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First published online September 28, 2007; 10.1104/pp.107.103176 Plant Physiology 145:786-800 (2007) © 2007 American Society of Plant Biologists Cytokinins in the Bryophyte Physcomitrella patens: Analyses of Activity, Distribution, and Cytokinin Oxidase/Dehydrogenase Overexpression Reveal the Role of Extracellular Cytokinins1,[W]
Biozentrum Klein Flottbek, Universität Hamburg, D–22609 Hamburg, Germany (K.v.S., M.F.N., H.B.); Institute of Experimental Botany, Academy of Sciences of the Czech Republic, CZ–16502 Prague 6, Czech Republic (P.I.D., V.M.); and Laboratory of Growth Regulators, Palack
Ultra-performance liquid chromatography-tandem mass spectrometry was used to establish the cytokinin profile of the bryophyte Physcomitrella patens (Hedw.) B.S.G.; of 40 analyzed cytokinins, 20 were detected. cis-Zeatin-riboside-O-glucoside, N6-(  2-isopentenyl)adenosine-5'-monophosphate (iPRMP), and trans-zeatin-riboside-O-glucoside were the most abundant intracellular cytokinins. In addition, the aromatic cytokinins N6-benzyladenosine (BAR), N6-benzyladenine, meta-, and ortho-topolin were detected. Unexpectedly, the most abundant extracellular cytokinin was the nucleotide iPRMP, and its identity was confirmed by quadrupole time-of-flight mass spectrometry. The effects of overexpressing a heterologous cytokinin oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) gene (AtCKX2 from Arabidopsis [Arabidopsis thaliana]) on the intracellular and extracellular distribution of cytokinins was assessed. In cultures of CKX-transformed plants, ultra-performance liquid chromatography-tandem mass spectrometry measurements showed that there were pronounced reductions in the extracellular concentrations of N6-(2-isopentenyl)adenine (iP) and N6-(2-isopentenyl)adenosine (iPR), but their intracellular cytokinin concentrations were only slightly affected. In vitro and in vivo measured CKX activity was shown to be strongly increased in the transformants. Major phenotypic changes observed in the CKX-overexpressing plants included reduced and retarded budding, absence of sexual reproduction, and abnormal protonema cells. In bud-induction bioassays with wild-type Physcomitrella, the nucleotides iPRMP, trans-zeatin-riboside-5'-monophosphate, BAR monophosphate, and the cis-zeatin forms cZ and cZR had no detectable effects, while the activities displayed by other selected cytokinins were in the following order: iP > tZ > N6-benzyladenine > BAR > iPR > tZR > meta-topolin > dihydrozeatin > ortho-topolin. The results on wild type and CKX transgenics suggest that extracellular iP and iPR are the main cytokinins responsible for inducing buds in the bryophyte Physcomitrella. Cytokinin profile is discussed regarding the evolution of cytokinin biosynthetic pathways.
Cytokinins play important roles as growth-regulating compounds in plants (Kieber, 2002  ). External applications of cytokinins to mosses have been shown to induce bud formation and, thus, the transition from filamentous, protonemic growth to the formation of gametophores (Bopp and Brandes, 1964; Reski and Abel, 1985). However, knowledge of the endogenous cytokinin profiles of mosses is incomplete, and it is unclear how their intracellular and extracellular distributions regulate developmental processes. We have therefore attempted to establish the cytokinin profile of Physcomitrella patens, a model organism for plant development and metabolism studies (Cove et al., 2006).
Naturally occurring cytokinins are N6-substituted adenine derivatives bearing either an isoprenoid or an aromatic side chain. Isoprenoid forms include N6-(
Cytokinin bases and their corresponding ribosides and nucleotides can be interconverted, usually in reactions catalyzed by purine metabolizing enzymes (for review, see Chen, 1997
The adenine moiety of cytokinins can also be glycosylated, resulting in the formation of either N7- or N9-glucosides, which are much less active than the unglycosylated forms (Letham et al., 1983
Two cytokinin biosynthesis pathways are known to be present in plants. The direct de novo synthesis of free cytokinins is catalyzed by adenylate isopentenyltransferases (IPTs), which preferentially alkylate ADP and ATP to the corresponding cytokinin nucleotides (Kakimoto, 2001
CKX activity was first demonstrated by Paces et al. (1971)
Overexpression of Arabidopsis AtCKX1 or AtCKX2 has contrasting effects in the shoots and roots of tobacco plants. In experiments reported by Werner et al. (2001)
As AtCKX2 expressed in transgenic tobacco, compared to other AtCKX isoforms, was shown to highly increase the degradation of iP-type cytokinins (Galuszka et al., 2007 The moss Physcomitrella represents an example of an evolutionarily primitive land plant. As mosses generally are regarded as living fossils, the analysis of their cytokinin physiology sheds light on the evolution of cytokinin-mediated growth regulation. We present a comprehensive analysis of the intracellular and extracellular distributions of cytokinins in moss, together with data on their activity, and the influence of cytokinin deficiency on its developmental processes.
Generation of CKX-Overexpressing Plants cDNA encoding the AtCKX2 gene from Arabidopsis was placed under the control of the rice (Oryza sativa) actin1 promoter to construct the expression vector, designated pHP_act1_AtCKX2. After polyethylene glycol (PEG)-mediated transformation of Physcomitrella protoplasts, transformants resistant to G418 were selected. From 30 lines the plants tCKX7 and tCKX16 were selected for further characterization at the molecular, metabolic, and phenotypic levels. The integration of the transgene into high Mr genomic DNA of Physcomitrella was confirmed by Southern blotting (data not shown), and the transcription of the transgene was shown by reverse transcription (RT)-PCR analysis, using specific primers for the AtCKX2 gene (Fig. 1 ).
In Vitro CKX Activity The CKX activities in wild-type, tCKX16, and tCKX7 plants were compared in radiometric assays using a copper-imidazole buffer (Table I ). The CKX specific activity was up to approximately 27-fold higher in tissue extracts of the transformants than in corresponding wild-type extracts. Furthermore, the specific activity was up to 157-fold higher in protein preparations from the culture media of the transformants than in corresponding wild-type preparations, indicating that a considerable proportion of the recombinant CKX was secreted into the culture medium.
In Vivo Cytokinin Metabolism
To assess the effects of the transformation on cytokinin catabolism, the radiolabeled cytokinins N6-benzyl[2-3H]adenine ([2-3H]BA), N6-( As a control, [2-3H]BA, which is a very poor substrate for CKX, was applied to wild-type and tCKX7 cultures at a concentration of 20 pmol/mL and its metabolism was monitored in the culture medium by HPLC coupled to online liquid scintillation counting. During 8 h of incubation, no significant differences in [2-3H]BA metabolization were found between the wild type and tCKX7 (Fig. 2A ). However, when [2-3H]iP (20 pmol/mL), a preferred substrate of CKX, was applied, dramatic differences in its metabolization were found (Fig. 2B). The amount of external [2-3H]iP in the media of wild-type and tCKX16 cultures decreased within 8 h to 75% and 25% of initial levels, respectively, while in tCKX7 cultures the concentration fell extremely rapidly, to below the detection limit within an hour. Thus, tCKX7 and tCKX16 have greatly enhanced in vivo degradation capacity for [2-3H]iP. The degradation capacities of the wild type, tCKX16, and tCKX7 per unit mass of wet protonema (including the retained medium) were found to be 3, 10, and 91 pmol h–1 g–1, respectively.
The riboside [2-3H]iPR was applied at a higher concentration (60 pmol/mL) to allow quantifiable amounts of labeled metabolites to be extracted from the tissues. Monitoring the external [2-3H]iPR concentration, again a stronger depletion was observed in the media of the transformant cultures (63%–77% decrease after 8 h of incubation) than in the wild-type culture medium (15% after 8 h; Fig. 2C). To assess the effects of CKX overexpression with respect to the conversion products [2-3H]iP and [2-3H]iPR nucleotides, detailed analyses of culture media and tissue extracts were carried out. In wild-type culture medium, the extracellular metabolite [2-3H]iP accumulated to concentrations of up to 15 pmol/mL within 20 h, while in the media of both of the transformants no [2-3H]iP was detectable after 20 h (data not shown), probably because any formed was rapidly degraded. After 20 h of incubation with [2-3H]iPR, no radioactivity was found in the extracellular fractions of cytokinin nucleotides (data not shown). The distribution of radioactivity among the extractable tissue-bound [2-3H]iPR metabolites is presented in Table II . Relatively small intracellular amounts of the [2-3H]iPR substrate (7.5%) remained after 20 h in wild-type tissues, while in the tCKX16 and tCKX7 transformants no detectable tissue-bound [2-3H]iPR remained after 20 and 4 h. Similarly, the main metabolite [2-3H]iP was detectable in wild-type tissues, but not in tCKX7 and tCKX16 tissues, after 20 h incubation.
The relative proportions of cytokinin nucleotides were also clearly reduced in the transformant cultures, and no [2-3H]iPRMP [N6-( 2-isopentenyl)adenosine-5'-monophosphate, iPRMP] was found in tCKX7 cultures. As a result of CKX overexpression, the metabolism of [2-3H]iPR was directed, as expected, toward the degradation products, which accounted for 75% to 100% of the extractable radioactivity in the transformants, but only for at most 53% in the wild type. In summary, the results of the labeling studies clearly demonstrate the functional overexpression of the heterologous CKX under in vivo conditions, revealing dramatic reductions in the levels of labeled cytokinin bases, ribosides, and nucleotides.
The contents of more than 40 isoprenoid and aromatic cytokinins in the tissues and culture media of the three investigated lines was monitored by sensitive ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS). Liquid cultures were washed with fresh medium at day 0 and harvested at three time points (days 0, 10, and 20) over a cultivation period of 20 d. Changes in the distribution of extracellular and intracellular cytokinins were monitored, thus allowing to estimate the amount of released cytokinins over the time course. All genotypes were grown and sampled in triplicate. Mean values and SDs of the amounts of the cytokinins found in the tissues and media are shown in Figure 3, A and B , respectively, and the results are summarized in Table III and Supplemental Table S3 (see also Supplemental Tables S1 and S2).
Cytokinin Profile in Wild-Type Tissue The profiling revealed that all groups of isoprenoid cytokinins, such as iP-, tZ-, cZ-, and DHZ-type cytokinins, are present in Physcomitrella tissue, and that all of these groups were represented by detectable amounts of the bases, ribosides, and nucleotides, except DZRMP. In addition, O-glucosides of both the bases (tZOG and cZOG) and ribosides (cZROG and tZROG) of the cZ and tZ types of hydroxylated cytokinins were found (Fig. 3A; Table III; Supplemental Table S3).
The most abundant intracellular cytokinins were cZROG, followed by iPRMP, tZROG, cis-zeatin-riboside-5'-monophosphate (cZRMP), trans-zeatin-riboside-5'-monophosphate (tZRMP), cZOG, and iPR, for which maximum concentrations found were 646, 332, 170, 72, 68, 52, and 36 pmol/g dry weight (DW), respectively. All other detected cytokinins were present at concentrations Interestingly, the aromatic cytokinin bases BA, meta-topolin (mT), and ortho-topolin (oT) were also detected, but N6-benzyladenosine (BAR) was the only aromatic cytokinin riboside found, and no nucleotides of aromatic cytokinins were detectable. Intracellular concentrations of most cytokinins (e.g. iPRMP) were highest at the beginning of the sampling period. However, concentrations of some metabolites (e.g. cZR and DZR) did not significantly change during cultivation, and levels of one (tZ) increased between days 10 and 20. Most of the decrease in intracellular cytokinin contents was probably due to the release of cytokinins into the fresh culture medium in which the tissue was suspended at day 0 (Fig. 3A).
Although strong effects of CKX overexpression on the exogenously applied tritiated cytokinins [2-3H]iPR and [2-3H]iP were observed in the short-term labeling experiments (see Fig. 2), it had a much less pronounced influence on endogenously produced cytokinins, and the intracellular content of cZR was even higher in the tCKX7 and tCKX16 transformants than in the wild type. The compound showing the most significant decrease was tZROG (especially in tCKX16), and concentrations of most of the other cytokinin metabolites showed only nonsignificant tendencies to be lower in the CKX overexpressors (Fig. 3A).
The UPLC-MS/MS analysis of the medium of wild-type cultures revealed that all groups of isoprenoid cytokinins detected in the tissue were also present in the wild-type medium. However, concentrations of the cytokinins cZRMP, DHZRMP, DHZR, and DHZ were below their respective detection limits (for abbreviations, see Supplemental List S1). The concentrations of the extracellular cytokinins ranged between 0.08 and 16 pmol per 100 mL of medium. The major extracellular cytokinin was iPRMP (maximum, 16 pmol/100 mL in wild-type medium), followed by iP (10 pmol/100 mL) and iPR (3 pmol/100 mL). All other cytokinin forms were present at concentrations lower than 1 pmol/100 mL (Fig. 3B). The identity of iPRMP in Physcomitrella medium was strongly supported using a combination of capillary liquid chromatography (CapLC module) and mass spectrometric analysis with a Q-Tof micro hybrid quadrupole time-of-flight mass spectrometer, enabling high resolution identification of cytokinin derivatives (Supplemental Fig. S1). Taken together, the corresponding fragmentation pattern and the exact mass confirmed the presence of iPRMP as a major cytokinin in Physcomitrella culture media. The only aromatic cytokinin in the media was oT, detected in trace quantities at day 0; concentrations of all other aromatic cytokinins and oT at all other sampling times were below the detection limit. In contrast to the intracellular fractions, the extracellular cytokinins mostly accumulated over time; the only deviations from this pattern were that concentrations of iP declined between days 10 and 20, and concentrations of cZRMP generally remained stable throughout the sampling period (Fig. 3B).
The influence of CKX overexpression on extracellular cytokinin levels was most pronounced for the iP-type derivatives; iPR concentrations were approximately 19-fold lower in tCKX7 medium than in wild-type medium (0.17 versus 3.3 pmol per 100 mL), and iP concentrations were 55- and 30-fold reduced in tCKX7 and tCKX16, respectively (0.19 and 0.35 pmol per 100 mL in the tCKX7 and tCKX16 media, respectively, and 10.6 pmol per 100 mL in the wild-type medium, on day 10, when the concentrations were maximal; Fig. 3B). In addition, extracellular concentrations of the cytokinin nucleotide iPRMP were reduced in both of the transformant cultures. Unexpectedly, however, extracellular concentrations of some cytokinins (including tZROG, cZROG, cZRMP, and cZR) were higher in cultures of the transformants than in wild-type cultures. In summary, it can be concluded that CKX overexpression also affects levels of endogenously produced cytokinins. The strongest reductions were found for iP and iPR in the culture medium.
Cytokinin-deficient transformants showed numerous phenotypical changes at various levels. (1) Following protoplast transformation using the AtCKX2-carrying construct (pHP_act1_AtCKX2), far fewer stable transgenic lines were obtained than from protoplasts transformed with the control plasmid pHP23 (two and 20, respectively, from approximately 106 protoplasts). Clearly, therefore, expression of the AtCKX2 gene has a strongly negative influence on protoplast vitality and/or regeneration capacity.
(2) At the protonema level, the morphology of the filaments was strongly altered in both the tCKX7 and tCKX16 transformants. The morphology of the chloronema of the transgenics was abnormal and irregular (Fig. 5A), mainly due the occurrence of a high number of cells that were substantially shorter (31–58 µm, versus
(3) Budding was impaired in untreated tCKX7 and tCKX16 protonemata growing in both liquid and agar culture, the time until buds appeared being longer and the numbers of buds produced lower in the transformant cultures than in the wild-type cultures (Fig. 4B ).
(4) Although tCKX7 and tCKX16 transformants produced gametophores, no sexual reproduction was observed. Since the formation of neither archegonia nor antheridia was observed, the transition to the sporophytic generation seems to be blocked at the level of gametangia development (data not shown). In general, the development of gametophores was delayed and, moreover, they were often smaller than wild-type gametophores (Fig. 4A). Since exogenously applied cytokinins are known to induce bud formation in moss protonema, we applied iP to assess whether the increased cytokinin degradation in the transformants influenced the budding response. The transformants formed significantly fewer buds than the wild type, even when iP was applied at concentrations of 500 nmol/L (Supplemental Fig. S3). The tCKX7 transformant displayed an especially weak budding response, concentrations of 50 nmol/L or more being required to induce any detectable response. Control assays with BA revealed comparable budding responses for the wild type and CKX transformants (data not shown).
The activities of selected representatives of the various types of cytokinins from the broad spectrum of 20 endogenous cytokinins detected in the UPLC-MS/MS analysis (Fig. 3, A and B) were assayed in a bud-induction bioassay with wild-type cultures. The most potent bud-inducing cytokinin was found to be iP, followed in order of decreasing activity by tZ, BA, BAR, iPR, and tZR. The cytokinins DHZ, mT, and oT exhibited very weak activities, while cZ, cZR, and the nucleotides iPRMP, tZRMP, and BAR monophosphate (BARMP) showed no activity at all (Fig. 5).
Since the discovery of cytokinins, their functions and roles in plant development have mainly been assessed by exogenously administering cytokinins or by creating IPT-transgenic plants with increased rates of cytokinin biosynthesis. However, since the identification of CKX genes (Houba-Hérin et al., 1999 ; Morris et al., 1999), new approaches for evaluating the role of cytokinins have become available, resulting from the possibility to experimentally decrease the endogenous cytokinin contents in CKX-overexpressing plants.
Here, we used the rice actin1 promoter (Wang et al., 1992
In vitro enzyme assays (Table I) showed that CKX specific activity was up to 27-fold higher in the tissues of the transformants than in wild-type tissues, and up to 157-fold higher in concentrated protein preparations from their culture media. Similarly, significantly higher stimulation of CKX activity in the media than in the cells has been reported for Saccharomyces cerevisiae cultures expressing the AtCKX2 gene (Werner et al., 2001
Cultures were fed with radiolabeled cytokinins to confirm that the capacity of the transformants to degrade cytokinins had been enhanced, as intended. In a control experiment, [2-3H]BA, which has been reported to be either a nonsubstrate (Armstrong, 1994
The increased cytokinin breakdown capacity of the CKX transformants was also confirmed by the analysis of tissue extracts. After 20 h of incubation, neither the applied substrate [2-3H]iPR nor its metabolic product [2-3H]iP were detectable in the transformants (Table II). This is in accordance with the higher amount of degradation products found in the transformants and indicates that their reductions in concentrations of cytokinin metabolites were indeed due to enhanced CKX activity. In the experiments with tCKX7 cultures, all of the extractable radioactivity was found in the fractions of degradation products. Differences between the total and extractable amounts of intracellular radiolabeling showed that only 6 pmol of the labeling remained unextracted in wild-type tissues, compared to 149 pmol and 157 pmol in tCKX16 and tCKX7 tissues, respectively (data not shown). We presume that significant amounts of purine-like degradation products were fixed in macromolecules such as RNAs in the transformants, which are not extractable by the method employed. Thus, the dramatically higher amounts of nonextractable radioactivity in the CKX transformants further highlight their enhanced capacity to degrade cytokinins.
Unexpectedly, levels of radiolabeled iP nucleotides were found to be reduced in the extracts of the transformants (Table II), although cytokinin nucleotides are not, reportedly, substrates for CKX (Armstrong, 1994
Furthermore, the lower amounts of iP nucleotides found in the transformants may reflect the fact that fewer cytokinins were available for nucleotide-forming reactions, especially via adenosine kinase (von Schwartzenberg et al., 1998
After radiolabeling with [2-3H]iP or [2-3H]iPR, no Z-type cytokinins could be detected, indicating that trans-hydroxylation of free iP-type cytokinins by cytochrome P450 monooxygenase-like enzymes (Takei et al., 2004
In this work we present a comprehensive determination of intracellular and extracellular cytokinins in axenic cell cultures of a bryophyte and relate the results obtained to CKX overexpression. The UPLC-MS/MS measurements of endogenous cytokinins reflect their steady-state levels, as governed by their integrated rates of biosynthesis, interconversion, and breakdown.
The analyses revealed that Physcomitrella contains at least 20 different cytokinins, far more than previously reported in any mosses (Bopp, 1990
All groups of cytokinins occurred as bases, ribosides, and nucleotides (Supplemental Table S3, although levels of DHZRMP were mostly below the detection limit [data not shown]). The members of the Z family (both trans- and cis-isomers) were also present in the forms of O-glucosides (Fig. 3, A and B), indicating that the O-glycosylation mechanism, whereby the hormonal activity of Zs is "tuned" (Veach et al., 2003
Ördög et al. (2004)
For multicellular green algae like Cladophora capensis and Ulva spec, a prevalence of both iP- and cZ-type cytokinins was found by HPLC-MS-based studies (Stirk et al., 2003
Since tZ-type cytokinins have been shown to be generated by adenylate IPTs and cZ-type cytokinins by tRNA-IPTs in Arabidopsis (Miyawaki et al., 2006 In this work we also report for the first time in a bryophyte the occurrence of the aromatic cytokinins BA(R), mT, and oT (Fig. 3A), the biosynthesis of which is so far unclear.
To include polar cytokinin compounds in the analysis, we developed an extraction protocol involving freeze drying of the culture medium instead of the commonly used solid-phase extraction method. Remarkably, the predominant extracellular cytokinins were found to be nucleotides, predominantly represented by iPRMP (Fig. 3B; Supplemental Table S3). tZRMP and cZRMP were also found in the medium, but only in minor quantities.
The presence of extracellular cytokinin nucleotides was confirmed by Q-Tof MS, which provided both structural identification and highly accurate mass determinations (Supplemental Fig. S1). The detection of considerable concentrations of extracellular cytokinin nucleotides in Physcomitrella is in accordance with the previously reported occurrence of tZRMP and iPRMP in media of tobacco cell suspension cultures (Motyka et al., 2003 The accumulation of extracellular nucleotides seems to be a rather slow process since during the short-term labeling experiments using [2-3H]iPR and [2-3H]iP no radioactive monophosphates were detected in the culture medium. Since the tissue used for inoculation was washed and suspended in fresh medium at day 0, little or no nucleotides were detected at the starting point of the culture. During the following 20 d, the wild-type cultures accumulated 16 pmol iPRMP in 100 mL of medium, which we presume was released from the large pool of intracellular nucleotides (see Fig. 3, A and B). The alternative possibility, that they were formed from appropriate base or riboside precursors in the culture medium by external activities of adenine phosphoribosyl transferase or adenosine kinase, seems unlikely since these enzymes have not been detected in culture media to date. The fact that, like most other cytokinins, the internal iPRMP concentration decreased during the culture period (Fig. 3, A and B) suggests that cytokinin nucleotides are released from the cells. However, their transport mechanism is unclear.
No detailed mass spectrometric analysis of natural cytokinins and concomitant assessment of their hormonal activity have previously been reported for Physcomitrella or any other bryophyte. One of the main advantages of mosses as experimental models for plant hormone research is that they can be grown in suspension culture and no artificial systems like callus or organ cultures need to be employed. We tested the biological activities of bases and/or ribosides of all cytokinin groups detected in the UPLC-MS/MS analysis (Fig. 3) using the cytokinin bioassay, with slight modifications, described for Funaria hygrometrica by Hahn and Bopp (1968)
Although the nucleotide iPRMP was the dominant cytokinin in the culture medium, it had no detectable bud-inducing activity, and neither did tZRMP and BARMP. However, it should be mentioned that tZRMP has been shown to be bound by the Arabidopsis AHK3 receptor, indicating that cytokinin nucleotides may have signaling functions (Spichal et al., 2004 In addition, cZ and cZR, which are major cytokinins in Physcomitrella tissue (Fig. 3), exhibited no detectable bud-inducing activity. Thus, these and the other inactive cytokinins appear to play no role in the morphogenetic process of bud development in Physcomitrella (Fig. 5).
Surprisingly large amounts of cZ-type cytokinins, especially O-glycosylated riboside or base forms (Fig. 3, A and B), were found in Physcomitrella. Although cZ has been demonstrated to bind to receptors (Spichal et al., 2004
Assuming that the biogenesis of cZ-type cytokinins in Physcomitrella is generally, as recently demonstrated in Arabidopsis (Miyawaki et al., 2006
The main differences in endogenously produced cytokinins between wild-type and transgenic cultures were in the extracellular concentrations of iPR and iP, which were significantly reduced in the transformants (Fig. 3B). The reductions in the extracellular iP level were most pronounced at days 10 and 20. The findings of reduced iP and iPR contents in the medium are consistent with the results obtained in the labeling experiments using tritiated iP and iPR, in which rapid depletion of the extracellular label was observed (Fig. 2).
Concentrations of other endogenously produced cytokinins showed fewer obvious reductions and often only nonsignificant tendencies to decline. Indeed, both intracellular and extracellular cZR contents were even higher in the transformant cultures than in the wild-type cultures, implying that cZ-type cytokinins are probably resistant to AtCKX2 attack in this system, although cZ-type cytokinins are generally believed to be substrates for CKX (Armstrong, 1994
Our enzymatic studies on Physcomitrella CKX from crude extracts of untransformed wild-type tissue revealed that cZ is degraded at rates up to 7-fold and 4.5-fold higher than tZ and iP, respectively. In contrast, in assays with tCKX7 and tCKX16 protein extracts, cZ appears to be a much poorer CKX substrate than tZ and iP, and the order of CKX-catalyzed cleavage rates appears to be inverted: iP > tZ > cZ (S. Gajdo
The contents of the aromatic cytokinins BA, BAR, mT, and oT were also not reduced in tCKX7 and tCKX16 cultures (Fig. 3). This finding is in agreement with the low degradation capacity of CKX toward BA (Laloue and Fox, 1989
In the moss bud-induction assays, the number of buds induced on protonema is proportional to the hormonal activity in the culture medium (Bopp and Brandes, 1964
In addition, extracellular iP and iPR are likely to be the main bud-inducing cytokinins in natural conditions in Physcomitrella, since concentrations of these hormones were most strongly reduced in cultures of the cytokinin-deficient transformants and both iP and iPR were found to have strong bud-inducing capacity in the Physcomitrella bioassay (Fig. 5). Extracellular iP and iPR also seem to have great importance for protonema development since cell morphology was altered under iP and iPR deficiency (Fig. 4; Supplemental Fig. S2). In cytokinin-overproducing Physcomitrella plants, the so-called ove mutants, major changes in hormone composition in the extracellular space have also been observed, principally accumulations of iP and iPR, which correlated with strong overproduction of buds (Wang et al., 1980
Plant Culture
Wild-type Physcomitrella patens (Hedw.) B.S.G. was maintained on solid agar medium using the ABC medium described by Knight et al. (1988)
For the determination of budding frequency, the less turbid Knop medium was used (Hahn and Bopp, 1968
Liquid cultures used for cytokinin profiling and metabolism studies were grown in a medium described by Wang et al. (1980)
cDNA of the AtCKX2 gene of Arabidopsis (Arabidopsis thaliana; accession no. AF303978) cloned into the vector pCR-Blunt II-topo (Invitrogen) was provided as a gift by T. Schmülling (FU-Berlin). The vector pBAS_GFP (Zeidler et al., 1999
The AtCKX2 cDNA was isolated from the vector pCR-Blunt II-topo-AtCKX2 by digestion with KpnI, and the 1,600-bp AtCKX2 fragment was blunted and ligated into the pBAS vector backbone. Since the resulting vector pBAS_AtCKX2 did not contain a plant-selective marker, the entire expression cassette (3,330 bp) with the actin1 promoter, AtCKX2, and terminator was isolated from pBAS_AtCKX2 by XbaI/HindIII digestion and then blunt-end ligated into the NdeI site of the vector pHP23_
The construct pHP_act1_AtCKX2 was transferred into Physcomitrella protoplasts derived from liquid cultures by PEG-mediated transformation according to Schaefer et al. (1991)
The presence of the construct pHP_act1_AtCKX2 in genomic DNA of the Physcomitrella transformants tCKX7 and tCKX16 was demonstrated by PCR using the primers 5'-ATCATCAGCAAGGTTATTGACAC-3' and 5'-TCATCGCCGACATACGATTG-3'; at an annealing temperature of 55°C, a 307-bp fragment of AtCKX2 was amplified.
The in vivo metabolization of the radiolabeled cytokinins [2-3H]BA,[2-3H]iP, and [2-3H]iPR was analyzed as described by von Schwartzenberg et al. (2003)
The CKX from Physcomitrella cells (plant material equivalent to approximately 1.3–1.5 g DW) was extracted and partially purified using the method of Chatfield and Armstrong (1986)
The CKX activity was determined by in vitro assays in which the conversion of [2-3H]iP to [2-3H]adenine in the copper-imidazole-sensitized technique described by Chatfield and Armstrong (1987) Each CKX determination was performed in two independent biological samples and repeated three times. Results of one representative determination are presented. Statistical variation of results are expressed as the average ± SD.
Samples of both the culture media (100 mL) and tissues were freeze-dried and stored at –20°C until LC-MS analysis. The procedure used for cytokinin analysis was a modified form of the method described by Faiss et al. (1997) The cytokinin fractions were analyzed using an ACQUITY UPLC ultra-performance liquid chromatograph (Waters), equipped with a BEH C18 (1.7 µm; 2.1 x 150 mm) column, linked to a Quattro micro API (Waters MS Technologies) triple quadrupole mass spectrometer equipped with an electrospray interface. The purified samples were dissolved in 15 µL MeOH/H2O (30/70, v/v) and 10 µL of each sample was injected into the chromatographic system. The analytes were eluted with a 10-min binary linear gradient, composed of 15 mM ammonium formate (pH 4.0, A) and methanol (B) starting at an A:B ratio of 1:9 (v/v) and finishing at a 1:1 ratio of A:B (flow rate 0.25 mL/min, column temperature 40°C), with retention times for the monitored compounds ranging from 2.50 to 6.50 min. The analytes were quantified by multiple reaction monitoring of [M+H]+ and the appropriate product ion. For selective MRM experiments, optimal conditions were as follows: capillary voltage 0.6 kV, source/desolvation gas temperature 100°C/350°C, cone/desolvation gas 2.0/550 L/h, LM/HM resolution 12.5, ion energy 1 0.3 V, ion energy 2 1.5 V, entrance 2.0 V, exit 2.0 V, multiplier 650 eV. The dwell time, cone voltage, and collision energy corresponding to exact diagnostic transition were optimized for each cytokinin. On the basis of the observed retention times, which appeared to be sufficiently constant, the chromatographic run was split into eight retention windows. The dwell time of each MRM channel was calculated to provide 16 scan points per peak, during which time the inter channel delay was 0.1 s. In MRM mode, the limit of detection for most of the cytokinins was lower than 5.0 fmol and the linear range was at least five orders of magnitude. The identity of all measured cytokinin metabolites was verified by comparison of the mass spectra and chromatographic retention times with those of authentic standards.
A CapLC module (Waters) capillary liquid chromatography system equipped with a reversed-phase (Symmetry C18, 0.3x150 mm, 5 µm; Waters) column coupled to a hybrid Q-Tof micro mass analyzer (Waters MS Technologies) was used for high resolution identification and confirmation of iPRMP. Following injection, cytokinins were eluted with a 25-min binary linear gradient, again composed of 15 mM ammonium formate (pH 4.0, A) and methanol (B) starting at an A:B ratio of 1:9 (v/v) and finishing at a 1:1 ratio of A:B, but with a flow-rate of 5 µL/min and column temperature of 35°C. Electrospray ionization in the positive ion mode was performed using the following parameters: source block/desolvation temperature, 90°C/200°C; capillary/cone voltage, 2,500/30 V; and spray/cone gas flow (N2), 50/250L/h. In the full-scan mode, data were acquired in the mass range of 50 to 500 D, with a cycle time of 28 µs, a scan time of 1.0 s, and a collision energy of 4 V. For the MS/MS experiments, analytes were fragmented with the collision cell filled with argon gas and collision energies of 15, 20, and 25 V. For the exact mass determination experiments, a lock spray was used for external calibration with a mixture of 0.1 M NaOH/10% formic acid (v/v) and acetonitrile (1:1:8 by volume) as a reference. Accurate masses were calculated and used for the determination of the elementary composition and structure of the analytes with fidelity
Undifferentiated protonemic tissues of 7-d-old Physcomitrella liquid cultures were rinsed with sterile water and used to inoculate petri dishes with Knop-agar medium (Hahn and Bopp, 1968
The following materials are available in the online version of this article.
We thank Petra Amakorová (Olomouc), Marie Korecká (Prague), Susanne Bringe, Jutta Krüger, and Vera Schwekendiek (Hamburg) for skillful technical assistance. M.F. acknowledges funding from the University of Hamburg's "Graduiertenförderung" program. The authors thank Mark Held (University of Waterloo, Canada) and Heinz Hahn (University of Hamburg) for critically reading a previous version of this manuscript, and Peter Schulz and Radka Vanková (Prague) for contributions in earlier parts of this project. The authors further acknowledge Thomas Schmülling (FU-Berlin) for providing the AtCKX2 cDNA. Received June 6, 2007; accepted September 21, 2007; published September 28, 2007.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant no. DFG Schw687/4); the Ministry of Education, Youth and Sports of the Czech Republic (grant no. MSM6198959216); the Grant Agency of the Czech Republic (grant no. 522/06/0703); and the Grant Agency of the Academy of Sciences of the Czech Republic (grant no. IAA600380701). 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Klaus von Schwartzenberg (kvschwartzenberg{at}botanik.uni-hamburg.de).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.103176 * Corresponding author; e-mail kvschwartzenberg{at}botanik.uni-hamburg.de.
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ABSTRACT
RESULTS
30 pmol/g DW. 
60 µm usually) but wider (diameter, up to 48 µm, versus <33 µm usually) than wild-type cells (Supplemental Fig. S2). However, although the material was clonal, not all protonema cells of the CKX transformants displayed altered cell morphology, and the size of a substantial proportion of the cells remained unaffected by cytokinin deficiency, with dimensions comparable to the wild type. 
ová, V. Motyka, and K. von Schwartzenberg, unpublished data). 
olcová B, Novák O, Strnad M, Friedeck
ková R, 
apková V, Petrá
