Miroslav Kamínek

Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction .

A method for separation of cytokinins from auxin and abscisic acid, which allows further separation of cytokinin ribotides from cytokinin bases, ribosides and glucosides and their purification on a single Oasis MCX column was developed. Due to the mixed reversed-phase and cation-exchange mode of the Oasis MCX sorbent the cationic cytokinin bases, ribosides and glucosides as well as the anionic auxin, abscisic acid and cytokinin ribotides are retained and can be sequentially eluted by solvents containing different concentrations of methanol and ammonium hydroxide. Characteristics of the method are high recoveries of analyzed phytohormones and their sufficient purity for quantification by HPLC-ELISA (RIA) or HPLC-MS.

Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants

Cytokinins (CKs) are plant hormones affecting numerous developmental processes. Zeatin and its derivatives are the most important group of isoprenoid CKs. Zeatin occurs as two isomers: while trans-zeatin (transZ) was found to be a bioactive substance, cis-zeatin (cisZ) was reported to have a weak biological impact. Even though cisZ derivatives are abundant in various plant materials their biological role is still unknown. The comprehensive screen of land plants presented here suggests that cisZ-type CKs occur ubiquitously in the plant kingdom but their abundance might correlate with a strategy of life rather than with evolutionary complexity. Changing levels of transZ and cisZ during Arabidopsis ontogenesis show that levels of the two zeatin isomers can differ significantly during the life span of the plant, with cisZ-type CKs prevalent in the developmental stages associated with limited growth. A survey of the bioassays employed illustrates mild activity of cisZ and its derivatives. No cis↔trans isomerization, which would account for the effects of cisZ, was observed in tobacco cells and oat leaves. Differences in uptake between the two isomers resulting in distinct bioactivity have not been detected. In contrast, cisZ and transZ have a different metabolic fate in oat and tobacco. Analysis of a CK-degrading enzyme, cytokinin oxidase/dehydrogenase (CKX), reveals that Arabidopsis possesses two isoforms, AtCKX1 expressed in stages of active growth, and AtCKX7, both of which have the highest affinity for the cisZ isomer. Based on the present results, the conceivable function of cisZ-type CKs as delicate regulators of CK responses in plants under growth-limiting conditions is hypothesized.

Changes in Cytokinin Content and Cytokinin Oxidase Activity in Response to Derepression of ipt Gene Transcription in Transgenic Tobacco Calli and Plants

Metabolic control of cytokinin oxidase by its substrate was investigated in planta using wild-type (WT) and conditionally ipt gene-expressing transgenic (IPT) tobacco (Nicotiana tabacum L.) callus cultures and plants. The derepression of the tetracycline (Tc)-dependent ipt gene transcription was followed by a progressive, more than 100-fold increase in total cytokinin content in IPT calli. The activity of cytokinin oxidase extracted from these calli began to increase 16 to 20 h after gene derepression, and after 13 d it was 10-fold higher than from Tc-treated WT calli. An increase in cytokinin oxidase activity, as a consequence of elevated cytokinin levels, was found in detached leaves (8-fold after 4 d) and in roots of intact plants (4-fold after 3 d). The partially purified cytokinin oxidase from WT, repressed IPT, and Tc-derepressed IPT tobacco calli exhibited similar characteristics. It had the same broad pH optimum (pH 6.5–8.5), its activity in vitro was enhanced 4-fold in the presence of copper-imidazole, and the apparent Km(N6-[[delta]2iso-pentenyl]adenine) values were in the range of 3.1 to 4.9 [mu]M. The increase in cytokinin oxidase activity in cytokinin-overproducing tissue was associated with the accumulation of a glycosylated form of the enzyme. The present data indicate the substrate induction of cytokinin oxidase activity in different tobacco tissues, which may contribute to hormone homeostasis.

Senescence‐Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.

Senescence-induced ectopic expression of the A. tumefaciens ipt gene in wheat delays leaf senescence, increases cytokinin content, nitrate influx, and nitrate reductase activity, but does not affect grain yield

The manipulation of cytokinin levels by senescence-regulated expression of the Agrobacterium tumefaciens ipt gene through its control by the Arabidopsis SAG12 (senescence-associated gene 12) promoter is an efficient tool for the prolongation of leaf photosynthetic activity which potentially can affect plant productivity. In the present study, the efficiency of this approach was tested on wheat (Triticum aestivum L.)-a monocarpic plant characterized by a fast switch from vegetative to reproductive growth, and rapid translocation of metabolites from leaves to developing grains after anthesis. When compared with the wild-type (WT) control plants, the SAG12::ipt wheat plants exhibited delayed chlorophyll degradation only when grown under limited nitrogen (N) supply. Ten days after anthesis the content of chlorophyll and bioactive cytokinins of the first (flag) leaf of the transgenic plants was 32% and 65% higher, respectively, than that of the control. There was a progressive increase in nitrate influx and nitrate reductase activity. However, the SAG12::ipt and the WT plants did not show differences in yield-related parameters including number of grains and grain weight. These results suggest that the delay of leaf senescence in wheat also delays the translocation of metabolites from leaves to developing grains, as indicated by higher accumulation of ((15)N-labelled) N in spikes of control compared with transgenic plants prior to anthesis. This delay interferes with the wheat reproductive strategy that is based on a fast programmed translocation of metabolites from the senescing leaves to the reproductive sinks shortly after anthesis.

Farnesylation Directs AtIPT3 Subcellular Localization and Modulates Cytokinin Biosynthesis in Arabidopsis

Cytokinins regulate cell division and differentiation as well as a number of other processes implicated in plant development. The first step of cytokinin biosynthesis in Arabidopsis (Arabidopsis thaliana) is catalyzed by adenosine phosphate-isopentenyltransferases (AtIPT). The enzymes are localized in plastids or the cytoplasm where they utilize the intermediate dimethylallyl-diphosphate from the methylerythritolphosphate or mevalonic acid pathways. However, the regulatory mechanisms linking AtIPT activity and cytokinin biosynthesis with cytokinin homeostasis and isoprenoid synthesis are not well understood. Here, we demonstrate that expression of AtIPT3, one member of the adenosine AtIPT protein family in Arabidopsis, increased the production of specific isopentenyl-type cytokinins. Moreover, AtIPT3 is a substrate of the protein farnesyl transferase, and AtIPT3 farnesylation directed the localization of the protein in the nucleus/cytoplasm, whereas the nonfarnesylated protein was located in the plastids. AtIPT3 gain-of-function mutant analysis indicated that the different subcellular localization of the farnesylated protein and the nonfarnesylated protein was closely correlated with either isopentenyl-type or zeatin-type cytokinin biosynthesis. In addition, mutation of the farnesyl acceptor cysteine-333 of AtIPT3 abolishes cytokinin production, suggesting that cysteine-333 has a dual and essential role for AtIPT3 farnesylation and catalytic activity.

Cytokinin biosynthesis: a black box?

The biosynthetic origin of cytokinins (CKs) in plants has been debated ever since the recognition of CKs as plant hormones. Although several possible biosynthetic pathways have been suggested, none of the data published to date are conclusive. The enzymes involved in CK biosynthesis have not yet been purified and characterized in detail, nor have plant genes encoding CK biosynthetic enzymes been identified.

Purine cytokinins: a proposal of abbreviations

Contributions presented at the International Symposium on Auxins and Cytokinins in Plant Development (Prague July 26–30, 1999) as well as the editorial preparation of this special issue of Plant Growth Regulation showed a lack of uniformity in cytokinin nomenclature and corresponding abbreviations. It is difficult to unify the cytokinin nomenclature because of the relatively complicated systematic names, which force plant physiologists to use semi-systematic as well as trivial names. However, it may be possible to reach a consensus about cytokinin abbreviations. In contrast to the large number of synonyms used widely for individual cytokinins (e.g. more than 25 synonyms were reported for 6-benzylaminopurine [1]) far fewer abbreviations have appeared in the literature. The original, as well as revised, abbreviations and symbols recommended by IUPAC-IUB Commission on Biochemical Nomenclature in 1967 [2] and 1970 [3], respectively, for nucleic acids and their components including cytokinins have not been widely adopted by plant physiologists and biochemists. The most likely reason for this has been the ambiguous meaning and complicated typing due to the use of three-lettersymbols for adenine and adenosine, which serve as a basis for construction of cytokinin abbreviations using superscripts for locants and both hyphens and apostrophes for denotation of nucleotides. Significant improvement in this respect was achieved in the system introduced by Letham [4] and modified by Letham and Palni [5]. This system combines the use of abbreviations for systematic or trivial names of cytokinin bases (e.g. Z for transzeatin, BA for 6-benzylaminopurine and KIN for kinetin) with abbreviations of substitutents on the Figure 1. Scheme of cytokinin molecule and purine ring numbering. X1 – X5, substituents.

Different effects of two brassinosteroids on growth, auxin and cytokinin content in tobacco callus tissue

Two synthetic brassinosteroids, 24-epibrassinolide (24-epiBR) and 2α,3α, 17β-trihydroxy-5α-androstan-6-one (THA-BR), exhibit different effects on growth of tobacco callus tissue. When added to a culture medium containing growth-limiting amounts of auxin, 24-epiBR reduced and THA-BR increased the fresh weight yield of tissue up to 53% and 207%, respectively, after 6 weeks of cultivation. The stimulatory and inhibitory effects of the two brassinosteroids on tissue growth occurred over a broad range of concentrations without a pronounced maximum corresponding to the ‘yes or no’ type of response. Different effects of 24-epiBR and THA-BR on tissue growth were inversely proportional to the content of endogenous cytokinins. Maximum contents of predominant cytokinins N6-(△2-isopentenyl)adenine (iP) and trans-zeatin (Z) in tissues supplied with 24-epiBR in growth-inhibiting concentrations were up to 3.7 fold and 3.4 fold higher, respectively, as compared to tissues grown on media containing growth-stimulating concentrations of THA-BR. Stimulation of tissue growth by THA-BR correlated with content of endogenous IAA and an inverse correlation was found between the content of endogenous IAA and cytokinins in tissues supplied with 24-epiBR. THA-BR exhibited weak cytokinin-like activity in a bioassay based on stimulation of growth of lateral buds of pea while 24-epiBR was inactive. Results indicate that the qualitatively different effects of the two brassinosteroids on growth of tobacco tissue may reflect their different influence on content of endogenous cytokinin.

Cytokinin oxidase from auxin- and cytokinin-dependent callus cultures of tobacco (Nicotiana tabacum L.)

Cytokinin oxidase was extracted and partially purified from auxin- and cytokinin-dependent callus tissue of tobacco (Nicotiana tabacum L. cv. Wisconsin 38). The activity of the enzyme preparation was examined using an assay based on the conversion of tritiated N6-(Δ2-isopentenyl)adenine ([2,8-3H]iP) to adenine. Cytokinin oxidase exhibited a temperature optimum at 45–50°C and a relatively high pH optimum (8.5–9.0). The apparent Km value of the enzyme was 4.3 μM for iP. On the basis of the substrate competition assays, iP was determined to be the preferred substrate of the enzyme. Substrate competition was also observed with zeatin and the cytokinin-active urea derivative Thidiazuron. Cytokinins bearing saturated isoprenoid side chains or cyclic side chain structures, as well as auxins and abscisic acid, had no effect on the conversion of [2,8-3H]iP. The cytokinin oxidase exhibited increased activity in the presence of copper-imidazole complex in the reaction mixture. Under optimal concentrations of copper (15 mM CuCl2) and imidazole (100 mM), the enzyme activity was enhanced ca. 40-fold. Under these conditions the pH optimum was lowered to pH 6.0, whereas the temperature optimum, the apparent Km value, and the substrate specificity were not altered. Most of the enzyme moiety did not bind to the lectin concanavalin A. The characteristics of cytokinin oxidase presented here suggest that a novel molecular form of the enzyme, previously identified and characterized in Phaseolus lunatus callus cultures (Kamínek and Armstrong (1990) Plant Physiol 93:1530), also occurs in cultured tobacco tissue.