The genomes of charophyte green algae, close relatives of land plants, typically do not show signs of developmental regulation by phytohormones. However, scattered reports of endogenous phytohormone production in these organisms exist. We performed a comprehensive analysis of multiple phytohormones in Viridiplantae, focusing mainly on charophytes. We show that auxin, salicylic acid, ethylene and tRNA-derived cytokinins including cis-zeatin are found ubiquitously in Viridiplantae. By contrast, land plants but not green algae contain the trans-zeatin type cytokinins as well as auxin and cytokinin conjugates. Charophytes occasionally produce jasmonates and abscisic acid, whereas the latter is detected consistently in land plants. Several phytohormones are excreted into the culture medium, including auxin by charophytes and cytokinins and salicylic acid by Viridiplantae in general. We note that the conservation of phytohormone biosynthesis and signaling pathways known from angiosperms does not match the capacity for phytohormone biosynthesis in Viridiplantae. Our phylogenetically guided analysis of established algal cultures provides an important insight into phytohormone biosynthesis and metabolism across Streptophyta.

• MeSH
• Biological Evolution MeSH
• Chlorophyta metabolism   genetics   MeSH
• Cyclopentanes metabolism   MeSH
• Cytokinins * metabolism   MeSH
• Ethylenes metabolism   MeSH
• Phylogeny * MeSH
• Abscisic Acid metabolism   MeSH
• Salicylic Acid metabolism   MeSH
• Indoleacetic Acids * metabolism   MeSH
• Oxylipins metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators * metabolism   MeSH
• Signal Transduction MeSH
• Viridiplantae metabolism   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cyclopentanes MeSH
• Cytokinins * MeSH
• ethylene MeSH Browser
• Ethylenes MeSH
• jasmonic acid MeSH Browser
• Abscisic Acid MeSH
• Salicylic Acid MeSH
• Indoleacetic Acids * MeSH
• Oxylipins MeSH
• Plant Growth Regulators * MeSH

The field of plant hormonomics focuses on the qualitative and quantitative analysis of the hormone complement in plant samples, akin to other omics sciences. Plant hormones, alongside primary and secondary metabolites, govern vital processes throughout a plant's lifecycle. While active hormones have received significant attention, studying all related compounds provides valuable insights into internal processes. Conventional single-class plant hormone analysis employs thorough sample purification, short analysis and triple quadrupole tandem mass spectrometry. Conversely, comprehensive hormonomics analysis necessitates minimal purification, robust and efficient separation and better-performing mass spectrometry instruments. This review summarizes the current status of plant hormone analysis methods, focusing on sample preparation, advances in chromatographic separation and mass spectrometric detection, including a discussion on internal standard selection and the potential of derivatization. Moreover, current approaches for assessing the spatiotemporal distribution are evaluated. The review touches on the legitimacy of the term plant hormonomics by exploring the current status of methods and outlining possible future trends.

• Keywords
• Hormonomics, Internal standard, Liquid chromatography, Mass spectrometry, Matrix effect, Metabolomics, Omics, Plant hormone, Solid phase extraction,
• Publication type
• Journal Article MeSH
• Review MeSH

Auxins are a group of phytohormones that play a key role in plant growth and development, mainly presented by the major member of the family - indole-3-acetic acid (IAA). The levels of free IAA are regulated, in addition to de novo biosynthesis, by irreversible oxidative catabolism and reversible conjugation with sugars and amino acids. These conjugates, which serve as inactive storage forms of auxin and/or degradation intermediates, can also be oxidized to form 2-oxindole-3-acetyl-1-O-ß-d-glucose (oxIAA-glc) and oxIAA-amino acids (oxIAA-AAs). Until now, only oxIAA conjugates with aspartate and glutamate have been identified in plants. However, detailed information on the endogenous levels of these and other putative oxIAA-amino acid conjugates in various plant species and their spatial distribution is still not well understood but is finally getting more attention. Herein, we identified and characterized two novel naturally occurring auxin metabolites in plants, namely oxIAA-leucine (oxIAA-Leu) and oxIAA-phenylalanine (oxIAA-Phe). Subsequently, a new liquid chromatography-tandem mass spectrometry method was developed for the determination of a wide range of IAA metabolites. Using this methodology, the quantitative determination of IAA metabolites including newly characterized oxIAA conjugates in roots, shoots and cotyledons of four selected plant models - Arabidopsis thaliana, pea (Pisum sativum L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) was performed to compare auxin metabolite profiles. The distribution of various groups of auxin metabolites differed notably among the studied species as well as their sections. For example, oxIAA-AA conjugates were the major metabolites found in pea, while oxIAA-glc dominated in Arabidopsis. We further compared IAA metabolite levels in plants harvested at different growth stages to monitor the dynamics of IAA metabolite profiles during early seedling development. In general, our results show a great diversity of auxin inactivation pathways among angiosperm plants. We believe that our findings will greatly contribute to a better understanding of IAA homeostasis.

• Keywords
• 2-oxindole-3-acetic acid, HPLC-MS/MS, auxin conjugates, auxin metabolism, catabolism, indole-3-acetic acid, quantitative analysis,
• Publication type
• Journal Article MeSH

A number of scientific reports published to date contain data on endogenous levels of various phytohormones in potato (Solanum tuberosum L.) but a complete cytokinin profile of potato tissues, that would include data on all particular molecular forms of cytokinin, has still been missing. In this work, endogenous levels of all analytically detectable isoprenoid cytokinins, as well as the auxin indole-3-acetic acid (IAA), and abscisic acid (ABA) have been determined in shoots and roots of 30 day old in vitro grown potato (cv. Désirée). The results presented here are generally similar to other data reported for in vitro grown potato plants, whereas greenhouse-grown plants typically contain lower levels of ABA, possibly indicating that in vitro grown potato is exposed to chronic stress. Cytokinin N-glucosides, particularly N7-glucosides, are the dominant cytokinin forms in both shoots and roots of potato, whereas nucleobases, as the bioactive forms of cytokinins, comprise a low proportion of cytokinin levels in tissues of potato. Differences in phytohormone composition between shoots and roots of potato suggest specific patterns of transport and/or differences in tissue-specific metabolism of plant hormones. These results represent a contribution to understanding the hormonomics of potato, a crop species of extraordinary economic importance.

• MeSH
• Cytokinins analysis   metabolism   MeSH
• Stress, Physiological MeSH
• Plant Roots metabolism   MeSH
• Abscisic Acid analysis   metabolism   MeSH
• Indoleacetic Acids analysis   metabolism   MeSH
• Plant Growth Regulators analysis   metabolism   MeSH
• Solanum tuberosum growth & development   metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Plant Shoots metabolism   MeSH
• Chromatography, High Pressure Liquid MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokinins MeSH
• indoleacetic acid MeSH Browser
• Abscisic Acid MeSH
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH

BACKGROUND AND AIMS: The metabolism of cytokinins (CKs) and auxins in vascular plants is relatively well understood, but data concerning their metabolic pathways in non-vascular plants are still rather rare. With the aim of filling this gap, 20 representatives of taxonomically major lineages of cyanobacteria and algae from Cyanophyceae, Xanthophyceae, Eustigmatophyceae, Porphyridiophyceae, Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Zygnematophyceae and Klebsormidiophyceae were analysed for endogenous profiles of CKs and auxins and some of them were used for studies of the metabolic fate of exogenously applied radiolabelled CK, [3H]trans-zeatin (transZ) and auxin ([3H]indole-3-acetic acid (IAA)), and the dynamics of endogenous CK and auxin pools during algal growth and cell division. METHODS: Quantification of phytohormone levels was performed by high-performance or ultrahigh-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS/MS, UHPLC-MS/MS). The dynamics of exogenously applied [3H]transZ and [3H]IAA in cell cultures were monitored by HPLC with on-line radioactivity detection. KEY RESULTS: The comprehensive screen of selected cyanobacteria and algae for endogenous CKs revealed a predominance of bioactive and phosphate CK forms while O- and N-glucosides evidently did not contribute greatly to the total CK pool. The abundance of cis-zeatin-type CKs and occurrence of CK 2-methylthio derivatives pointed to the tRNA pathway as a substantial source of CKs. The importance of the tRNA biosynthetic pathway was proved by the detection of tRNA-bound CKs during the course of Scenedesmus obliquus growth. Among auxins, free IAA and its oxidation catabolite 2-oxindole-3-acetic acid represented the prevailing endogenous forms. After treatment with [3H]IAA, IAA-aspartate and indole-3-acetyl-1-glucosyl ester were detected as major auxin metabolites. Moreover, different dynamics of endogenous CKs and auxin profiles during S. obliquus culture clearly demonstrated diverse roles of both phytohormones in algal growth and cell division. CONCLUSIONS: Our data suggest the existence and functioning of a complex network of metabolic pathways and activity control of CKs and auxins in cyanobacteria and algae that apparently differ from those in vascular plants.

• Keywords
• Cytokinin, algae, auxin, cyanobacteria, cytokinin 2-methylthioderivatives, cytokinin oxidase/dehydrogenase, indole-3-acetic acid, metabolism, tRNA, trans-zeatin,
• MeSH
• Chlorophyta chemistry   metabolism   physiology   MeSH
• Cytokinins analysis   metabolism   MeSH
• Phylogeny MeSH
• Spectrometry, Mass, Electrospray Ionization methods   MeSH
• Homeostasis physiology   MeSH
• Indoleacetic Acids analysis   metabolism   MeSH
• Plant Growth Regulators analysis   metabolism   MeSH
• Cyanobacteria chemistry   metabolism   physiology   MeSH
• Streptophyta chemistry   metabolism   physiology   MeSH
• Tandem Mass Spectrometry methods   MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokinins MeSH
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH

The herbicide 2,4-D exhibits an auxinic activity and therefore can be used as a synthetic and traceable analog to study auxin-related responses. Here we identified that not only exogenous 2,4-D but also its amide-linked metabolite 2,4-D-Glu displayed an inhibitory effect on plant growth via the TIR1/AFB auxin-mediated signaling pathway. To further investigate 2,4-D metabolite conversion, identity and activity, we have developed a novel purification procedure based on the combination of ion exchange and immuno-specific sorbents combined with a sensitive liquid chromatography-mass spectrometry method. In 2,4-D treated samples, 2,4-D-Glu and 2,4-D-Asp were detected at 100-fold lower concentrations compared to 2,4-D levels, showing that 2,4-D can be metabolized in the plant. Moreover, 2,4-D-Asp and 2,4-D-Glu were identified as reversible forms of 2,4-D homeostasis that can be converted to free 2,4-D. This work paves the way to new studies of auxin action in plant development.

• MeSH
• Arabidopsis drug effects   growth & development   metabolism   MeSH
• F-Box Proteins genetics   metabolism   MeSH
• Herbicides pharmacology   MeSH
• Homeostasis MeSH
• Plant Roots drug effects   growth & development   metabolism   MeSH
• 2,4-Dichlorophenoxyacetic Acid pharmacology   MeSH
• Indoleacetic Acids metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Receptors, Cell Surface genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators pharmacology   MeSH
• Seedlings drug effects   growth & development   metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• F-Box Proteins MeSH
• Herbicides MeSH
• 2,4-Dichlorophenoxyacetic Acid MeSH
• Indoleacetic Acids MeSH
• Arabidopsis Proteins MeSH
• Receptors, Cell Surface MeSH
• Plant Growth Regulators MeSH
• TIR1 protein, Arabidopsis MeSH Browser

Cytokinins (CKs) are phytohormones regulating plant growth and development as well as response to the environment. In order to evaluate their function in heat stress (HS) responses, the effect of CK elevation was determined during three types of HS - targeted to shoots, targeted to roots and applied to the whole plant. The early (30min) and longer term (3h) responses were followed at the hormonal, transcriptomic and proteomic levels in Arabidopsis transformants with dexamethasone-inducible expression of the CK biosynthetic gene isopentenyltransferase (ipt) and the corresponding wild-type (Col-0). Combination of hormonal and phenotypic analyses showed transient up-regulation of the CK/abscisic acid ratio, which controls stomatal aperture, to be more pronounced in the transformant. HS responses of the root proteome and Rubisco-immunodepleted leaf proteome were followed using 2-D gel electrophoresis and MALDI-TOF/TOF. More than 100 HS-responsive proteins were detected, most of them being modulated by CK increase. Proteome and transcriptome analyses demonstrated that CKs have longer term positive effects on the stress-related proteins and transcripts, as well as on the photosynthesis-related ones. Transient accumulation of CKs and stimulation of their signal transduction in tissue(s) not exposed to HS indicate that they are involved in plant stress responses.

• Keywords
• Abscisic acid, Arabidopsis thaliana, cytokinin, heat stress, isopentenyltransferase, proteome.,
• MeSH
• Alkyl and Aryl Transferases physiology   MeSH
• Arabidopsis drug effects   metabolism   physiology   MeSH
• Cytokinins physiology   MeSH
• Dexamethasone pharmacology   MeSH
• Plant Roots metabolism   physiology   MeSH
• Abscisic Acid physiology   MeSH
• Proteomics MeSH
• Heat-Shock Response physiology   MeSH
• Gene Expression Regulation, Plant physiology   MeSH
• Plant Growth Regulators physiology   MeSH
• Signal Transduction drug effects   physiology   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Gene Expression Profiling MeSH
• Plant Shoots metabolism   physiology   MeSH
• Hot Temperature 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
• Abscisic Acid MeSH
• Plant Growth Regulators MeSH

Plant hormones act as chemical messengers in the regulation of myriads of physiological processes that occur in plants. To date, nine groups of plant hormones have been identified and more will probably be discovered. Furthermore, members of each group may participate in the regulation of physiological responses in planta both alone and in concert with members of either the same group or other groups. The ideal way to study biochemical processes involving these signalling molecules is 'hormone profiling', i.e. quantification of not only the hormones themselves, but also their biosynthetic precursors and metabolites in plant tissues. However, this is highly challenging since trace amounts of all of these substances are present in highly complex plant matrices. Here, we review advances, current trends and future perspectives in the analysis of all currently known plant hormones and the associated problems of extracting them from plant tissues and separating them from the numerous potentially interfering compounds.

• MeSH
• Plant Growth Regulators * chemistry   isolation & purification   MeSH
• Plants chemistry   MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Plant Growth Regulators * MeSH

Polar auxin transport (PAT) plays key roles in the regulation of plant growth and development. Flavonoids have been implicated in the inhibition of PAT. However, the active flavonoid derivative(s) involved in this process in vivo has not yet been identified. Here, we provide evidence that a specific flavonol bis-glycoside is correlated with shorter plant stature and reduced PAT. Specific flavonoid-biosynthetic or flavonoid-glycosylating steps were genetically blocked in Arabidopsis thaliana. The differential flavonol patterns established were analyzed by high-performance liquid chromatography (HPLC) and related to altered plant stature. PAT was monitored in stem segments using a radioactive [(3)H]-indole-3-acetic acid tracer. The flavonoid 3-O-glucosyltransferase mutant ugt78d2 exhibited a dwarf stature in addition to its altered flavonol glycoside pattern. This was accompanied by reduced PAT in ugt78d2 shoots. The ugt78d2-dependent growth defects were flavonoid dependent, as they were rescued by genetic blocking of flavonoid biosynthesis. Phenotypic and metabolic analyses of a series of mutants defective at various steps of flavonoid formation narrowed down the potentially active moiety to kaempferol 3-O-rhamnoside-7-O-rhamnoside. Moreover, the level of this compound was negatively correlated with basipetal auxin transport. These results indicate that kaempferol 3-O-rhamnoside-7-O-rhamnoside acts as an endogenous PAT inhibitor in Arabidopsis shoots.

• Keywords
• Arabidopsis thaliana, flavonol biosynthesis, flavonol glycoside, flavonol glycosyltransferases, plant growth, polar auxin transport,
• MeSH
• Arabidopsis growth & development   metabolism   MeSH
• Biological Transport MeSH
• Phenotype MeSH
• Kaempferols metabolism   physiology   MeSH
• Indoleacetic Acids metabolism   MeSH
• Plant Shoots genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Kaempferols MeSH
• Indoleacetic Acids MeSH

The molecular basis of cellular auxin transport is still not fully understood. Although a number of carriers have been identified and proved to be involved in auxin transport, their regulation and possible activity of as yet unknown transporters remain unclear. Nevertheless, using single-cell-based systems it is possible to track the course of auxin accumulation inside cells and to specify and quantify some auxin transport parameters. The synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) are generally considered to be suitable tools for auxin transport studies because they are transported specifically via either auxin influx or efflux carriers, respectively. Our results indicate that NAA can be metabolized rapidly in tobacco BY-2 cells. The predominant metabolite has been identified as NAA glucosyl ester and it is shown that all NAA metabolites were retained inside the cells. This implies that the transport efficiency of auxin efflux transporters is higher than previously assumed. By contrast, the metabolism of 2,4-D remained fairly weak. Moreover, using data on the accumulation of 2,4-D measured in the presence of auxin transport inhibitors, it is shown that 2,4-D is also transported by efflux carriers. These results suggest that 2,4-D is a promising tool for determining both auxin influx and efflux activities. Based on the accumulation data, a mathematical model of 2,4-D transport at a single-cell level is proposed. Optimization of the model provides estimates of crucial transport parameters and, together with its validation by successfully predicting the course of 2,4-D accumulation, it confirms the consistency of the present concept of cellular auxin transport.

• MeSH
• Biological Transport MeSH
• Cells, Cultured MeSH
• 2,4-Dichlorophenoxyacetic Acid chemistry   metabolism   MeSH
• Indoleacetic Acids chemistry   metabolism   MeSH
• Naphthalenes chemistry   metabolism   MeSH
• Plant Growth Regulators chemistry   metabolism   MeSH
• Nicotiana chemistry   cytology   metabolism   MeSH
• Models, Theoretical MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2,4-Dichlorophenoxyacetic Acid MeSH
• Indoleacetic Acids MeSH
• Naphthalenes MeSH
• naphthalene MeSH Browser
• Plant Growth Regulators MeSH