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Research article Tobacco cells transformed with the fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application Petra Suchomelova ´-Mas ˇkova ´ a , Ondrˇej Nova ´k b , Helena Lipavska ´ a, * a Department of Plant Physiology, Faculty of Science, Charles University in Prague, Vini cna ´ 5, 128 44 Prague 2, Czech Republic b Laboratory of Growth Regulators, Palacky ´ University & Institute of Experimental Botany, Academy of Sciences of the Czech Republic, S ˇ lechtitelu ˚ 11, Olomouc, Czech Republic Received 31 March 2007 Available online 29 April 2008 Abstract In plants, the G 2 /M control of cell cycle remains an elusive issue as doubts persist about activatory dephosphorylationdin other eukaryotes provided by CDC25 phosphatase and serving as a final all-or-nothing mitosis regulator. We report on the effects of tobacco (Nicotiana tabacum L., cv. Samsun) transformation with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on cell characteristics. Transformed cell sus- pension cultures showed higher dry mass accumulation during the exponential phase and clustered more circular cell phenotypes compared to chains of elongated WT cells. Similar cell parameters, as in the transformants, can be induced in WT by cytokinins. Spcdc25 cells, after cyto- kinin treatment, showed giant cell clusters and growth inhibition. In addition, Spcdc25 expression led to altered carbohydrate status: increased starch and soluble sugars with higher sucrose:hexoses ratio, inducible in WT by cytokinin treatment. Taken together, the Spcdc25 transformation had a cytokinin-like effect on studied characteristics. However, endogenous cytokinin determination revealed markedly lower cytokinin levels in Spcdc25 transformants. This indicates that the cells sense Spcdc25 expression as an increased cytokinin availability, manifested by changed cell morphology, and in consequence decrease endogenous cytokinin levels. Clearly, the results on cell growth and morphology are consistent with the model of G 2 /M control including cytokinin-regulated activatory dephosphorylation. Nevertheless, no clear link is obvious between Spcdc25 transformation and carbohydrate status and thus the observed cytokinin-like effect on carbohydrate levels poses a problem. Hence, we propose that Spcdc25-induced higher CDK(s) activity at G 2 /M generates a signal-modifying carbohydrate metabolism to meet high energy and C de- mands of forthcoming cell division. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Carbohydrate status; cdc25; Cell cycle; Cell morphology; Cytokinin; Nicotiana tabacum; Schizosaccharomyces pombe 1. Introduction Temporal and spatial control of the cell cycle is essential for initiation and maintenance of meristems and for regulation of organogenesis, therefore it represents one of the most inten- sively studied processes in contemporary plant biology. The basic mechanisms of cell cycle are conserved among all eukaryotes and a lot of knowledge has been acquired of yeast and animal model organisms. The study of the plant cell cycle, however, lags behind, partly because of the existence of unique regulatory pathways not described for other eukaryotes. As in all eukaryotic organisms, plant cell cycle progression is governed by cyclin-dependent kinase (CDK) activities. In yeasts, a single CDK associated with different cyclins regu- lates the progression through the cell cycle, while in mam- mals, several distinct CDKs function at different stages of the cell cycle. In plants, the transition through G 1 /S and G 2 / M control points is regulated by CDKA and CDKA/B kinase Abbreviations: BAP, 6-benzylaminopurine; CDK, cyclin-dependent kinase; NSS, non-structural soluble saccharide; PPB, preprophase band. * Corresponding author. Tel.: þ420 22 195 1680; fax: þ420 22 195 1704. E-mail address: [email protected] (H. Lipavska ´). 0981-9428/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.04.017 Available online at www.sciencedirect.com Plant Physiology and Biochemistry 46 (2008) 673e684 www.elsevier.com/locate/plaphy

Tobacco cells transformed with the fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application

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Page 1: Tobacco cells transformed with the fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application

Available online at www.sciencedirect.com

Plant Physiology and Biochemistry 46 (2008) 673e684www.elsevier.com/locate/plaphy

Research article

Tobacco cells transformed with the fission yeast Spcdc25 mitoticinducer display growth and morphological characteristics as well

as starch and sugar status evocable by cytokinin application

Petra Suchomelova-Maskova a, Ondrej Novak b, Helena Lipavska a,*

a Department of Plant Physiology, Faculty of Science, Charles University in Prague, Vini�cna 5, 128 44 Prague 2, Czech Republicb Laboratory of Growth Regulators, Palacky University & Institute of Experimental Botany, Academy of Sciences of the Czech Republic,

Slechtitelu 11, Olomouc, Czech Republic

Received 31 March 2007

Available online 29 April 2008

Abstract

In plants, the G2/M control of cell cycle remains an elusive issue as doubts persist about activatory dephosphorylationdin other eukaryotesprovided by CDC25 phosphatase and serving as a final all-or-nothing mitosis regulator. We report on the effects of tobacco (Nicotiana tabacumL., cv. Samsun) transformation with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on cell characteristics. Transformed cell sus-pension cultures showed higher dry mass accumulation during the exponential phase and clustered more circular cell phenotypes compared tochains of elongated WT cells. Similar cell parameters, as in the transformants, can be induced in WT by cytokinins. Spcdc25 cells, after cyto-kinin treatment, showed giant cell clusters and growth inhibition. In addition, Spcdc25 expression led to altered carbohydrate status: increasedstarch and soluble sugars with higher sucrose:hexoses ratio, inducible in WT by cytokinin treatment. Taken together, the Spcdc25 transformationhad a cytokinin-like effect on studied characteristics. However, endogenous cytokinin determination revealed markedly lower cytokinin levels inSpcdc25 transformants. This indicates that the cells sense Spcdc25 expression as an increased cytokinin availability, manifested by changed cellmorphology, and in consequence decrease endogenous cytokinin levels. Clearly, the results on cell growth and morphology are consistent withthe model of G2/M control including cytokinin-regulated activatory dephosphorylation. Nevertheless, no clear link is obvious between Spcdc25transformation and carbohydrate status and thus the observed cytokinin-like effect on carbohydrate levels poses a problem. Hence, we proposethat Spcdc25-induced higher CDK(s) activity at G2/M generates a signal-modifying carbohydrate metabolism to meet high energy and C de-mands of forthcoming cell division.� 2008 Elsevier Masson SAS. All rights reserved.

Keywords: Carbohydrate status; cdc25; Cell cycle; Cell morphology; Cytokinin; Nicotiana tabacum; Schizosaccharomyces pombe

1. Introduction

Temporal and spatial control of the cell cycle is essentialfor initiation and maintenance of meristems and for regulationof organogenesis, therefore it represents one of the most inten-sively studied processes in contemporary plant biology. Thebasic mechanisms of cell cycle are conserved among all

Abbreviations: BAP, 6-benzylaminopurine; CDK, cyclin-dependent kinase;

NSS, non-structural soluble saccharide; PPB, preprophase band.

* Corresponding author. Tel.: þ420 22 195 1680; fax: þ420 22 195 1704.

E-mail address: [email protected] (H. Lipavska).

0981-9428/$ - see front matter � 2008 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.plaphy.2008.04.017

eukaryotes and a lot of knowledge has been acquired of yeastand animal model organisms. The study of the plant cell cycle,however, lags behind, partly because of the existence ofunique regulatory pathways not described for othereukaryotes.

As in all eukaryotic organisms, plant cell cycle progressionis governed by cyclin-dependent kinase (CDK) activities. Inyeasts, a single CDK associated with different cyclins regu-lates the progression through the cell cycle, while in mam-mals, several distinct CDKs function at different stages ofthe cell cycle. In plants, the transition through G1/S and G2/M control points is regulated by CDKA and CDKA/B kinase

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674 P. Suchomelova-Maskova et al. / Plant Physiology and Biochemistry 46 (2008) 673e684

activities, respectively. For activation of CDK, the presence ofa regulatory cyclin subunit is necessary: D-type cyclins at G1/S and A- and B-type cyclins, eventually D-type cyclins asCDKB partners, at G2/M (for review see e.g. [5,15]). Besidesthese, the modulation of CDK/cyclin complex activity is pro-vided by CDK inhibitors (predominantly active at G1/S) andreversible (de)phosphorylation on conserved CDK aminoacid residues (Thr 160e167, Thr 14 and Tyr 15). For fully ac-tive CDK/cyclin entering mitosis, the complex has to be phos-phorylated at Thr160e167 (accomplished by CDK-activatingkinase) and dephosphorylated at Thr14 and Tyr15. In yeastthis dephosphorylation, provided by Cdc25 phosphatase, isthe most important regulation mechanism representing thefinal positive regulator of mitosis [25]. Some time ago inhibi-tory kinase Wee1 was characterised, exhibiting a large cellphenotype in plants with the dominant-negative allele of thisgene [38,42]. During the last decade, efforts to find plantactivatory G2/M phosphatase were not fully successful; theonly cdc25 orthologue has been identified in unicellular algaOstreococcus tauri [16]. Although a Cdc25-like catalytic pro-tein subunit with in vitro CDK-binding and activatory capabil-ity has recently been identified in higher plants (Arabidopsisand rice), the gene is not able, unlike the algal one, to comple-ment yeast cdc25 mutant strains and its mutation exhibits noobvious phenotype in plants [17,37]. Recently, Boudolf et al.[3] hypothesised that in higher plants the control of mitosisonset might have been evolutionarily replaced by the CDKBkinase pathway and the cdc25 gene eventually was lost, orthat the phosphatase responsible for CDK activation is com-pletely unrelated to eukaryotic Cdc25. All these pieces ofknowledge strongly argue for further study of transgenicplants expressing foreign cdc25 gene.

The cell division process is very sensitive to extra- and intra-cellular signals influencing mainly transition through controlpoints. At G1/S the rate-limiting factor is the actual level ofD cyclin, whose expression is under the control of phytohor-mones, especially cytokinins, and sucrose [29,30]. Cytokinin,together with auxin, induced the expression and probably activ-ity of CDK throughout the cell cycle, and cytokinins alsorepressed the expression of some CDK inhibitors [28]. Thestimulatory cytokinin and auxin effects at G2/M have beendocumented for mitotic cyclins expressions as well [28].Cytokinins, however, seemed to be indispensable for entryinto mitosis [18,53]. One of the reasons could be that cytokininstimulates dephosphorylation of CDK supposedly throughactivation of plant CDK phosphatase [53]. The authors demon-strated that fission yeast Cdc25 was able to dephosphorylateplant CDK and that the primary signal for plant CDK activatorydephosphorylation at mitosis entry came from cytokinin [53].

Many studies have been devoted to the investigation of theeffect of fission yeast Spcdc25 expression on plant growth anddevelopment. Bell et al. [2] in glasshouse experiments withday-neutral tobacco constitutively expressing fission yeastSpcdc25 gene showed, besides others, an earlier onset and en-hanced intensity of flowering. Recently, we reported a markedpositive effect of sucrose on flowering induction in day-neutraltobacco plants in vitro, and the remarkable impact of fission

yeast Spcdc25 expression on the flowering gradient, status ofapical meristem and overall floral capacity [44]. McKibbinet al. [19] demonstrated in Spcdc25 plants a higher amountof lateral root primordia composed of smaller cells. We foundout that the fission yeast Spcdc25 expression exhibits a cytoki-nin-like effect on de novo organ formation on tobacco stemsegments of transformed plants manifested by earlier andmore abundant shoot formation and restricted root inductionas well as cytokinin-independent shoot regeneration in trans-genic material [41]. Moreover, Orchard et al. [26] observedin Spcdc25-expressing BY-2 cells the earlier onset of mitosisin transgenic lines together with a smaller cell size phenotypeaccompanied by extremely low cytokinin levels in transgeniccells. However, such long-living cultures could exhibit a veryspecific phenotype caused at least partially by epigeneticchanges with the potential risk of changes in cell cycle duration(see e.g. [36,53]). We established newly derived tobacco(Nicotiana tabacum, cv. Samsun) cell cultures expressingfission yeast Spcdc25 to determine the effect of mitotic activa-tor on cellular morphogenesis and growth characteristics asopposed to wild-type cell cultures. Another aim of this studywas to examine to what extent it is possible to mimic the effectof transformation in wild-type cultures by exogenous cytokininapplication.

2. Methods

2.1. Plant material

Cell suspension cultures were derived from internode stemsegments of Nicotiana tabacum, cv. Samsun, wild type (WT)and three independent lines transformed with cdc25 cDNAfrom Schizosaccharomyces pombe (Spcdc25) under the 35SCaMV promoter (lines A, C and F) [2].

2.2. Cultivation

2.2.1. Cultivation mediumMacro- and micronutrients [13], Fe chelate according to [23],

vitamins [51], enriched with 30 g l�1 sucrose, 100 mg l�1

inositol, 1 g l�1 casein hydrolysate, 1 mg l�1 a-naphthylaceticacid (NAA) and 1 mg l�1 2,4-dichlorphenoxyacetic acid (2,4-D).

2.2.2. Cultivation conditionsOrbital shaker 125 rpm, 25 �C, 16 h photoperiod, irradiance

25 mmol.m�2 s�1. Subcultivation interval: 3 weeks.

2.2.3. Experimental variantsBasic medium without cytokinin or supplemented with 0.1,

1 and 2 mg l�1 6-benzylaminopurine (BAP), cells sampled onthe 12th or 14th day of cultivation (exponential phase).

2.3. Cytometry

Sampled cells were stained by Hoechst 33258 dye and mi-totic cells were microphotographed (Olympus BX51, DigitalCamera Apogee U4000) and cell size and shape measured

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675P. Suchomelova-Maskova et al. / Plant Physiology and Biochemistry 46 (2008) 673e684

using image analysis (Lucia G version 4.82). Cell size was de-termined as a cell area documented in the picture; cell shapewas quantified using the parameter of circularity (4p * area/perimeter * perimeter) and elongation (max. feret /min. feret).

2.4. Growth parameters

Dry weight was determined and relative growth rates ((dryweight of sample � dry weight of inoculum)/dry weight of in-oculum) were quantified. Inoculum dry weight quantificationwas based on dry weight/fresh weight ratio in parallel samples.

2.5. Non-structural soluble saccharide (NSS) contentdetermination

The samples were freeze-dried, then boiled with 80% meth-anol (75 �C, 15 min), the solvent evaporated, and the residuedissolved in Milli-Q ultrapure water (Millipore, Bedford,MA, USA). Then the samples were purified by centrifugationand filtration. The content of soluble NSS was detected usinghigh-performance liquid chromatography (HPLC) with refrac-tometric detection (Spectra Physics, refractometer Shodex RI-71), pre-column: Hema-Bio 1000 QþSB; column: IEX Pbform (Watrex, Czech Republic). For details see [43].

2.6. Starch content determination

Starch granules were histochemically detected using iodinesolution; for photodocumentation see Section 2.3. For bio-chemical determination, the starch was enzymatically hydro-lysed by a-amylase and amyloglucosidase and the glucosecontent was measured by the HPLC. For details see [40].

2.7. Cytokinin content determination

The procedure used for cytokinin analysis was a modificationof the method described by Novak et al. [24]. Frozen plant ma-terial (w2.5 g fresh weight) was extracted overnight in Bieleskibuffer. Deuterium-labelled CK internal standards (OlchemimLtd., Czech Republic) were added, each at 5 pmol per sampleto check the recovery during purification and to validate the de-termination. The extracts were purified using combined cation(SCX-cartridge), anion (DEAE-Sephadex-C18-cartridge) ex-changer and immunoaffinity chromatography (IAC) based onwide-range specific monoclonal antibodies against cytokinins[6]. This resulted in three fractions: (1) the free bases, ribosidesand N-glycosides (fraction B), (2) a nucleotide fraction (NT)and (3) an O-glucoside fraction (OG). The metabolic eluatesfrom the IAC columns were evaporated to dryness and storedat �20 �C until further analyses. CK fractions were quantifiedby ultra performance liquid chromatography (UPLC) (AcquityUPLC�; Waters, Milford, MA, USA) coupled to a Quattromicro API (Waters, Milford, MA, USA) triple quadrupolemass spectrometer equipped with an electrospray interface.The purified samples were injected onto a C18 reversed-phasecolumn (BEH C18; 1.7 mm; 2.1 � 50 mm; Waters) and elutedwith a linear gradient (0 min, 10% B; 0e8 min, 50% B; flow-

rate of 0.25 ml min�1) of 15 mM ammonium formate (pH4.0, A) and methanol (B). Quantitation was obtained by multi-ple reaction monitoring (MRM) of [M þ H]þ and the appropri-ate product ion. In MRM mode, the limit of quantification(LOQ) for measured cytokinins was in range 1.0e10.0 fmol,and the linear range was at least five orders of magnitude.

2.8. Statistical evaluation

For statistical evaluation the NCSS 6.0. software was em-ployed, analysis of variance (one-way ANOVA), KruskaleWallis multiple comparison Z-value and TukeyeKramer tests,at the reliability level a ¼ 0.05 or 0.01.

3. Results

3.1. Growth and morphological characteristics

The cell suspension cultures, derived from stem segmentsof tobacco lines (A, C and F), independently transformedwith fission yeast Spcdc25 gene, were used for determinationof the changes in cell characteristics.

For evaluation of relative growth rates (dry weight at sam-pling time related to inoculum dry weight) the cultures onthe14th day after subcultivation (the exponential growth phase)were selected. At this stage all tested Spcdc25 transgenic cellsuspension cultures reached significantly higher growth ratescompared to the wild-type (WT) cell cultures (Fig. 1A). Never-theless, time curves of relative growth rates showed that theexponential phase of WT cultures tended to be longer andthe maximum relative biomass accumulation of WT cultureswas finally comparable to transgenic ones (data not shown).

The cell morphology and cell size were evaluated on the 14thday after subcultivation and only mitotic cells were measured tocompare cell parameters. For quantitative measurements two-dimensional image analysis was employed. Surprisingly, cellsof tobacco lines A, C and F exhibited variable mitotic cellsize. When compared with wild type, the average cell area ofSpcdc25 lines’ mitotic cells was similar or even increased(Fig. 2).

In contrast, the transformation with Spcdc25 resulted inmarked changes in cell shape. The evaluation of the param-eter of circularity and elongation proved a more circular (iso-diametric) cell phenotype of transformed cells (Fig. 3A,B).In all Spcdc25 transgenic lines the value of circularity wasdefinitely significantly higher and the parameter of elonga-tion lower than it was established for the WT cells. Aneven more pronounced effect of tobacco transformationwith fission yeast Spcdc25 gene was found with regard tocell arrangement. WT cells commonly formed chains(Fig. 4A) as was documented for other plant cell cultures,e.g. model tobacco BY-2 cell culture. On the contrary, trans-formed cells were mostly arranged in clusters of differentsize (Fig. 4B,D) as we also documented earlier for BY-2[26]; and in some cases cell chains of doublets were ob-served in the cultures (Fig. 4B, in a slit). To strengthen thereliability of observed phenomena we also evaluated Spcdc25

Page 4: Tobacco cells transformed with the fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application

de

c

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B

controltransformant Atransformant Ctransformant F

Fig. 1. Relative growth rates of tobacco cell suspension cultures on media with or without cytokinin addition. The evaluation of relative growth rates (dry weight

mass at sampling time (t) related to calculated inoculum dry weight (0); (dry masst � dry mass0)/dry mass0 of control and transgenic lines A, C and F was per-

formed 14 days (the end of exponential growth phase) after the transfer to the fresh medium (modified liquid Heller medium (1 mg l�1 2,4-D and 1 mg l�1 NAA)

without cytokinin (A) or supplied with 0.1, 1 or 2 mg l�1 BAP (B)). Significant differences between variants are indicated by different letters (n ¼ 6e20;

P < 0.05).

676 P. Suchomelova-Maskova et al. / Plant Physiology and Biochemistry 46 (2008) 673e684

tobacco internode segment-derived calli cultivated on solidmedia as well as cell suspension cultures cultivated on rollerenabling reduction of mechanical strains caused by mixing,necessary for aeration of cultures, and detected that similarcell characteristics prevailed (data not shown). The idea tomake use of synchronised cell cultures proved unfeasible be-cause of cluster-arrangement of transgenic cells preventingefficient synchronisation agent removal.

3.2. Starch and sugar content

When evaluating morphological cell characteristics (onthe14th day after subcultivation) we observed plenty of col-ourless bodies in the cytosol of Spcdc25 transgenic cells.None or only few such bodies of small size were found inWT cells. Histochemical tests with Lugol solution provedthem to be starch granules. The representative photographsof stained cells are documented in the Fig. 5A, B and D.All detected bodies in transgenic cells are stainable by kaliumiodide while in the WT cells low number of small light browncoloured starch grains were found. Although the results ofhistochemical detection are rather persuasive we supple-mented the observations with a more precise biochemical de-termination of starch levels quantified by HPLC. The amountof glucose obtained after enzymatic hydrolysis with a-amy-lase and amyloglucosidase is presented in Fig. 6. As is clearlyvisible, on the14th day after the transfer to the fresh medium,there were significantly higher or even manifold higher

glucose levels in transgenic lines samples compared to theWT ones.

To understand the carbohydrate context of changed starchlevels we also compared the soluble carbohydrate levels, bothin WTas well as in transgenic cells. The same trend as for starchwas observed as regards sugar levels. The total soluble carbohy-drate amounts were slightly or significantly higher in studiedtransgenic lines in contrast to WT cells (Fig. 7A). The most re-markable difference was found in sucrose:hexoses ratio. In thetransgenic cells the sucrose prevailed while WT cells containedmainly hexoses.

3.3. The effect of cytokinin treatment

Some of the above-described changes in transgenic cultures(morphological shifts as well as changes in saccharide levels)are referred to be achieved by exogenous cytokinin treatment.Besides these, we published earlier [41] that, in the system ofde novo regeneration on internode stem segments, Spcdc25expression induces a cytokinin-like shift in the organogenicresponse. Hence, we wondered whether the application ofcommonly used stable cytokinin (6-benzylaminopurine(BAP)) could simulate the effect of Spcdc25 transformationin the WT cell cultures and what would be the effects of thecytokinin treatment in transgenic cell lines. As the tobaccocell cultures used in this study were independent of exogenouscytokinin, the possible effect of cytokinin treatment seemed tobe quite easily interpretable.

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a

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controltransformant Atransformant Ctransformant F

Fig. 2. Cell area in tobacco cell suspension cultures on media without cytoki-

nin. Tobacco cells of control and transgenic lines A, C and F cultivated in

modified liquid Heller media (1 mg l�1 2,4-D and 1 mg l�1 NAA). The anal-

ysis was performed 14 days after transfer to the fresh medium. The maximum

size of mitotic cells was quantified as cell area using image analysis Lucia G.

Significant differences between variants are indicated by different letters

(n ¼ 90e110; P < 0.05).

677P. Suchomelova-Maskova et al. / Plant Physiology and Biochemistry 46 (2008) 673e684

3.3.1. Growth rates and cell characteristicsThe treatment of cultures with exogenous cytokinin led to

different responses in WT and transgenics. While WT cellson the medium with 1 mg l�1 BAP showed growth similar totransformed cells cultivated on cytokinin-free medium(Fig. 1B), in transformants a more or less dramatic drop ofgrowth was registered. Cytokinin supply to transformed cellseven at very low concentrations (0.1 mg l�1) caused mostlya rapid decrease of biomass accumulation (Fig. 1B) and expo-sure of Spcdc25 transgenic cultures to higher cytokinin con-centration (1 mg l�1) led to an even more pronounced dropin the growth. In wild-type cell cultures a twofold increasein cytokinin concentration (2 mg l�1) led to only moderatechanges in growth rates (Fig. 1B) but a further increase in cy-tokinin application into the medium to 5 and 10 mg l�1 did notpromote growth, and actually inhibited it (data not shown).

It is often stressed that cytokinin levels have a close rela-tionship with auxin levels. For BY-2 cells it was publishedthat the cultures in medium where auxin was omitted showedsimilar characteristics as those after cytokinin application [21].

Therefore, to change the endogenous auxin:cytokinin bal-ance we transferred the cultures into a growth regulator-freemedium (omitting both cytokinin and auxin). The differencebetween the responses was dramatic. Compared to the WT cul-tures (reaching 47% of auxin supported culture, Fig. 1A) the

drop in growth rates in the transgenic line was much more pro-nounced (5%). Cell characteristics were also at the centre of ourinterest. By exposure of cells to 1 mg l�1 BAP for 14 days, WTcells failed to form regular chains, divided chaotically and in-stead, they formed clumps of variable size composed of morecircular cells (Fig. 4C) similar to those of Spcdc25 transformedcells cultivated without BAP. Sometimes cell chains of doubletswere observed to be similar to transgenic line A. Transformedcells in BAP-containing medium, on the contrary, establishedgiant clusters of scarcely viable cells.

3.3.2. Starch and sugar contentsSimilarly, to complete the characterisation of cultures under

cytokinin treatment, we measured the starch and soluble sugarscontents. The BAP application to wild-type cells again causedsimilar changes to those induced by the Spcdc25 transformation.In BAP-treated WT cells the amount and size of starch grainsincreased (Fig. 5C), and the rise in starch levels was alsoconfirmed by biochemical determination of starch-originatingglucose levels (Fig. 6). Simultaneously we proved higher totalsoluble carbohydrate levels in exogenous cytokinin influencedWT cells together with sucrose:hexoses ratio shifted tosucrose, regardless of the BAP concentrations (1 mg l�1 or2 mg l�1 BAP) (Fig. 7B).

3.4. Endogenous cytokinin levels

The results of a cytokinin-like effect of Spcdc25 transforma-tion led us to check the endogenous cytokinin contents in trans-genics to clarify whether actual changes in cytokinin levels orother regulatory mechanisms are responsible for the observedphenomena. For cytokinin determination, the cell cultures ofWT and transformant C on the 12th day of subcultivationwere used, similarly as for cell characteristics and growth mea-surements. Although the total cytokinin levels are relativelylow in both variants (Fig. 8A), it is clearly apparent that thelevels are significantly lower in transgenics, reaching onlyone third of the WT cell levels. The graph presenting levelsof individual cytokinins shows that the vast majority of cytoki-nins (over 90%) are cis-zeatin derivatives. In the samples therewere comparable levels of trans-zeatin, slightly elevated levelsof dihydrozeatin-type cytokinins in transformant C and slightlyelevated levels of isopentenyladenine-type cytokinins in WT.Moreover, the levels of phosphates of all three types of cytoki-nins were also higher in WT (Fig. 8B).

4. Discussion

4.1. The cell cultures of all fission yeast Spcdc25transformed tobacco lines under study exhibited higherdry mass accumulation during the exponential phase incomparison to the wild type (WT)

There is a wealth of evidence that any change in cell cyclegene expression can cause a shift in cell cycle progression (seeSection 1), consequently altering the rate of cell division. Inyeast, the cdc25 mitotic activator overexpression accelerates

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a

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controltransformant Atransformant Ctransformant F

A B

controltransformant Atransformant Ctransformant F

Fig. 3. Cell shape in tobacco cell suspension cultures on media without cytokinin. Tobacco cells of control and transgenic lines A, C and F were cultivated in

modified liquid Heller media (1 mg l�1 2,4-D and 1 mg l�1 NAA). The analysis was performed 14 days after the transfer to the fresh medium. The shape of mitotic

cells was quantified as parameter of circularity (A) and elongation (B) using image analysis Lucia G. Significant differences between variants are indicated by

different letters (n ¼ 90e110; P < 0.05).

678 P. Suchomelova-Maskova et al. / Plant Physiology and Biochemistry 46 (2008) 673e684

the entry of cells into mitosis [31]. Thus, in plant cell culturesexpression of Spcdc25 under strong promoter might be mani-fested as the acceleration of the cell cycle conducive smallercell size phenotype and/or the shortening of G2 phase withcompensatory prolongation of the G1 phase. In fission yeast,cdc25 overexpression led to a decrease in mitotic cell lengththrough G2 shortening, nevertheless the overall cell cycle du-ration was similar to WT [31]. Orchard et al. [26], however,found out in BY-2 cells expressing Spcdc25 that the G2 phasewas shortened, the mitotic cell size reduced and although therewas a compensatory prolongation of the G1 phase, the cell cy-cle duration was still reduced. The observed enhanced drymass accumulation in the Spcdc25 cultures under study(Fig. 1A) is then in accordance with assumed cell divisionacceleration.

4.2. Transformed cells exhibited a clustered, morecircular cell phenotype as compared to elongated tochain-like arranged WT cells

Further, we focused on cell size and shape determination toconfirm the hypothesis that these characteristics are altered asa result of Spcdc25 expression. Given that Spcdc25 expressionleads to G2 phase shortening, the premature entry into mitosiscould cause a chaotic organisation of cell division. This as-sumption was proven by the observed cell culture phenotype

exhibiting low organisation (eventual appearance of cellchains of doublets) (Fig. 4A,B,D). The finding is fully inaccordance with the results obtained by Orchard et al. [26]in Spcdc25 transformed BY-2 cells where sister filamentswere documented. In addition we observed a significant alter-ation in cell shape that was quantified by measurements ofcircularity and elongation parameters, also arguing for acceler-ation of the entry into mitosis and differently organised cellplane building (Fig. 3A,B). This corresponds well with thedata on fission yeast wee1� mutants that formed very short,almost round cells [35].

On the contrary, we repeatedly did not detect smaller cells intransgenic cell cultures (Fig. 2) as Orchard et al. [26] describedfor BY-2 Spcdc25 cultures. One possible explanation for thisdiscrepancy could be that G1 phase compensatory prolongationis deepened so that the overall cell cycle duration is similar toWT as in the case of fission yeast cdc25 overexpression [31] orother mechanisms regulating cell size at mitosis function in ourplant material. Interestingly, Sorrell et al. [36] indicated thatdifferent results for D-cyclin expression profile in BY-2 cellscompared to other plant species could be caused by specificproperties of this culture, set by its longevity and ‘‘immortal-ity’’ related to changed phenotype/genotype. This suggestionled us to speculate that the results achieved with the model sys-tem, as BY-2 is, could in some cases differ from those obtainedwith other tobacco cell cultures/lines.

Page 7: Tobacco cells transformed with the fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application

Fig. 4. Cell arrangement in tobacco cell suspension cultures. Tobacco cells of control and transgenic lines A and C were cultivated in modified liquid Heller media

(1 mg l�1 2,4-D and 1 mg l�1 NAA) without cytokinin (A, B and D) or control supplied with 1 mg l�1 BAP (C). The analysis was performed 14 days after the

transfer to the fresh medium. The arrangements of cells are documented as microphotographs under UV light in combination with Nomarski differential contrast.

In the slit, an alternative cell arrangement of transgenic line A occasionally forming chains of doublets. Bar ¼ 100 mm.

Fig. 5. Histochemical detection of starch in tobacco cell suspension cultures. Tobacco cells of control and transgenic lines A and C were cultivated in modified

liquid Heller media (1 mg l�1 2,4-D and 1 mg l�1 NAA) without cytokinin (A, B and D) or control supplied with 1 mg l�1 BAP (C). The analysis was performed

14 days after the transfer to the fresh medium. The starch was visualised by Lugol solution in native preparations. Bar ¼ 100 mm.

Page 8: Tobacco cells transformed with the fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application

a

ab

b

c

0

10

20

30

40

50

60

70

non-treated 1mg.l-1 BAP

gluc

ose

cont

ent

[µg/

mg

dry

wei

ght]

controltransformant Atransformant C

Fig. 6. Starch content in tobacco cell suspension cultures. Tobacco cells of

control and transgenic lines A and C were cultivated in modified liquid Heller

media (1 mg l�1 2,4-D and 1 mg l�1 NAA) without cytokinin or supplied with

1 mg l�1 BAP. The analysis was performed 14 days after the transfer to the

fresh medium. The amount of starch was quantified as glucose content ob-

tained after enzymatic splitting of starch with a-amylase and amyloglucosi-

dase. Significant differences between variants are indicated by different

letters (n ¼ 6; P < 0.05).

680 P. Suchomelova-Maskova et al. / Plant Physiology and Biochemistry 46 (2008) 673e684

4.3. Cytokinin application to WT cells induced similarchanges in growth and morphological parameters tothose induced by the Spcdc25 transformation

Changes in cell morphology resembling those found inSpcdc25 transformants have been shown to be inducible withcytokinin application. Petrasek [27] found out that applicationof 4.5 mM 6-benzylaminopurine (BAP) to the cytokinin-independent tobacco culture VBI-0 changed the filamentousphenotype to a spherical one. This phenomenon strengthenedwith proceeding subcultures. The effect has been ascribed toindirect impact of BAP on the cytoskeleton, especially microtu-bules [32]. Based on our results in the context of contemporarymodels of cytokinin-induced regulation of G2/M transition,another explanation of observed phenomena in VBI-0 cells isat our disposal. BAP application to tobacco cultures could drivethe cells faster into mitosis, which results in disorganised build-ing of the cell division plane. Numerous data show a closerelationship of cell cycle machinery to the cytoskeleton. Ithas been shown that cyclin-dependent kinase A (CDKA) co-localises with mitotic structures, e.g. preprophase band(PPB), anaphase spindle and phragmoplast [48]. Microinjectionof purified active CDK/cyclin B complexes to stamen hair ofTradescantia accelerated prophase progression and induced rapiddestabilisation of the PPB [14]. Moreover, the presence of non-degradable mitotic cyclin B1 in tobacco led to reorganisation ofmicrotubules in phragmoplast as well as failure in organising

cortical microtubules, resulting in an isodiametric shape ofepidermal cells [50]. Ectopic expression of cyclin B2 in alfalfa,besides acceleration of the entry into mitosis, shortened thetime-window of the appearance of the PPB, an important factorfor the orientation of cell division [49].

Hence, the application of exogenous cytokinin (commonlyused 6-benzylaminopurine (BAP)) was tested. The aim was toverify the idea that the Spcdc25-expression-caused changeswere of cytokinin-inducible nature in WT cells under study.The results validated the assumption as the WT cells aftercytokinin treatments failed to form chains and were insteadarranged in clusters or chains of cell doublets (Fig. 4C).

4.4. After BAP application, Spcdc25 transformed cellsshowed inhibition of growth and establishment of giantclusters of cells with markedly reduced viability

Providing that the influence of Spcdc25 expression andcytokinin application have an additive nature, we could expectthat cytokinin treatment would further deepen Spcdc25-induced morphology characteristics. To summarise the resultsfrom experiments with cytokinin application, we state that anycytokinin supply that is stimulatory for WT cultures, the trans-genic cells sense as inhibitory (presumably like ‘‘cytokininoverdose’’) (Fig. 1B). The effect of auxin depletion from themedium can be interpreted similarly.

4.5. Are the results discussed above in harmony with thecontemporary model of the regulation at G2/M transitionin plants?

The question is difficult to answer as (1) the introduced geneis not of plant origin, and in particular, (2) a consensus has notbeen reached as regards the phosphorylation/dephosphoryla-tion regulation of plant CDK activity and especially phospha-tase responsible for CDK activation at G2/M. CDKs in higherplants are most probably, similarly as in other eukaryotes, neg-atively controlled by phosphorylation at G2 because tyrosinephosphorylation has been detected unambiguously undercytokinin deprivation, osmotic stress or DNA damage [34,53]and shown to participate in the control of cell/organ size [10].

Further, orthologues of the Wee1 kinase have been de-scribed [11,38,47]. In maize and Arabidopsis, Wee1 kinasecan phosphorylate CDKA in vitro [33,42]. In tomato, impair-ing the expression of Wee1 kinase results in reduction in plantand fruit size which was on a molecular level correlated withincrease of CDKA activity originating from a decrease of theamount of Tyr 15 phosphorylated CDKA [10].

Recently, the first plant cdc25 orthologue has been identifiedin unicellular alga Ostreococcus tauri [16]. It encodes a protein,which can complement the fission yeast cdc25 mutant. Soonafter, in a higher plant (Arabidopsis), a small isoform of tyro-sine phosphatase was identified [17,37]. The sequence includesa catalytic domain of cdc25-like protein; nevertheless, it seemsto function as a heterodimer whose regulatory subunit has notbeen revealed so far. However, cdc25 expression was not en-hanced in rapidly dividing compared with non-proliferative

Page 9: Tobacco cells transformed with the fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application

a

ab

b

0

20

40

60

80

100

120

140

control transformant A transformant C

[µg/

mg

dry

wei

ght]

fructoseglucosesucrose

A

abb

0

20

40

60

80

100

120

140

1 mg.l-1 BAP 2 mg.l-1 BAP

control

B

Fig. 7. Non-structural soluble carbohydrate levels in tobacco cell suspension cultures. Tobacco cells of control and transgenic lines A and C were cultivated in

modified liquid Heller media (1 mg l�1 2,4-D and 1 mg l�1 NAA) without cytokinin (A) or supplied with 1 or 2 mg l�1 BAP (B). The analysis was performed

14 days after the transfer to the fresh medium. Significant differences between variants are indicated by different letters (n ¼ 9e10; P < 0.05).

681P. Suchomelova-Maskova et al. / Plant Physiology and Biochemistry 46 (2008) 673e684

Arabidopsis tissues [37]. Failure to fully identify the planthomologue of cdc25 led recently [3] to the suggestion that tyro-sine dephosphorylation in plants might be achieved by a phos-phatase unrelated to Cdc25. Another interesting hypothesispresented in the same article even proposes that the Cdc25-con-trolled onset of mitosis might have been evolutionarily replacedby a B-type CDK-dominated pathway, eventually resulting in

Fig. 8. Cytokinin levels in tobacco cell suspension cultures on media without cytoki

C was performed 12 days after the transfer to the fresh media (modified liquid He

dogenous cytokinins and (B) average amount of individual cytokinins. Limit of q

ratios of 10:1 (LOQ 5.0 fmol for tZ, cZ, BAP, BAPR; LOQ 2.5 fmol for tZR, c

dHZROG, dHZRMP, iP, iPR, iPRMP). Significant differences between variants ar

the loss of the cdc25 gene [3]. This model features CDKB phos-phorylating Arabidopsis CDK inhibitor ICK2/KRP2, whichthen dissociates from CDKA, enabling the latter to drive cellsinto mitosis. Nevertheless, it is necessary to keep in mind thatbefore mitosis, CDKA undergoes activatory dephosphorylationand inhibition of this action (e.g. by application of lovastatin,an inhibitor of isoprenoid-type cytokinin synthesis) causes

nin. The analysis of endogenous cytokinin levels of control and transgenic line

ller media with 1 mg l�1 2,4-D and 1 mg l�1 NAA). (A) Total amount of en-

uantifications for individual cytokinins were determined from signal-to-noise

ZR, cZ9G, cZOG, cZROG, cZRMP; LOQ 1 fmol for dHZ, dHZR, dHZOG,

e indicated by different letters (n ¼ 5; P < 0.01).

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682 P. Suchomelova-Maskova et al. / Plant Physiology and Biochemistry 46 (2008) 673e684

an arrest of the cells in G2 phase (see e.g. [18]). This argument,together with the knowledge of Sorrell et al. [39] and Orchardet al. [26] who showed that the G2/M specific CDKB is mostlikely phosphoregulated at the same evolutionarily conservedamino acid residues as CDKA, strongly supports the importantrole of CDK activatory dephosphorylation in plants.

Further, the local expression of the Spcdc25 gene in to-bacco leaves resulted in modulation of cell division patterns[52]. Spcdc25 expression induces a small cell size in theroot meristems of tobacco plants [19] and in tobacco BY-2cells [26], and shortens G2 phase of the BY-2 cell cyclethrough a premature peak of CDKB-kinase activity andbypasses a cytokinin requirement at G2/M [26]. Hence, the re-sults indicate that Spcdc25 functions as a mitotic inducer in theplant cell cycle and recognises G2/M CDK(s) as substrates.Moreover, Sorrell et al. [37] reported on Arabidopsis cdc25 in-ducing a short cell length when expressed in fission yeast.

Cdc25 plant homologue or protein, fulfilling in plants thesame role, is proposed to be activated by cytokinins (e.g.[53]). In the event we accept this concept, it is easy to under-stand that Spcdc25 expression induced changes in cell divisionmanifested by altered morphological characteristics thatmimic the cytokinin application. Moreover, our previous re-sults demonstrated a cytokinin-like effect of fission yeastSpcdc25 expression on de novo organ formation in tobaccostem segments manifested by earlier and more abundant shootformation and restricted root induction [41]. Taken together,our results achieved either on organ or on cellular level areconsistent with the model of cell cycle control proposing thecytokinin regulation of CDK activatory dephosphorylationthrough so far unknown protein action.

4.6. Transgenic cultures showed changes incarbohydrate accumulation; increased starch andsoluble sugar levels with higher sucrose proportion inthe sugar spectrum

When the cell characteristics were followed we noticed theenhanced presence of small objects in transformed cells thatby histochemical tests proved to be starch bodies(Fig. 5A,B,D). The biochemical quantification supported thedifferences between starch amounts of control and transgenics(Fig. 6) and motivated us to compare the levels of endogenoussoluble carbohydrates as the carbohydrate metabolism func-tions as a network of negative and positive feedback signallingpathways precisely regulating the levels of individual carbohy-drates, in particular the balance between soluble carbohydratesand starch levels (e.g. [22]). The observed change in sucrose:-hexoses ratio (Fig. 7A) further pointed to the carbohydratemetabolism disturbance resulting from Spcdc25 expression.

4.7. BAP application to WT cells caused a shiftin carbohydrate levels towards those found inSpcdc25 transformants

It is well known that there exists a mutual relationship be-tween cytokinin levels and the carbohydrate status of a plant

[4,7,9]. For cytokinin-independent BY-2 cell cultures it hasbeen published that cytokinin application as well as the absenceof auxin in the medium results in amyloplast accumulation[20,21]. Cytokinin application enhanced the expression ofplasma membrane hexose uptake carriers in Chenopodiumrubrum cell suspension cultures, thus improving carbohydratesupply to the cells [1]. Therefore, the increase in starch deposi-tion as well as higher soluble carbohydrate levels after cytoki-nin application (Figs. 5C, 6 and 7B) seems to be in accordancewith literature data. In contrast, the question arises how toexplain the similar changes induced by Spcdc25 expression.

4.8. The Spcdc25 expression resulted in a decrease ofendogenous cytokinin levels

When discussing the results on growth and cellular charac-teristics presented here as well as those on de novo organformation [41], we pointed to the fact that in all cases weobserved a cytokinin-like effect of Spcdc25 expression. Webased our explanation on the idea of Spcdc25 acting as a mi-totic inducer simulating cytokinin activation of plant cdc25-like phosphatase, and thus having an impact on cell-division-dependent processes resembling cytokinin application. AsSpcdc25 operates downstream of the cytokinin action, we pro-posed that constitutive Spcdc25 expression could be sensed bythe plant as high cytokinin availability and in consequencea compensatory decrease in cytokinin levels could be expected[41]. This assumption was later supported by the results ob-tained with Spcdc25 transformed BY-2 cells [26]. However,when we searched for the explanation of morphologicalchanges together with the observed changes in carbohydratemetabolism, it appeared that the question of Spcdc25 influenceon the cytokinin levels in plants had to be reconsidered. Never-theless, the measurement of cytokinin levels revealed a markeddecrease in total cytokinins resulting from cdc25 expression(Fig. 8A). The transformants had significantly lower levels ofcis-zeatin derivatives. Minor elevation of dihydrozeatin-type cy-tokinins in transformant was fully compensated by elevated con-tent of isopentenyladenine-type cytokinins in WT (Fig. 8B).Thus, the results verified the original hypothesis [41].

4.9. If there is no increase in cytokinin levels, how do weexplain cytokinin-like changes in carbohydratemetabolism induced by Spcdc25 expression?

It seems that other mechanisms must operate that are respon-sible for Spcdc25-induced changes as regards carbohydratestatus besides those proposed for changes in cell morphology.It is now well documented that sugar levels contribute to the de-cision of the cell to continue in the cell cycle (e.g. via positivemodulation of D cyclin(s) levels, and the negative regulation ofinhibitor levels, e.g. [12,28,30]). We propose that the relation-ship between carbohydrate metabolism and cell cycle is notonly unidirectional, i.e. sugar level is a component of a complexsystem controlling cell cycle progression, but instead therelationship is bidirectional. That means that also the decisionto continue in the cell cycle and especially to enter mitosis

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generates a signal aimed to prepare the cell for subsequenthighly C and energy demanding cell division and therefore, be-sides others, it changes carbohydrate status. Hence, we proposethat a higher proportion of active CDK, resulting from Spcdc25expression, emanates the signal to metabolic pathways includ-ing the carbohydrate metabolism. The question remainswhether some of the enzymes/proteins/compounds, involvedin regulation of carbohydrate balance, could be the directsubstrate of CDK or whether the control is more indirect. Tosupport this hypothesis the detailed study of downstreamCDK phosphorylation pathway would be necessary.

Some encouragement for this idea has arrived only recentlyfrom an analysis of cell cycle-related protein complexes inA. thaliana [46] presenting, besides others, interactions be-tween CDKA;1 and components of carbohydrate metabolism(Rubisco subunit binding-protein, and especially with cyto-plasmic phosphoglucomutase). In addition, Geelen et al. [8]referred to an interaction between trehalose-6-P-synthase pro-tein and CDKA. In both cases, the data are interpreted as themechanism of how carbohydrate status modifies cell cycleprogress. In connection with our results we propose that itcan be interpreted in the opposite sense, i.e. that the CDKA in-teraction generates a signal modulating carbohydrate metabo-lism in response to cell cycle progression. The unique studyof CDK substrates in yeast showed that there exist proteinsinvolved in saccharide biosynthesis and transport that are regu-lated by CDK activity [45]. So it is tempting to speculatewhether some of the published data on the influence of cytoki-nins on carbohydrate metabolism do not refer to cytokininactions mediated by some components of cell cycle regulation.

4.10. Conclusion

Although a lot of work is needed to answer all the arisingquestions precisely, we would like to propose some possibleregulatory pathways indicated by the results achieved withSpcdc25 transformants. The constitutive expression ofSpcdc25 probably leads to a higher proportion of active mi-totic CDK compared to the wild type. Consequently, signallingpathways downstream from CDK are more active as well, andinfluence the processes directly dependent on cell division.This status might be sensed by the cell or whole plant as in-creased cytokinin availability, which results in a compensatorydecrease in cytokinin levels. In addition we propose that en-hanced activity of CDK(s) at G2/M might generate a signalmodifying carbohydrate metabolism to support the require-ments of forthcoming cell division. Our results bring more in-sight to the plant cell cycle regulation and pose new questionsabout complexity and interconnection of regulatory networksbetween the cell cycle and carbohydrate metabolism in plants,that need further complex investigations.

Acknowledgements

The authors wish to thank to Ivana Macha�ckova, RadomıraVankova and Tomas Masek for fruitful discussions and PetraAmakorova for skilful technical assistance during cytokinin

analysis. This work was supported by the Ministry of Educa-tion, Youth and Sports of the Czech Republic (MSM0021620858 and MSM 6198959216) and by the Grant Agencyof Charles University (GAUK 207/2005).

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