Josef Holík

A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction

BackgroundWe have developed a new analytical approach for isolation and quantification of cytokinins (CK) in minute amounts of fresh plant material, which combines a simple one-step purification with ultra-high performance liquid chromatography–fast scanning tandem mass spectrometry.ResultsPlant tissue samples (1–5 mg FW) were purified by stop-and-go-microextraction (StageTip purification), which previously has only been applied for clean-up and pre-concentration of peptides. We found that a combination of two reverse phases and one cation-exchange phase, was the best tool, giving a total extraction recovery higher than 80%. The process was completed by a single chromatographic analysis of a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) in 24.5 minutes using an analytical column packed with sub-2-microne particles. In multiple reaction monitoring mode, the detection limits ranged from 0.05 to 5 fmol and the linear ranges for most cytokinins were at least five orders of magnitude. The StageTip purification was validated and optimized using samples of Arabidopsis thaliana seedlings, roots and shoots where eighteen cytokinins were successfully determined.ConclusionsThe combination of microextraction with one-step high-throughput purification provides fast, effective and cheap sample preparation prior to qualitative and quantitative measurements. Our procedure can be used after modification also for other phytohormones, depending on selectivity, affinity and capacity of the selected sorbents.

Comparison of oriented and random antibody immobilization in immunoaffinity chromatography of cytokinins

Immunosorbents for the plant hormones cytokinins prepared by random antibody immobilization (to Affi-Gel 10) and by oriented approach via oxidized carbohydrate moieties on the Fc region (to Affi-Gel Hz or hydrazide derivative of Perloza MT 200) have been compared. Both approaches yielded immunosorbents with high dynamic capacity (ca. 5-10 nmol ml gel-1). Oriented antibody immobilization did not exhibit crucial effects in the case of low-molecular-mass cytokinins. Antibodies immobilized via a spacer to Affi-Gel 10 have probably enough conformational freedom to enable good accessibility to cytokinins. The sorbents were used in analysis of endogenous cytokinins in maize seeds. In phosphatase treated samples trans-zeatin and its riboside were predominant.

Antioxidant enzymatic protection during tobacco leaf ageing is affected by cytokinin depletion

Plant ageing and senescence are associated with increased levels of reactive oxygen species. Level of cytokinins, the apparent inhibitors of plant senescence, is controlled by their irreversible degradation catalysed by cytokinin oxidase/dehydrogenase (CKX). We investigated the CKX activity, cytokinin concentration, and activities of antioxidative enzymes in tobacco (Nicotiana tabacum L. cv. Samsun NN) overexpressing the Arabidopsis gene for AtCKX2, targeted for extracellular secretion pathway. The control and AtCKX2 plants differed substantially in their phenotypes. When the lowest leaves in controls became yellow all leaves in AtCKX2 tobacco still remained green. Activities of antioxidant enzymes decreased with leaf age in both tobacco plants except for ascorbate peroxidase (APX) in the old leaves and glutathione reductase (GR) in young leaves. Enhancement of GR activity at all leaf stages, an increase of superoxide dismutase and a decline of catalase in young leaves, as well as an increase of APX in the oldest leaves were observed in AtCKX2 plant compared to control. Similar changes were detected after determination of isoenzymes on zymograms. It is evident that AtCKX2 plants had postponed onset of senescence despite the significantly lowered level of cytokinins. Enhanced antioxidant protection, especially in the oldest leaves, could subsidise this phenomenon.

Emission of climate relevant volatile organochlorines by plants occurring in temperate forests.

Chlorine, one of the most abundant elements in nature, undergoes a complex biogeochemical cycle in the environment, involved in the formation of volatile organochlorines (VOCls), which in turn can contribute to environmental problems, contaminate natural ecosystems, and are of public health concern. Several industrial and natural sources of VOCls have already been identified; however, data - particularly on the natural sources - are still scarce. The aim of this study was to investigate the diversity of emission of VOCls from soil and several undergrowth plants collected in temperate spruce forest ecosystem and the effect of salting on the VOCl emission of plants. Undergrowth plants were found to emit chloroform (CHCl 3 ) in the range of 2.2-201 pmol/day/g dry weight (DW), tetra-chloromethane (CCl 4 ) 0-23.5 pmol/day/g DW, and tetrachloroethene (C 2 Cl 4 ) 0-13.5 pmol/day/g DW; the average emission rates were about 10 times higher than that of soil (2.9-47.2; 0-5.8; 0-3.6 pmol/day/g DW of CHCl 3 ; CCl 4 ; C 2 Cl 4 emission, respectively). Addition of sodium chloride solution in most cases caused an increase in the emission of CHCl 3 and caused a species specific - effect on the emissions of CCl 4 and C 2 Cl 4 . The results suggest that the emission of VOCls from spruce forest contribute to the atmospheric input of reactive chlorine; however, on a global scale it is only a minor net source.

Changes in cytokinin levels and metabolism in tobacco (Nicotiana tabacum L.) explants during in vitro shoot organogenesis induced by trans-zeatin and dihydrozeatin

The uptake and metabolism of trans-zeatin and/or dihydrozeatin, in correlation with cytokinin oxidase/dehydrogenase (CKX) and β-glucosidase activity, were studied in leaf segments derived from wild-type (WT) and transgenic (T) tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) during in vitro induction of shoot organogenesis. T explants harbored the maize gene Zm-p60.1β-glucosidase. Higher levels of shoot regeneration were observed on T explants in the early stages of cultivation. In WT explants, the content of cytokinin (CK)-O- and N-glucosides increased. In T explants, a higher content of Z-9-riboside and Z-9-riboside-5′-monophosphate and higher CKX activity during the early stage of cultures were found. A positive correlation was obtained for bioactive CK content and the organogenic response in T explants. Our results indicate a connection between the organogenic capacity of tobacco explants, metabolism of endogenous CKs and uptake of exogenous CKs from the cultivation medium.

Endogenous Phytohormones in Spontaneously Regenerated Centaurium erythraea Rafn. Plants Grown In Vitro

Phytohormones are important regulators of numerous developmental and physiological processes in plants. Spontaneous morphogenesis of the common centaury (Centaurium erythraea Rafn.) is possible on nutrition medium without addition of any plant growth regulator depending solely on endogenous phytohormone levels. Thus, this plant species represents a very good model system for the investigation of numerous physiological processes under phytohormonal control in vitro. We analysed the total amount of endogenous cytokinins (CKs) including the contents of their individual groups in shoots and roots of C. erythraea plants grown in vitro. The total amount of endogenous CKs was 1.4 times higher in shoots than in roots. Inactive or weakly active N-glucosides found to predominate in both organs of centaury plants, whereas free bases and O-glucosides represented only a small portion of the total CK pool. Consequently, centaury roots showed higher IAA content as well as IAA/free CK base ratios compared to shoots. Centaury tissues also showed increased levels of “stress hormones”. In contrast to SA, considerably higher levels of ABA were found in centaury shoots than in roots. Our results could serve as a basis for understanding and elucidating spontaneous de novo shoot organogenesis and further plant regeneration of C. erythraea in vitro.

Changes in cytokinin content and altered cytokinin homeostasis in AtCKX1 and AtCKX2-overexpressing centaury (Centaurium erythraea Rafn.) plants grown in vitro

The plant hormones cytokinins (CKs) regulate a number of physiological processes. Their homeostasis is controlled by the rate of de novo synthesis and the rate of catabolism. The aim of this work was to analyze the content of total as well as individual groups of endogenous CKs in AtCKX1 and AtCKX2-overexpressing centaury (Centaurium erythraea Rafn.) plants grown in vitro. Transgenic CKX plants represent a suitable model system for studying physiological and morphological processes controlled by CKs. In this work we clearly demonstrate a significant effect of AtCKX transgenes on CK metabolism in transgenic centaury plants. However, shoots and roots of only one AtCKX1 line and three AtCKX2 lines with a significant reduction of bioactive CKs were obtained. We also show that changes in the CKs metabolism considerably affected endogenous indole-3-acetic acid (IAA) levels in plant tissues. All analyzed transgenic AtCKX centaury lines exhibited decreased amount of endogenous IAA in shoots as well as in roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all analyzed AtCKX centaury transformants.

Preparation of tritiated oostatic peptides for study of radioactivity incorporation in flesh fly Neobellieria bullata

Summary.A series of insect oostatic peptides containing 3,4-dehydroproline in the C-terminal part or inside of the peptide chain was synthesized and tritiated by addition of 3H2 to double bond of 3,4-dehydroproline residue. 3H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl β-hydrogens. In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [3H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[3H] Pro-Ala-OH, [3H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[3H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[3H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro5-[3H] Pro-OH and H-Asp-[3H] Pro-OH. These peptides made possible a highly sensitive comparative study on radioactivity incorporation into head and ovaries of the flesh fly Neobellieria bullata, which revealed this process to proceed differently. The reasons of the found differences are discussed.

Oostatic peptides containing d-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with d-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The d-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either d-aspartic acid or d-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the d-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the d-amino acid residues in the corresponding peptides. The introduction of the non-coded d-amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.

Study of oostatic peptide uptake and metabolism in developing ovaries of the flesh fly, Neobellieria bullata.

Abstract The uptake and metabolism of the oostatic pentapeptide analogue of trypsin modulating oostatic factor (TMOF), H-Tyr-Asp-Pro-Ala-Pro-OH (5P), in ovaries of Neobellieria bullata (Parker) (Diptera: Sarcophagidae) were analyzed during their developmental stages. During selected stages of yolk deposition, the fate of [3HPro3]5P after its in vivo injection was compared to its uptake after in vitro incubation of dissected ovaries. The ovaries were analyzed from 30 s to 180 min after incubation. A detection sensitivity of 60–100 fmol of the labeled 5P was achieved using radio-high performance liquid chromatography. While the uptake of the applied radioactivity strongly depended on the stage of vitellogenesis, especially for the in vitro experiment, degradation of 5P was very quick and independent of whether the label was injected or incubated with the ovaries, regardless of the developmental stage of ovaries. No tracers of 5P were detected at 30 s after applying the labeled 5P in all tests.