Tomáš Hluska

Publications (3) View all

• Article: Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: heterologous expression, purification and properties.

  Marta Kowalska, Petr Galuszka, Jitka Frébortová, Marek Šebela, Tibor Béres, Tomáš Hluska, Mária Šmehilová, Kristin D Bilyeu, Ivo Frébort
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  ABSTRACT: The catabolism of cytokinins is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalyzing the irreversible cleavage of N(6)-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX, EC 1.5.99.12). CKXs also show low cytokinin oxidase activity, but molecular oxygen is a comparatively poor electron acceptor. The CKX gene family of Arabidopsis thaliana comprises seven members. Four code for proteins secreted to the apoplast, the remainder are not secreted. Two are targeted to the vacuoles and one is restricted to the cytosol. This study presents the purification and characterization of each of these non-secreted CKX enzymes and substrate specificities are discussed with respect to their compartmentation. Vacuolar enzymes AtCKX1 and AtCKX3 were produced in Pichia pastoris and cytosolic enzyme AtCKX7 was expressed in Escherichia coli. The recombinant proteins were purified by column chromatography. All enzymes preferred synthetic electron acceptors over oxygen, namely potassium ferricyanide and 2,3-dimetoxy-5-methyl-1,4-benzoquinone (Q(0)). In slightly acidic conditions (pH 5.0), N(6)-(2-isopentenyl)adenine 9-glucoside (iP9G) was the best substrate for AtCKX1 and AtCKX7, whereas AtCKX3 preferentially degraded N(6)-(2-isopentenyl)adenine 9-riboside-5'-monophosphate (iPMP). Moreover, vacuolar AtCKX enzymes in certain conditions degraded N(6)-(2-isopentenyl)adenine di- and triphosphates two to five times more effectively than its monophosphate.
  Phytochemistry 12/2010; 71(17-18):1970-8. · 3.35 Impact Factor
• Article: Evolution of cytokinin biosynthesis and degradation.

  Ivo Frébort, Marta Kowalska, Tomás Hluska, Jitka Frébortová, Petr Galuszka
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  ABSTRACT: Cytokinin hormones are important regulators of development and environmental responses of plants that execute their action via the molecular machinery of signal perception and transduction. The limiting step of the whole process is the availability of the hormone in suitable concentrations in the right place and at the right time to interact with the specific receptor. Hence, the hormone concentrations in individual tissues, cells, and organelles must be properly maintained by biosynthetic and metabolic enzymes. Although there are merely two active cytokinins, isopentenyladenine and its hydroxylated derivative zeatin, a variety of conjugates they may form and the number of enzymes/isozymes with varying substrate specificity involved in their biosynthesis and conversion gives the plant a variety of tools for fine tuning of the hormone level. Recent genome-wide studies revealed the existence of the respective coding genes and gene families in plants and in some bacteria. This review summarizes present knowledge on the enzymes that synthesize cytokinins, form cytokinin conjugates, and carry out irreversible elimination of the hormones, including their phylogenetic analysis and possible variations in different organisms.
  Journal of Experimental Botany 02/2011; 62(8):2431-52. · 5.36 Impact Factor
• Article: Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation to cytokinin levels.

  Sárka Vyroubalová, Katerina Václavíková, Veronika Turecková, Ondrej Novák, Mária Smehilová, Tomás Hluska, Ludmila Ohnoutková, Ivo Frébort, Petr Galuszka
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  ABSTRACT: Plant hormones, cytokinins (CKs), have been for a long time considered to be involved in plant responses to stress. However, their exact roles in processes linked to stress signalization and acclimatization to adverse environmental conditions are unknown. In this study, expression profiles of the entire gene families of CK biosynthetic and degradation genes in maize (Zea mays) during development and stress responses are described. Transcript abundance of particular genes is discussed in relation to the levels of different CK metabolites. Salt and osmotic stresses induce expression of some CK biosynthetic genes in seedlings of maize, leading to a moderate increase of active forms of CKs lasting several days during acclimatization to stress. A direct effect of CKs to mediate activation of stress responses does not seem to be possible due to the slow changes in metabolite levels. However, expression of genes involved in cytokinin signal transduction is uniformly down-regulated within 0.5 h of stress induction by an unknown mechanism. cis-Zeatin and its derivatives were found to be the most abundant CKs in young maize seedlings. We demonstrate that levels of this zeatin isomer are significantly enhanced during early stress response and that it originates independently from de novo biosynthesis in stressed tissues, possibly by elevated specific RNA degradation. By enhancing their CK levels, plants could perhaps undergo a reduction of growth rates maintained by abscisic acid accumulation in stressed tissues. A second role for cytokinin receptors in sensing turgor response is hypothesized besides their documented function in CK signaling.
  Plant physiology 08/2009; 151(1):433-47. · 6.53 Impact Factor