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
- cytokininy analýza metabolismus MeSH
- fyziologický stres MeSH
- kořeny rostlin metabolismus MeSH
- kyselina abscisová analýza metabolismus MeSH
- kyseliny indoloctové analýza metabolismus MeSH
- regulátory růstu rostlin analýza metabolismus MeSH
- Solanum tuberosum růst a vývoj metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- výhonky rostlin metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hormonal dynamics after Plasmodiophora brassicae infection were compared in two Brassica napus cultivars-more resistant SY Alister and more sensitive Hornet, in order to elucidate responses associated with efficient defense. Both cultivars responded to infection by the early transient elevation of active cytokinins (predominantly cis-zeatin) and auxin indole-3-acetic acid (IAA) in leaves and roots, which was longer in Hornet. Moderate IAA levels in Hornet roots coincided with a high expression of biosynthetic gene nitrilaseNIT1 (contrary to TAA1, YUC8, YUC9). Alister had a higher basal level of salicylic acid (SA), and it stimulated its production (via the expression of isochorismate synthase (ICS1)) in roots earlier than Hornet. Gall formation stimulated cytokinin, auxin, and SA levels-with a maximum 22 days after inoculation (dai). SA marker gene PR1 expression was the most profound at the time point where gall formation began, in leaves, roots, and especially in galls. Jasmonic acid (JA) was higher in Hornet than in Alister during the whole experiment. To investigate SA and JA function, SA was applied before infection, and twice (before infection and 15 dai), and JA at 15 dai. Double SA application diminished gall formation in Alister, and JA promoted gall formation in both cultivars. Activation of SA/JA pathways reflects the main differences in clubroot resistance.
- MeSH
- aminohydrolasy genetika MeSH
- Brassica napus růst a vývoj metabolismus parazitologie MeSH
- cyklopentany analýza MeSH
- cytokininy analýza MeSH
- intramolekulární transferasy genetika MeSH
- kořeny rostlin růst a vývoj metabolismus parazitologie MeSH
- kyseliny indoloctové analýza MeSH
- listy rostlin růst a vývoj metabolismus parazitologie MeSH
- nemoci rostlin parazitologie MeSH
- odolnost vůči nemocem MeSH
- oxylipiny analýza MeSH
- Plasmodiophorida patogenita MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin analýza MeSH
- rostlinné proteiny genetika MeSH
- vývojová regulace genové exprese MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
The methodology described here represents an improved strategy for analysis of a broad range of stress-related plant hormones including jasmonates, salicylic acid, abscisic acid, and auxin metabolites. The method conditions are optimized in order to reduce the background effect of complicated plant matrix, allow effective preconcentration and thus perform highly sensitive profiling of multiple plant hormones by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).
- MeSH
- cyklopentany analýza MeSH
- kyselina abscisová analýza MeSH
- kyselina salicylová analýza MeSH
- kyseliny indoloctové analýza MeSH
- oxylipiny analýza MeSH
- regulátory růstu rostlin analýza MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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, [(3)H]trans-zeatin (transZ) and auxin ([(3)H]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 [(3)H]transZ and [(3)H]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 [(3)H]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.
- MeSH
- Chlorophyta chemie metabolismus fyziologie MeSH
- cytokininy analýza metabolismus MeSH
- fylogeneze MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- homeostáza fyziologie MeSH
- kyseliny indoloctové analýza metabolismus MeSH
- regulátory růstu rostlin analýza metabolismus MeSH
- sinice chemie metabolismus fyziologie MeSH
- Streptophyta chemie metabolismus fyziologie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
Parallel determination of auxin and cytokinin levels within plant organs and tissues represents an invaluable tool for studies of their physiological effects and mutual interactions. Thanks to their different chemical structures, auxins, cytokinins and their metabolites are often determined separately, using specialized procedures of sample purification, extraction, and quantification. However, recent progress in the sensitivity of analytical methods of liquid chromatography coupled to mass spectrometry (LC-MS) allows parallel analysis of multiple compounds. Here we describe a method that is based on single step purification protocol followed by LC-MS separation and detection for parallel analysis of auxins, cytokinins and their metabolites in various plant tissues and cell cultures.
- MeSH
- chromatografie kapalinová MeSH
- cytokininy analýza chemie izolace a purifikace MeSH
- kyseliny indoloctové analýza chemie izolace a purifikace MeSH
- metabolomika metody MeSH
- regulátory růstu rostlin analýza MeSH
- rostlinné extrakty analýza chemie izolace a purifikace MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Bryophytes represent a very diverse group of non-vascular plants such as mosses, liverworts and hornworts and the oldest extant lineage of land plants. Determination of endogenous phytohormone profiles in bryophytes can provide substantial information about early land plant evolution. In this study, we screened thirty bryophyte species including six liverworts and twenty-four mosses for their phytohormone profiles in order to relate the hormonome with phylogeny in the plant kingdom. METHODOLOGY: Samples belonging to nine orders (Pelliales, Jungermanniales, Porellales, Sphagnales, Tetraphidales, Polytrichales, Dicranales, Bryales, Hypnales) were collected in Central and Northern Bohemia. The phytohormone content was analysed with a high performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS). PRINCIPAL FINDINGS: As revealed for growth hormones, some common traits such as weak conjugation of both cytokinins and auxins, intensive production of cisZ-type cytokinins and strong oxidative degradation of auxins with abundance of a major primary catabolite 2-oxindole-3-acetic acid were pronounced in all bryophytes. Whereas apparent dissimilarities in growth hormones profiles between liverworts and mosses were evident, no obvious trends in stress hormone levels (abscisic acid, jasmonic acid, salicylic acid) were found with respect to the phylogeny. CONCLUSION: The apparent differences in conjugation and/or degradation strategies of growth hormones between liverworts and mosses might potentially show a hidden link between vascular plants and liverworts. On the other hand, the complement of stress hormones in bryophytes probably correlate rather with prevailing environmental conditions and plant survival strategy than with plant evolution.
- MeSH
- Bryophyta klasifikace metabolismus MeSH
- cyklopentany analýza metabolismus MeSH
- cytokininy analýza metabolismus MeSH
- fylogeneze MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- kyselina abscisová analýza metabolismus MeSH
- kyselina salicylová analýza metabolismus MeSH
- kyseliny indoloctové analýza metabolismus MeSH
- oxylipiny analýza metabolismus MeSH
- regulátory růstu rostlin analýza metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Stress-induced changes in phytohormone metabolite profiles have rapid effects on plant metabolic activity and growth. The jasmonates (JAs) are a group of fatty acid-derived stress response regulators with roles in numerous developmental processes. To elucidate their dual regulatory effects, which overlap with those of other important defence-signalling plant hormones such as salicylic acid (SA), abscisic acid (ABA) and indole-3-acetic acid (IAA), we have developed a highly efficient single-step clean-up procedure for their enrichment from complex plant matrices that enables their sensitive quantitative analysis using hyphenated mass spectrometry technique. The rapid extraction of minute quantities of plant material (less than 20mg fresh weight, FW) into cold 10% methanol followed by one-step reversed-phase polymer-based solid phase extraction significantly reduced matrix effects and increased the recovery of labile JA analytes. This extraction and purification protocol was paired with a highly sensitive and validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method and used to simultaneously profile sixteen stress-induced phytohormones in minute plant material samples, including endogenous JA, several of its biosynthetic precursors and derivatives, as well as SA, ABA and IAA.
- MeSH
- chromatografie kapalinová MeSH
- cyklopentany analýza chemie MeSH
- extrakce na pevné fázi MeSH
- fyziologický stres MeSH
- kyselina abscisová analýza chemie MeSH
- kyselina salicylová analýza chemie MeSH
- kyseliny indoloctové analýza MeSH
- listy rostlin chemie MeSH
- mastné kyseliny analýza chemie MeSH
- molekulární struktura MeSH
- oxylipiny analýza chemie MeSH
- regulátory růstu rostlin chemie izolace a purifikace fyziologie MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phytohormones are key regulators in various physiological processes of plant growth and development. Their chemical analyses, together with their genomics and proteomics, are an integral part of plant development studies. The information on hormone levels is often valuable for biologists dealing with any hormone-regulated processes. Plant tissue is a complex multicomponent mixture containing phytohormones in minute quantities (pg or ng/g fresh weight) along with many other related compounds with similar structures and/or physicochemical properties. Therefore, their analysis requires rapid, sensitive and sufficiently selective analytical methods. The significance of solid-phase extraction for purification and hyphenated techniques such as GC-MS, LC-MS and CE-MS for routine analyses of phytohormones is discussed.
- MeSH
- Brassica MeSH
- chemické techniky analytické MeSH
- chromatografie kapalinová MeSH
- cyklopentany analýza MeSH
- cytokininy analýza MeSH
- elektroforéza kapilární MeSH
- financování organizované MeSH
- fyziologie rostlin MeSH
- gibereliny analýza MeSH
- kyselina abscisová analýza MeSH
- kyseliny indoloctové analýza MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- rostliny MeSH
- steroidy MeSH
Plant hormones cytokinins, auxin (indole-3-acetic acid), and abscisic acid are central to regulation of plant growth and defence to abiotic stresses such as salinity. Quantification of the hormone levels and determination of their ratios can reveal different plant strategies to cope with the stress, e.g., suppression of growth or mobilization of plant metabolism. This chapter describes a procedure enabling such quantification. Due to the high variability of these hormones in plant tissues, it is advantageous to determine their content in the same sample. Reverse phase and ion exchange chromatography allows separation of the individual hormone fractions. Hormones as well as their metabolites can be identified and quantified by LC/MS.
- MeSH
- cytokininy analýza izolace a purifikace MeSH
- fyziologický stres * MeSH
- hmotnostní spektrometrie MeSH
- kořeny rostlin chemie metabolismus MeSH
- kyselina abscisová analýza izolace a purifikace MeSH
- kyseliny indoloctové analýza izolace a purifikace MeSH
- listy rostlin chemie metabolismus MeSH
- rostlinné extrakty chemie MeSH
- rostliny chemie metabolismus MeSH
- salinita * MeSH
- tolerance k soli fyziologie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
