Plant microgametogenesis involves stages leading to the progressive development of unicellular microspores into mature pollen. Despite the active and continuing interest in the study of male reproductive development, little is still known about the hormonomics at each ontogenetic stage. In this work, we characterized the profiles and dynamics of phytohormones during the process of microgametogenesis in four Nicotiana species (Nicotiana tabacum, Nicotiana alata, Nicotiana langsdorffii, and Nicotiana mutabilis). Taking advantage of advanced HPLC-ESI-MS/MS, twenty to thirty endogenous hormone derivatives were identified throughout pollen ontogenesis, including cytokinins, auxins, ABA and its derivatives, jasmonates, and phenolic compounds. The spectra of endogenous phytohormones changed dynamically during tobacco pollen ontogeny, indicating their important role in pollen growth and development. The different dynamics in the accumulation of endogenous phytohormones during pollen ontogenesis between N. tabacum (section Nicotiana) and the other three species (section Alatae) reflects their different phylogenetic positions and origin within the genus Nicotiana. We demonstrated the involvement of certain phytohormone forms, such as cis-zeatin- and methylthiol-type CKs, some derivatives of abscisic acid, phenylacetic and benzoic acids, in pollen development for the first time here. Our results suggest that unequal levels of endogenous hormones and the presence of specific derivatives may be characteristic for pollen development in different phylogenetic plant groups. These results represent the currently most comprehensive study of plant hormones during the process of pollen development.

• Keywords
• Nicotiana spp., hormonome, male gametophyte, ontogeny, phytohormones, pollen development,
• Publication type
• Journal Article 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 a class of phytohormones, signalling molecules specific to plants. They act as regulators of diverse physiological processes in complex signalling pathways. It is necessary for plants to continuously regulate cytokinin distribution among different organs, tissues, cells, and compartments. Such regulatory mechanisms include cytokinin biosynthesis, metabolic conversions and degradation, as well as cytokinin membrane transport. In our review, we aim to provide a thorough picture of the latter. We begin by summarizing cytokinin structures and physicochemical properties. Then, we revise the elementary thermodynamic and kinetic aspects of cytokinin membrane transport. Next, we review which membrane-bound carrier proteins and protein families recognize cytokinins as their substrates. Namely, we discuss the families of "equilibrative nucleoside transporters" and "purine permeases", which translocate diverse purine-related compounds, and proteins AtPUP14, AtABCG14, AtAZG1, and AtAZG2, which are specific to cytokinins. We also address long-distance cytokinin transport. Putting all these pieces together, we finally discuss cytokinin distribution as a net result of these processes, diverse in their physicochemical nature but acting together to promote plant fitness.

• Keywords
• ABCG14, AZG1, AZG2, PUP14, cytokinin distribution, cytokinin hydrophobicity, cytokinin transport, membrane transport,
• MeSH
• Arabidopsis metabolism   MeSH
• Biological Transport MeSH
• Cell Membrane metabolism   MeSH
• Cytokinins metabolism   MeSH
• Homeostasis MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Kinetics MeSH
• Plant Roots metabolism   MeSH
• Membrane Transport Proteins metabolism   MeSH
• Arabidopsis Proteins genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators metabolism   MeSH
• Signal Transduction physiology   MeSH
• Thermodynamics MeSH
• Plant Shoots metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Cytokinins MeSH
• Membrane Transport Proteins MeSH
• Arabidopsis Proteins MeSH
• Plant Growth Regulators 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

Cytokinin (CK) N-glucosides are the most abundant group of CK metabolites in many species; however, their physiological role in planta was for a long time perceived as irreversible storage CK forms only. Recently, a comprehensive screen showed that only vascular plants form CK N-glucosides in contrast to mosses, algae, and fungi. The formation of CK N-glucosides as biologically inactive CK conjugates thus represents an evolutionarily young mechanism for deactivation of CK bases. Even though CK N-glucosides are not biologically active themselves due to their inability to activate the CK perception system, new data on CK N-glucoside metabolism show that trans-zeatin (tZ) N7- and N9-glucosides are metabolized in vivo, efficiently releasing free CK bases that are most probably responsible for the biological activities observed in a number of bioassays. Moreover, CK N-glucosides' subcellular localization as well as their abundance in xylem both point to their possible plasma membrane transport and indicate a role also as CK transport forms. Identification of the enzyme(s) responsible for the hydrolysis of tZ N7- and N9-glucosides, as well as the discovery of putative CK N-glucoside plasma membrane transporter, would unveil important parts of the overall picture of CK metabolic interconversions and their physiological importance.

• Keywords
• UGT, cytokinin N-glucoside, cytokinin metabolism, cytokinin transport, isopentenyladenine N7-glucoside, isopentenyladenine N9-glucoside, zeatin N7-glucoside, zeatin N9-glucoside,
• Publication type
• Journal Article MeSH
• Review 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

Plant hormones cytokinins (CKs) are one of the major mediators of physiological responses throughout plant life span. Therefore, a proper homeostasis is maintained by regulation of their active levels. Besides degradation, CKs are deactivated by uridine diphosphate glycosyltransferases (UGTs). Physiologically, CKs active levels decline in senescing organs, providing a signal to nutrients that a shift to reproductive tissues has begun. In this work, we show CK glucosides distribution in Arabidopsis leaves during major developmental transition phases. Besides continuous accumulation of N-glucosides we detected sharp maximum of the glucosides in senescence. This is caused prevalently by N7-glucosides followed by N9-glucosides and specifically also by trans-zeatin-O-glucoside (tZOG). Interestingly, we observed a similar trend in response to exogenously applied CK. In Arabidopsis, only three UGTs deactivate CKs in vivo: UGT76C1, UGT76C2 and UGT85A1. We thereby show that UGT85A1 is specifically expressed in senescent leaves whereas UGT76C2 is activated rapidly in response to exogenously applied CK. To shed more light on the UGTs physiological roles, we performed a comparative study on UGTs loss-of-function mutants, characterizing a true ugt85a1-1 loss-of-function mutant for the first time. Although no altered phenotype was detected under standard condition we observed reduced chlorophyll degradation with increased anthocyanin accumulation in our experiment on detached leaves accompanied by senescence and stress related genes modulated expression. Among the mutants, ugt76c2 possessed extremely diminished CK N-glucosides levels whereas ugt76c1 showed some specificity toward cis-zeatin (cZ). Besides tZOG, a broader range of CK glucosides was decreased in ugt85a1-1. Performing CK metabolism gene expression profiling, we revealed that activation of CK degradation pathway serves as a general regulatory mechanism of disturbed CK homeostasis followed by decreased CK signaling in all UGT mutants. In contrast, a specific regulation of CKX7, CKX1 and CKX2 was observed for each individual UGT mutant isoform after exogenous CK uptake. Employing an in silico prediction we proposed cytosolic localization of UGT76C1 and UGT76C2, that we further confirmed by GFP tagging of UGT76C2. Integrating all the results, we therefore hypothesize that UGTs possess different physiological roles in Arabidopsis and serve as a fine-tuning mechanism of active CK levels in cytosol.

• Keywords
• Arabidopsis, GFP subcellular localization, cytokinin, glycosyltransferase, senescence,
• Publication type
• Journal Article 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