metabolism   MeSH
• Cyclopentanes analysis   metabolism   MeSH
• Cytokinins analysis   metabolism   MeSH
• Plant Roots growth & development   metabolism   MeSH
• Abscisic Acid analysis   metabolism   MeSH
• Salicylic Acid metabolism   MeSH
• Indoleacetic Acids analysis   metabolism   MeSH
• Oxylipins analysis   metabolism   MeSH
• Plant Growth Regulators analysis   metabolism   MeSH
• Salt Stress * MeSH
• In Vitro Techniques MeSH
• Plant Shoots growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cyclopentanes MeSH
• Cytokinins MeSH
• indoleacetic acid MeSH Browser
• jasmonic acid MeSH Browser
• Abscisic Acid MeSH
• Salicylic Acid MeSH
• Indoleacetic Acids MeSH
• Oxylipins MeSH
• Plant Growth Regulators MeSH

The endoplasmic reticulum (ER) is an extensive network of intracellular membranes. Its major functions include proteosynthesis, protein folding, post-transcriptional modification and sorting of proteins within the cell, and lipid anabolism. Moreover, several studies have suggested that it may be involved in regulating intracellular auxin homeostasis in plants by modulating its metabolism. Therefore, to study auxin metabolome in the ER, it is necessary to obtain a highly enriched (ideally, pure) ER fraction. Isolation of the ER is challenging because its biochemical properties are very similar to those of other cellular endomembranes. Most published protocols for ER isolation use density gradient ultracentrifugation, despite its suboptimal resolving power. Here we present an optimised protocol for ER isolation from Arabidopsis thaliana seedlings for the subsequent mass spectrometric determination of ER-specific auxin metabolite profiles. Auxin metabolite analysis revealed highly elevated levels of active auxin form (IAA) within the ER compared to whole plants. Moreover, samples prepared using our optimised isolation ER protocol are amenable to analysis using various "omics" technologies including analyses of both macromolecular and low molecular weight compounds from the same sample.

• Keywords
• auxin, density gradient centrifugation, endoplasmic reticulum, mass spectrometry, subcellular fractionation,
• MeSH
• Arabidopsis cytology   metabolism   MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Metabolome MeSH
• Metabolomics methods   MeSH
• Arabidopsis Proteins analysis   metabolism   MeSH
• Proteomics methods   MeSH
• Plant Cells MeSH
• Seedlings cytology   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• indoleacetic acid MeSH Browser
• Indoleacetic Acids MeSH
• Arabidopsis Proteins MeSH

Cytokinins are plant hormones, derivatives of adenine with a side chain at the N6-position. They are involved in many physiological processes. While the metabolism of trans-zeatin and isopentenyladenine, which are considered to be highly active cytokinins, has been extensively studied, there are others with less obvious functions, such as cis-zeatin, dihydrozeatin, and aromatic cytokinins, which have been comparatively neglected. To help explain this duality, we present a novel hypothesis metaphorically comparing various cytokinin forms, enzymes of CK metabolism, and their signalling and transporter functions to the comics superheroes Hulk and Deadpool. Hulk is a powerful but short-lived creation, whilst Deadpool presents a more subtle and enduring force. With this dual framework in mind, this review compares different cytokinin metabolites, and their biosynthesis, translocation, and sensing to illustrate the different mechanisms behind the two CK strategies. This is put together and applied to a plant developmental scale and, beyond plants, to interactions with organisms of other kingdoms, to highlight where future study can benefit the understanding of plant fitness and productivity.

• Keywords
• Hulk/Deadpool, aromatic cytokinins, cis-zeatin, cytokinin biosynthesis, cytokinin oxidase/dehydrogenase, cytokinin signalling, cytokinin transport, cytokinins, isopentenyl transferase,
• MeSH
• Arabidopsis metabolism   MeSH
• Models, Biological MeSH
• Biological Transport MeSH
• Biological Assay MeSH
• Cytokinins metabolism   MeSH
• Plant Physiological Phenomena * MeSH
• Glycosylation MeSH
• Hydrolysis MeSH
• Kinetics MeSH
• Kinetin metabolism   MeSH
• Oxidoreductases metabolism   MeSH
• Plant Growth Regulators metabolism   MeSH
• Plants metabolism   MeSH
• Signal Transduction * MeSH
• Protein Binding MeSH
• Zeatin analogs & derivatives   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• cytokinin oxidase MeSH Browser
• Cytokinins MeSH
• dihydrozeatin MeSH Browser
• Kinetin MeSH
• Oxidoreductases MeSH
• Plant Growth Regulators MeSH
• Zeatin MeSH

Cytokinins modulate a number of important developmental processes, including the last phase of leaf development, known as senescence, which is associated with chlorophyll breakdown, photosynthetic apparatus disintegration and oxidative damage. There is ample evidence that cytokinins can slow down all these senescence-accompanying changes. Here, we review relationships between the various mechanisms of action of these regulatory molecules. We highlight their connection to photosynthesis, the pivotal process that generates assimilates, however may also lead to oxidative damage. Thus, we also focus on cytokinin induction of protective responses against oxidative damage. Activation of antioxidative enzymes in senescing tissues is described as well as changes in the levels of naturally occurring antioxidative compounds, such as phenolic acids and flavonoids, in plant explants. The main goal of this review is to show how the biological activities of cytokinins may be related to their chemical structure. New links between molecular aspects of natural cytokinins and their synthetic derivatives with antisenescent properties are described. Structural motifs in cytokinin molecules that may explain why these molecules play such a significant regulatory role are outlined.

• Keywords
• antioxidant, antioxidant enzymes, antisenescent, cytokinin, derivative, genes, photosynthesis, plant defence, structure and activity relationship,
• MeSH
• Antioxidants chemistry   metabolism   MeSH
• Cytokinins chemistry   metabolism   MeSH
• Flavonoids analysis   MeSH
• Photosynthesis MeSH
• Plant Leaves chemistry   growth & development   physiology   MeSH
• Molecular Structure MeSH
• Plants chemistry   MeSH
• Plant Development MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Antioxidants MeSH
• Cytokinins MeSH
• Flavonoids 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

Auxins mediate various processes that are involved in plant growth and development in response to specific environmental conditions. Its proper spatio-temporal distribution that is driven by polar auxin transport machinery plays a crucial role in the wide range of auxins physiological effects. Numbers of approaches have been developed to either directly or indirectly monitor auxin distribution in vivo in order to elucidate the basis of its precise regulation. Herein, we provide an updated list of valuable techniques used for monitoring auxins in plants, with their utilities and limitations. Because the spatial and temporal resolutions of the presented approaches are different, their combination may provide a comprehensive outcome of auxin distribution in diverse developmental processes.

• Keywords
• auxin, auxin distribution, auxin signalling, auxin transport, direct visualization, indirect visualization, receptor, sensor,
• MeSH
• Arabidopsis metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Development physiology   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Indoleacetic Acids MeSH

Plant growth and development are critically influenced by unpredictable abiotic factors. To survive fluctuating changes in their environments, plants have had to develop robust adaptive mechanisms. The dynamic and complementary actions of the auxin and cytokinin pathways regulate a plethora of developmental processes, and their ability to crosstalk makes them ideal candidates for mediating stress-adaptation responses. Other crucial signaling molecules responsible for the tremendous plasticity observed in plant morphology and in response to abiotic stress are reactive oxygen species (ROS). Proper temporal and spatial distribution of ROS and hormone gradients is crucial for plant survival in response to unfavorable environments. In this regard, the convergence of ROS with phytohormone pathways acts as an integrator of external and developmental signals into systemic responses organized to adapt plants to their environments. Auxin and cytokinin signaling pathways have been studied extensively. Nevertheless, we do not yet understand the impact on plant stress tolerance of the sophisticated crosstalk between the two hormones. Here, we review current knowledge on the function of auxin and cytokinin in redirecting growth induced by abiotic stress in order to deduce their potential points of crosstalk.

• Keywords
• ROS, abiotic stress, adaptation, auxin, crosstalk, cytokinin, growth,
• MeSH
• Biological Transport MeSH
• Cytokinins metabolism   MeSH
• Adaptation, Physiological MeSH
• Stress, Physiological * MeSH
• Plant Physiological Phenomena * MeSH
• Gene Regulatory Networks MeSH
• Indoleacetic Acids metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators metabolism   MeSH
• Plants genetics   metabolism   MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Cytokinins MeSH
• Indoleacetic Acids MeSH
• Plant Growth Regulators 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