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
Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g-1 fresh weight). We have developed a simple microscale magnetic immunoaffinity-based method for selective one-step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one-step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30-80% depending on individual CKs. Immunoaffinity extraction using group-specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry-based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N9 -glucosides) in 1.0-mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.
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
Autoři prohloubili tematiku diferenciace SIRS a sepse s vyvážením imunitních prístupu a zdrženlivé a cílené antibiotické politiky z predchozího sdelení „Klinicko-mikrobiologické a imunologické aspekty péce o kardiochirurgické pacienty“ (7). Potvrdil se mimorádný význam nižších hladin prokalcitoninu pri odlišení cistého SIRS od sepse. Vyšetrení stavu celulární imunity (CI) je cenné pro hodnocení obrany proti infekci, posuzování systémového zánetu a eventuální imunointervenci. Na príkladech krátkodobého i delšího sledování pacientu se potvrdily dríve vytypované metody, jako je pocet obecných lymfocytu, subpopulací T-lymfocytu, CD a noveji stanovení intracelulárních cytokinu, zejména antiinflamatorního IL-10 a proinflamatorních TNFa, INFg a IL-2. Jsou zmíneny prípady úplné absence znaku CD, prognostické rysy CI, problémy s poznáním hyperinflamatorního SIRS a hyperCARS a objektivní potíže s hodnocením intervence Immodinem u jednotlivcu. Diagnostika stavu CI a eventuální imunointervence by se mely stát standardními soucástmi pooperacní i ostatní intenzivní péce ve spolupráci intenzivistu a imunologu.
The authors elaborated the theme Differentiation of SIRS – sepsis with balancing of the immune approaches and a reserved and target antibiotic policy from the previous report “Clinical, microbiological and immunological aspects of cardiosurgical patients care“ (7). They confirmed the extreme importance of lower procalcitonine levels for differentiation of clear SIRS from sepsis. Examination of cellular immunity (CI) condition is necessary for the evaluation of defence against infection, assessment of systemic inflammation and possible immunointervention. Short-time as well as longer monitoring of patients certified the earlier selected methods such as the number of common lymphocytes, subpopulations of T-lymphocytes, CD and newly determination of intracellular cytokines, especially anti-inflammatory IL-10 and pro-inflammatory TNF alpha, INF gamma and IL-2. The authors also mention cases of entire absence of CD signs, prognostic CI features, problems with recognition of hyperinflammatory SIRS and hyper CARS, and objective difficulties with evaluation of Immodin intervention in individuals. Diagnostics of CI condition and possible immunointervention should become a standard part of post-operational as well as other intensive care in cooperation of intensivists and immunologists.
- MeSH
- antibakteriální látky terapeutické užití MeSH
- biologické markery krev MeSH
- buněčná imunita účinky léků účinky záření MeSH
- cytokininy imunologie izolace a purifikace MeSH
- imunologické faktory terapeutické užití MeSH
- pooperační komplikace etiologie farmakoterapie krev MeSH
- sepse diagnóza etiologie krev MeSH
- syndrom systémové zánětlivé reakce diagnóza etiologie krev MeSH
- T-lymfocyty imunologie MeSH
- Publikační typ
- kazuistiky MeSH
