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Developmental Stage As a Possible Factor Affecting Cytokinin Content and Cytokinin Dehydrogenase Activity in Pinus Sylvestris

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Developmental Stage As a Possible Factor Affecting Cytokinin Content and Cytokinin Dehydrogenase Activity in Pinus Sylvestris BIOLOGIA PLANTARUM 51 (1): 193-197, 2007 BRIEF COMMUNICATION Developmental stage as a possible factor affecting cytokinin content and cytokinin dehydrogenase activity in Pinus sylvestris A.E. VALDÉS*, P. GALUSZKA**, B. FERNÁNDEZ*1, M.L. CENTENO*** and I. FRÉBORT** Departamento de Biología de Organismos y Sistemas, Universidad de Oviedo, c/ Catedrático Rodrigo Uría, s/n, E-33071 Oviedo, Spain* Faculty of Science, Palacký University, Šlechtitelů 11, CZ-78371 Olomouc, Czech Republic** Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana, León, Spain*** Abstract In the present study cytokinin dehydrogenase (CKX) activity was for the first time found in a conifer species, Pinus sylvestris. The activities were correlated with the endogenous cytokinin contents. Several enzyme substrates and two different electron acceptors were used to search for the enzyme activity in the extract from seeds, seedlings and plantlets. The highest specific activity was found in one-year-old plantlets with isopentenyladenine as the substrate and 2,6-dichlorophenolindophenol as the electron acceptor, at pH 8. An enhancement in the CKX specific activity corresponded to increasing contents of cytokinins, mainly isopentenyladenine and isopentenyladenosine, indicating that the enzyme activity is affected by the endogenous supply of cytokinins. CKX affinity for the ribosylated form of isopentenyladenine was dependent on the developmental stage, being higher in seeds than in seedlings, and not detectable in plantlets. The results are indicative of the presence of different isoenzymes throughout the development. Additional keywords: cytokinin metabolism, conifers, plant ageing. ⎯⎯⎯⎯ Tree maturation is modulated by intrinsic factors electron acceptor than oxygen in in vitro conditions, responsible for age-related changes, in which cytokinins classifying it as a dehydrogenase (EC 1.5.99.12) (Bilyeu (CKs) are the essential hormones involved throughout et al. 2001, Galuszka et al. 2001). The enzyme has a plant development, especially in the transition from the preferred specificity for the substrates isopentenyl- vegetative to the reproductive phase (Oka 2003). Control adenine (iP) and its ribosylated form (iPR), yielding of biologically active CKs occurs through a balance adenine or adenosine and 3-methyl-2-butenal as reaction between biosynthesis, interconversion among distinct products (McGaw and Horgan 1983). This substrate forms, transient inactivation by conjugation, and cata- specificity is relevant due to the participation of distinct bolic reactions resulting in a complete loss of biological forms of CKs in the plant growth and development with activity (Sakakibara and Takei 2002). Degradation of a differential implication dependent on the develop- these molecules occurs by N6-side-chain cleavage mental stage of the plant (Morris et al. 1990, Frugis et al. produced by the enzyme cytokinin dehydrogenase 2001, Zhang et al. 2003). (CKX), the only known plant enzyme which catalyses Previous results on ageing and maturation obtained the irreversible degradation of naturally occurring CKs. with Pinus showed that not all CKs are equally present CKX was believed to be a copper-containing amine throughout these processes, being reported the importance oxidase (EC 1.4.3.6) (Hare and Van Staden 1994), but of CKs with hydroxylated side-chains in adult states, further studies demonstrated the presence of a flavin whereas iP and iPR were associated with juvenile cofactor, as well as the enzymatic use of a different individuals (Valdés et al. 2002, 2003). These results, ⎯⎯⎯⎯ Received 11 May 2005, accepted 31 October 2005. Abbreviations: (2-OH)BA - N6-(2-hydroxybenzyl)adenine; CKX - cytokinin dehydrogenase; DCPIP - 2,6-dichlorophenolindophenol; DHZ - dihydrozeatin; DHZR - dihydrozeatin riboside; iP - N6-isopentenyladenine; iPR - N6-isopentenyladenosine; Q0 - 2,3-dimethoxy-5-methyl-1,4-benzoquinone; Z - zeatin; ZR zeatin riboside. Acknowledgements: This research was carried out with financial support from the commission of the European Communities, Agriculture and Fisheries (FAIR) specific RTD program, CT96-1445 and from the Grant Agency of the Czech Republic 522/03/0979. 1 Corresponding author; fax: (+34) 985104867, e-mail: [email protected] 193 A.E. VALDÉS et al. which indicate the physiological state of the plant, might substrate (0.15 mM) and an electron acceptor (0.5 mM) be representative of changes in the activity of some in 75 mM Tris-HCl buffer, pH 8, was incubated at 37 ºC degradative enzyme, such as the CKX, between juvenile for suitable period (1 - 5 h). The reaction was then and adult states. However, whether variations in the stopped by the addition of 0.3 cm3 40 % trichloroacetic cytokinin content are produced by enzymatic activity acid. After the addition of 0.2 cm3 of 4-aminophenol regulating interconversions between different cytokinin (solution in 6 % trichloroacetic acid), the spectrum over forms, or by differential de novo synthesis remains still the range 300 - 700 nm was scanned and the absorbance unknown. was measured at a specific wavelength (352 nm for iP) CKX genes have been identified and cloned in on a DU 7500 spectrophotometer (Beckman, Fullerton, several cereal species in addition to Arabidopsis and CA, USA). In order to select the best substrate for Dendrobium orchid (Bilyeu et al. 2001, Yang et al. 2003, monitoring CKX activity in coniferous species iP, iPR, Galuszka et al. 2004, Popelková et al. 2004), but zeatin (Z), zeatin riboside (ZR) and N6-(2-hydroxy- information about either the gene or the CKX protein in benzyl)adenine (2-OHBA) were tested. As electron other species is still hardly available. The objective of the acceptors 2,6-dichlorophenolindophenol (DCPIP) and current work was to investigate CKX activity in Pinus 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0) were used. sylvestris and to establish a method to be used in further Seeds, seedlings and plantlets were deep-frozen in experiments for monitoring CKX activity associated with liquid nitrogen and lyophilised prior to the extraction and developmental changes produced in the plant. purification of several CKs: the free bases Z, Pinus sylvestris L. imbibed seeds, 15-d-old seedlings dihydrozeatin (DHZ), iP and their ribosylated forms ZR, growing in a nursery in natural conditions and 1-year-old DHZR and iPR. Extraction and purification were plantlets also growing in a natural stand, were used for performed according to Fernández et al. (1995) cytokinin dehydrogenase (CKX) activity assays and combining overnight-liquid and solid phase extraction determinations of endogenous CKs content. Experiments with organic solvents and immunoaffinity chromato- were carried out with more than one thousand seeds, graphy techniques. Reverse-phase HPLC on a Kromasil more than one hundred seedlings and ten plantlets to 100 C18 - 5 μm (150 × 4.6 mm) was used for the have similar mass of each kind of material. Several separation of different CKs employing kinetin as internal independent pools for each sample were used, standard to corroborate chromatographic retention times. considering plant material from different individuals to The mobile phase was acetonitrile and triethylammonium avoid differences due to genotype. All material was acetate 40 mM, adjusted at pH 7. CKs were eluted from acquired from the Forest Administration of the Czech the column by means of a linear gradient from 5 to 20 % Republic, Šternberk, Czech Republic. acetonitrile (v/v) over 40 min. Collected fractions were For protein extraction frozen material was ground to tested by ELISA with polyclonal antibodies obtained powder with mortar and pestle in liquid nitrogen. Before according to Fernández et al. (1995). The radiolabelled allowed to thaw, the extraction of proteins was done with standard [8-14C]BA (2.0 GBq mmol-1) was added at the 0.5 M Tris-HCl buffer, pH 8, at 4 ºC, and repeated beginning of each analysis to determine purification stirring every 10 min for 1 h. After washing twice with losses. the same buffer to obtain higher protein recoveries, plant All hormonal analyses were carried out in three debris was removed by centrifugation at 10 000 g for independent samples with three repetitions each. 10 min. Polyvinylpyrrolidone [18 % (m/v)] was added to Deviation from normality and homogeneity of variance the supernatant to remove phenolic compounds, highly was tested with Shapiro-Wilk and Barlett-Box tests present in conifer extracts. After 15 min with every respectively. For parametric data one-way analysis of 5-min stirring, the pellet was removed by centrifugation variance (ANOVA) with Scheffé post-hoc comparison at 12 000 g, 10 min, 4 ºC. To concentrate the proteins, were used. The primary data on the iP, iPR and total CKs (NH4)2SO4 was slowly added to 70 % saturation to the were transformed by the square root to reach parametric supernatant within 1 h, at 4 ºC. Proteins were collected as assumptions. Non-parametric data were analysed by a precipitate by centrifugation at 12 000 g, and suspended means of Kruskal-Wallis and Mann-Whitney tests. The in the possible minimum volume of 0.1 M Tris-HCl level of significance was set at α = 0.05 for all tests. buffer and stored at 4 ºC. Protein content was estimated Pinus sylvestris exhibited an enzymatic activity by the method of Bradford with BSA as a standard capable of the side-chain cleavage of iP and iPR. The (Bradford 1976). activity was dependent on the plant tissue. In P. sylvestris The end-point enzymatic assay was performed using seeds, the values of specific activity with iPR were about the method of Libreros-Minotta and Tipton (1995) 85 % of those obtained with iP independently of the modified for the use of an electron acceptor (Frébort electron acceptor, while in the seedlings the activity with et al. 2002). Under acidic conditions the aldehyde iPR was only about 55 % of the activity with iP (Table 1). produced by cleaving a cytokinin forms with 4-amino- Assays using either Z, ZR or (2-OH)BA as substrates of phenol a coloured Schiff base ((4-hydroxyphenylimino)- the enzyme did not show any detectable activity.
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