Cytokinin-binding protein (70 kDa): localization in tissues and cells of etiolated maize seedlings...

Journal of Experimental Botany, Vol. 58, No. 10, pp. 2479–2490, 2007

doi:10.1093/jxb/erm108 Advance Access publication 21 June, 2007

RESEARCH PAPER

Cytokinin-binding protein (70 kDa): localization in tissuesand cells of etiolated maize seedlings and itsputative function

Fedor A. Brovko1, Victoria S. Vasil’eva1, Anna O. Shepelyakovskaya1, Svetlana Yu. Selivankina2,

Guzel R. Kudoyarova3, Alexander V. Nosov2, Dmitry A. Moshkov4, Alexander G. Laman1,

Khanafy M. Boziev1, Victor V. Kusnetsov2 and Olga N. Kulaeva2,*

1 Pushchino Branch of Ovchinnikov–Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences,pr. Nauki 6, Pushchino, Moscow region, 142290 Russia2 Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, Moscow,127276 Russia3 Institute of Biology, Ufa Research Center, Russian Academy of Sciences, pr. Oktyabrya 69, Ufa, 450054 Russia4 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, pr. Nauki 3, Pushchino,Moscow region, 142290 Russia

Received 22 December 2006; Revised 12 April 2007; Accepted 24 April 2007

Abstract

The distribution pattern of a 70 kDa cytokinin-binding

protein (CBP70) was studied in 4-d-old etiolated maize

seedlings (Zea mays L., cv. Elbrus). CBP70 was

detected in crude protein extracts of all root zones

and shoot parts by western blotting and by the

sandwich ELISA (enzyme-linked immunosorbent as-

say) technique, using a pair of monoclonal anti-CBP70

antibodies cross-reacting with non-overlapping protein

epitopes. The highest amount of CBP70 was found in

the root meristem, which corresponds to the concen-

tration in the meristem of zeatin, its riboside, nucleo-

tide, and 9N-glucoside. CBP70 accumulation was also

detected in other zones of cell division: in the root cap,

shoot apex, and vascular tissues, suggesting involve-

ment of the protein in the processes related to cell

proliferation. This suggestion was also supported by

CBP70 distribution in the root meristem: mitotically

inactive cells of the quiescent centre did not contain

a detectable amount of the protein. Stem cells adjoin-

ing the quiescent centre contained less CBP70 than

their daughter cells. Using monoclonal antibodies

against CBP70 for immunocytochemistry, the

presence of the protein in the cytoplasm and its

accumulation in nuclei and especially in nucleoli

was demonstrated; such a pattern was observed in all

cell types of seedlings. The subcellular distribution

pattern of CBP70 was analysed by immunogold

electron microscopy of the meristem and leaf cells;

CBP70 was localized in the cytoplasm and nucleo-

plasm, and its highest concentration was detected in

nucleoli. CBP70 was not detected in the vacuole and

cell wall. In the RNA polymerase I model system,

purified CBP70 mediated a trans-zeatin-dependent

activation of transcription in vitro, and anti-CBP70

monoclonal antibodies blocked this activation. Other

natural and synthetic physiologically active cytokinins

also activated transcript elongation in the model

system in the presence of CBP70. Adenine and in-

active analogues of cytokinins had no such effects.

These data suggest that CBP70 is a transcript elonga-

tion factor or a modulator of elongation factor activity

specifically mediating a cytokinin-dependent regula-

tion of transcription.

Key words: Cytokinin, cytokinin-binding protein, regulation of

transcript elongation, Zea mays L.

* To whom correspondence should be addressed: E-mail: [email protected]: BCIP, 3-bromo-4-chloro-5-indolyl phosphate; BSA, bovine serum albumin; CBP, cytokinin-binding protein; ELISA, enzyme-linkedimmunosorbent assay; mAb, monoclonal antibody; 2-ME, 2-mercaptoethanol; NBT, nitro blue tetrazolium; PMSF, phenylmethylsulphonyl fluoride; QC,quiescent centre; tZ, trans-zeatin.

ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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Introduction

Cytokinin was discovered as a factor inducing, togetherwith auxin, cell division in cell culture (Miller et al.,1955). Later, many other cytokinin activities in theregulation of plant growth and development were demon-strated (Mok and Mok, 2001). Recently, cytokinin in-volvement in the regulation of shoot and root meristemactivity was shown (Werner et al., 2003; Higuchi et al.,2004; Nishimura et al., 2004). Considerable progress inunderstanding the cytokinin mode of action was achieveddue to the discovery of membrane-bound cytokininreceptors (CRE1/AHK4/WOL, AHK2, and AHK3), whichbelong to the class of sensor histidine kinases of the two-component regulatory system (Inoue et al., 2001; Ueguchiat al., 2001), identification of the genes of the primaryresponse to cytokinin (ARR type-A genes) (Brandstatterand Kieber, 1998; Sakakibara et al., 1998; D’Agostinoet al., 2000), and transfactors of ARR type-B controllingexpression of the ARR type-A genes (Sakai et al., 2001).These discoveries led to the concept of a phosphatecascade in cytokinin signalling (Schmulling, 2001;Ferreira and Kieber, 2005; Sakakibara, 2006). However,more recently, analysis of triple Arabidopsis thalianamutants displaying damage to all three receptor histidinekinases suggested that cytokinin signalling might be morecomplex and involve other perception and transductionsystems (Higuchi et al., 2004; Nishimura et al., 2004).To this end, a search for other players in cytokinin signal

transduction in plant cells is of interest. Sensor histidinekinases are known to recognize cytokinin by its extracellu-lar CHASE domain and, therefore, are likely to be involvedin the perception of extracellular cytokinin. However,cytokinins can also penetrate into plant cells using purinetransporters (Burkle et al., 2003) and probably nucleosidetransporters (Hirose et al., 2005). In fact, cytokinins havebeen detected in different compartments of the plant cell,including the cytoplasm, nucleus, and chloroplasts (Hareand Van Staden, 1997; Benkova et al., 1999; Dewitte et al.,1999). This implies the occurrence of intracellular cyto-kinin targets or receptors. The presence of both membraneand intracellular (nuclear) receptors was found for auxin(Badescu and Napier, 2006). A number of receptors havenow been discovered for abscisic acid (Razem et al., 2006;Shen et al., 2006). Therefore, it is possible that cytokinins,along with membrane receptors, also interact with receptorsof other types. Cytokinin-binding proteins (CBPs), if linkedwith the control of transcription, are likely to be intra-cellular cytokinin targets. Such proteins with a molecularmass of 67 kDa (CBP67) were isolated from the cytosoland nuclei of mature barley leaves (Kulaeva et al., 2000)and senescent leaves of A. thaliana (Selivankina et al.,2004). The complex of these proteins with trans-zeatin (tZ)activated transcript elongation in test systems containingchromatin and RNA polymerase I, or nuclei isolated

from the leaves served as a source for CBP isolation(Selivankina et al., 2001, 2004). The correlation between cyto-kinin binding to CBP67 and their activities, in complex withthe protein, in transcription regulation (Kulaeva et al.,1998; Selivankina et al., 2001) suggested that CBP67scould be related to cytokinin intracellular perception andits signal transduction to the processes participating incontrol of transcription (Selivankina et al., 2004).A protein with a molecular mass of 70 kDa (CBP70)

was also isolated from etiolated maize seedlings. Thisprotein binds cytokinin with a high specificity and affinity(Brovko et al., 1996; Shepelyakovskaya et al., 2002) andshares immunodeterminants with CBP67 from barleyleaves (Kulaeva et al., 1998). Up to now, there is noevidence of the localization of such types of CBPs in planttissues and plant cells. A set of monoclonal mouse anti-bodies (mAbs) raised against maize CBP70 (Zagranichnayaet al., 1997) and the selection of a pair of them cross-reacting with non-overlapping epitopes of CBP70(Shepelyakovskaya et al., 2002) made it possible to analysethe protein in plant material by a very sensitive sandwichELISA (enzyme-linked immunosorbent assay) techniqueusing streptavidin–biotin detection. Immunocytochemistryand immunogold electron microscopy make it possible toanalyse CBP70 localization in seedlings.The aims of the present study were: (i) to elucidate the

distribution of CBP70 in etiolated maize seedlings and tocompare the distribution pattern of CBP70 with that ofendogenous cytokinins; (ii) to analyse CBP70 localizationin seedling cells; and (iii) to examine CBP70 activity inthe cytokinin-dependent regulation of transcription in vitroto extend the studies on CBP function.

Materials and methods

Plant materials and treatments

Maize (Z. mays L., cv. Elbrus) seeds were surface-sterilized andgerminated on moistened filter paper at 27 �C and 95% humidity indarkness. Four-day-old seedlings were sampled for immunocytol-ogy and CBP70 and cytokinin analyses. Barley (Hordeum vulgareL., cv. Viner) plants were grown in boxes with soil in a growthchamber at a 16 h photoperiod, 22/18 �C day/night temperature,222 lmol m�2 s�1 light intensity, and 70% relative humidity. Thefully expanded first leaves of 10-d-old plants were used forchromatin isolation.

CBP70 isolation

All steps of protein isolation were performed at 0–4 �C. A 100 galiquot of etiolated 5-d-old maize seedling shoots was homogenizedfor 10 s at 20 000 rpm with the Ultraturax T25 homogenizer (Ika,Germany) in buffer A (50 mM TRIS-HCl, pH 7.5, 2 mM MgCl2,2 mM EDTA-Na2, 50 mM KCl), containing 0.5 mM phenyl-methylsulphonyl fluoride (PMSF) and 5 mM 2-mercaptoethanol(2-ME). In preliminary experiments, it was shown that theseconditions for homogenization resulted in the highest CBP70 yield.The homogenate was filtered through Miracloth and centrifuged at10 000 g for 15 min. The protein fraction of the supernatant was

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purified by gel filtration chromatography on a column (5360 cm) ofSephadex G-25 Fine (Pharmacia, Sweden) equilibrated with bufferA. The eluted proteins were loaded on the DEAE-Sepharose column(1.6320 cm, Pharmacia, Sweden) equilibrated with buffer A. Thecolumn was washed with five bed volumes of buffer A, and CBP70was eluted with buffer A containing 200 mM KCl. Thereafter, theprotein was loaded on an adenosine Toyopearl (A-Toyopearl)column (1.6310 cm) attached to a trans-zeatin riboside Toyopearl(tZR-Toyopearl) column (2.6320 cm). Both columns were equili-brated with buffer A. Adenosine and tZR were immobilized onaminopropyl Toyopearl using a modified procedure of Erlanger andBeiser (1964). The A-Toyopearl column was used for removing theadenine-binding proteins and other unspecifically binding proteins.After sample application, the columns were washed with two bedvolumes of buffer A, the A-Toyopearl column was detached, thetZR-Toyopearl column was additionally washed with three bedvolumes of buffer A containing 500 mM KCl, and CBP70 waseluted with 6 M guanidine-HCl. Protein fractions were collected, andconcentrated using an 8MC Concentrator Cell with 30YM membrane(Amicon, USA). Finally, the protein solution was desalted on a PD10column equilibrated with buffer A and stored at –70 �C.

Preparation of crude protein extracts from plant tissues

Primary roots were sectioned as follows: division zone plus root cap(from 0 mm to 2 mm from the root tip), the elongation zone (from2 mm to 7 mm), the segment of the differentiation zone (from7 mm to 17 mm), and a 10 mm segment from the middle part of thelateral root formation zone. The mesocotyl, node, coleoptile, and thefirst leaf were excised from the shoot. Plant material (50–250 mg)was placed in the centrifuge tube, five volumes of A buffercontaining 1 mM PMSF and 8 mM 2-ME were added, and planttissue was homogenized by Ultraturax T25 at 20 000 rpm. Inpreliminary experiments, the optimum time for homogenization ofevery tissue was determined: 2–3 s for root tips, 4 s for thecoleoptiles, mesocotyls, and leaves, and 6 s for the lateral rootformation zones. The homogenate was centrifuged at 48 000 g and4 �C for 10 min. The supernatant was separated from low-molecular-weight compounds using Sephadex G-25 Fine mini-columns equilibrated with buffer A. Protein solutions were used forCBP70 analysis by immunoblotting and sandwich ELISA.

Electrophoresis and immunoblotting

Proteins from the crude extracts of plant tissues were analysed bySDS-PAGE (12% w/v) according to the method of Laemmli (1970).The samples of crude extracts loaded onto the gel were equalized inthe amounts of CBP70 (estimated by sandwich ELISA).Separated proteins were transferred to Hybond-C extra (Amersham,

UK) in a semi-dry electro blotter (Biometra, UK) according to themanufacturer’s recommendations using the Towbin buffer system(Towbin et al., 1979). Protein bands were visualized with 0.5%Ponceau S solution in 1 M acetic acid. The membrane was rinsedthree times with water and blocked with 1% bovine serum albumin(BSA) in PBS-T [phosphate-buffered saline containing 0.1% (v/v)Tween-20] overnight at 4 �C. Anti-CBP70 mouse mAb (100ng ml�1) in PBS-T was added for 1 h at room temperature. Anti-CBP70 mouse antibody was detected using secondary antibodyconjugated to alkaline phosphatase (Bio-Rad Laboratories, USA)according to the supplier’s recommendations. The enzyme colourreaction was developed with 3-bromo-4-chloro-5-indolyl phosphate(BCIP) and nitro blue tetrazolium (NBT).

Sandwich ELISA

The content of CBP70 in the crude extracts from the plant materialwas determined by sandwich ELISA using the mAbs Z-5 and Z-8

(Shepelyakovskaya et al., 2002). The mAb Z-8 was biotinylatedusing biotin N-hydroxysuccinimide ester (Harlow and Lane, 1988).The mAb Z-5 was dissolved in carbonate–bicarbonate buffer(100 mM NaHCO3/Na2CO3, pH 9.6, 10 lg ml�1) and incubated inmicrotitre 96-well IFA Multiwell plates overnight at 4 �C. Wellswere washed twice with PBS [20 mM Na2HPO4/NaH2PO4 and0.15 mM NaCl, pH 7.2, containing 1% (w/v) BSA] and incubatedat 37 �C for 1 h with the same solution. Serial 2-fold dilutions ofthe crude extracts were added to the corresponding wells andincubated overnight at 4 �C. The medium was then removed, andthe wells were washed five times with PBS-T. The biotinylatedmAb Z-8 (10 lg ml�1) was dissolved in PBS-T, added to each well,incubated for 2 h at room temperature, and wells were washed fivetimes with PBS-T. The streptavidin–peroxidase conjugate (Amersham,UK) (diluted 1:1000 in PBS-T) was then added and left to react for1 h at 37 �C, then the wells were washed four times with PBS-Tand twice with PBS. A 0.1 ml aliquot of the substrate solution[0.43 mg ml�1 o-phenylenediamine, 0.01% (v/v) H2O2, 0.02 Mcitric acid, and 0.05 mM Na2HPO4, pH 5.0] was added to each welland incubated for 20 min at 24 �C; the reaction was stopped with0.1 M H2SO4, and the plates were read with Titertek Multiscan(Flow Lab, UK) at 492 nm.

Immunocytochemistry

Root tips 2 mm long, nodes with 1 mm of adjacent organs(mesocotyl and leaves with coleoptile), and the parts of hypocotyland coleoptile 1 mm long of 4-d-old etiolated maize seedlings werecut off and fixed immediately in 4% (w/v) formaldehyde, 0.1%(w/v) glutaraldehyde, and 0.01% (w/v) picric acid in 20 mMsodium phosphate buffer (pH 7.4) for 8–12 h at 4 �C. Afterwashing in distilled water, the samples were dehydrated in gradedethanol (to 100%) and embedded in LR White (Sigma, USA).Polymerization was carried out under UV light for 12 h. Sectionsfrom embedded samples were cut with an Ultracut E ultramicro-tome (Reichert-Jung, Austria). For light microscopy, semi-thinsections (1–0.7 lm) were placed on glass slides coated with c-aminopropyltriethoxysilane. For electron microscopic observations,ultrathin sections (gold) were mounted onto Au grids.Prior to immunolabelling, sections were permeabilized with 0.5%

(v/v) Triton X-100 and incubated in 0.1% (w/v) NaBH4 toinactivate residual aldehydes. The sections were incubated in theblocking solution of BSA-G [3% (w/v) BSA and 0.5% (w/v) gelatinein PBS] for 1.5 h at 37 �C and then incubated at 4 �C overnightwith the primary mAbs against CBP70 (17 lg ml�1) diluted in theBSA-T solution [1% (w/v) BSA and 0.01% (v/v) Triton X-100 inPBS]. Slides were washed in the W solution [0.02% (w/v) NaN3,0.1% (v/v) Triton X-100, and 0.1 M glycine in PBS] for 10 minand incubated with the BSA-G solution for 20 min. Then thesections were incubated with biotinylated anti-mouse antibodies(Amersham, UK) in BSA-T for 6 h at 4 �C and washed in the Wsolution for 10 min. The reaction with the streptavidin–alkalinephosphatase conjugate (Amersham) in BSA-T was performed for1.5 h at 37 �C, slides were washed in the W solution for 10 min,then incubations with biotinylated anti-mouse antibodies andstreptavidin–phosphatase conjugate (for 1.5 h at 37 �C each) wererepeated. Sections were incubated with the substrate solution(3.8 mM BCIP, 4 mM NBT, 10 mM MgCl2, 150 mM NaCl,50 mM TRIS-HCl, pH 9.5) for 15 min. The reaction was stoppedby washing in 20 mM TRIS-HCl, pH 7.5. The stained dry sectionswere mounted in cedar wood oil, and images were obtained undera Univar microscope (Reichert-Jung, Austria).Three negative controls were used: (i) mouse anti-CBP70 mAbs

were omitted from material treatment; (ii) mouse anti-CBP70 mAbswere substituted by mouse mAbs raised against a protein which isabsent in plants (anti-Myc antibodies); and (iii) mouse anti-CBP70

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mAbs were substituted by mouse non-immune serum (5% mouseserum diluted by PBS). An equal low unspecific staining level wasdetected for all three controls under the chosen conditions ofimmunolabelling.

Immunolabelling procedure for electron microscopy

As mentioned above, ultrathin sections (gold) for electron micros-copy were mounted onto Au grids. Grids with sections of the rootmeristem and leaf tissues of 4-d-old etiolated maize seedlings wereincubated in a moisture chamber; most of the procedures werecarried out at room temperature. Initially, specimens were incubatedwith a drop of freshly prepared 1% (w/v) aqueous solution ofNaBH4 for 30 min, and then they were rinsed with distilled water(three times 5 min each). Unspecific antibody binding was blockedby incubation of specimens in the 5% solution of BSA (w/v) inPBST-500 (16.7 mM Na2HPO4, 3.3 mM KH2PO4, 500 mM NaCl,and 0.1% Tween-20, pH 7.4) for 20 min. The following reactionswere carried out using the same buffer; after each incubation,specimens were washed in PBST-500 with 50 mM glycine and0.5% Tween-20. Specimens were incubated with primary anti-CBP70 antibodies for 1 h at 37 �C. Control sections were incubatedwith non-immune serum. Then sections were incubated with thesolution of biotinylated anti-mouse antibodies (Amersham) (ata dilution of 1:100) for 1 h at room temperature, followed byincubation in the solution of streptavidin–gold (15 nm) (Amersham)(at a dilution of 1:100) under the same conditions. The labelledsections were post-stained in uranyl acetate and Reynold’s leadcitrate before examination with a Tesla TS-500 electron microscope(Czech Republic) (Vandenbosch, 1991).

Cytokinin determination

Cytokinins were extracted from plant tissues, purified, and separatedby thin-layer chromatography (TLC) as described earlier (Vysotskayaet al., 2001). Cytokinin-containing zones (based on standardpositions) were eluted with 0.1 M phosphate buffer, pH 7.4, for12 h, and the eluates were added directly to microtitre plate wells inserial dilutions. They were assayed using antibodies against zeatinriboside, which has been shown to be highly specific for severalzeatin derivatives (Kudoyarova et al., 1998). By this technique,zeatin nucleotide (RF 0–0.1), zeatin riboside (RF 0.4–0.5), and zeatin(RF 0.6–0.7) were successfully separated and assayed. More than90% recovery was obtained for zeatin, and its riboside and glucosidestandards using the described elution procedure. Recovery of zeatinnucleotide was not measured directly. However, the sum ofimmunoreactivity of the eluted fractions was not more than 20%lower than that of samples applied to the TLC plate, suggesting thatmost of the immunoreactive material was successfully recovered.

Chromatin isolation and measuring the RNA

polymerase I activity

Chromatin was isolated from barley leaves according to the protocoldescribed previously (Selivankina et al., 2004). Transcriptionelongation directed by chromatin-associated RNA polymerase Iwas measured in the reaction medium (100 ll) containing 50 mMTRIS-HCl, pH 8.0, 10 mM MgCl2, 10 mM 2-ME, 0.2 mM CTP,GTP, ATP, 0.01 mM UTP, and [3H]UTP (1.11 TBq mmol�1)(Amersham Biosciences, UK), and 10–50 lg of chromatin DNA.The reaction was carried out at 32 �C for 20 min and stopped bythe addition of an equal volume of cold 0.9% sodium pyrophos-phate in 10% trichloroacetic acid.

Protein assay

Protein content was estimated by the method of Esen (1978).

Determination of the number of cells

Plant material was macerated in 10% H2Cr2O7 at 60 �C for 20 min.The number of cells was counted in a Fuchs–Rosenthal haemo-cytometer as described by Brown and Rickless (1949).

Statistics

All experiments were performed three times with three replicationseach. Figures and tables present the mean values and their standarderrors (SE).

Results

CBP70 localization in 4-d-old maize seedlings

The distribution of CBP70 in 4-d-old etiolated maizeseedlings was analysed. For this purpose, crude proteinextracts were prepared from various root zones and shootorgans, and analysed by western blotting using an mAbagainst CBP70. The loaded samples were equalized in theamounts of CBP70 on the basis of sandwich ELISAresults. The CBP70 was detected in all root zones studied:the meristem, elongation, differentiation, and lateral rootformation zones (Fig. 1). In shoots, CBP70 was detectedin the mesocotyl, the node containing the shoot apex,coleoptile, and first leaf (Fig. 1). Crude protein extracts fromall these tissues and organs were analysed to quantify theCBP70 by using a sandwich ELISA. The results obtainedshowed that the highest content of CBP70 was in the rootmeristem (Fig. 2A, B, D) where it exceeded the content inother tissues and organs by 10–250 times (Fig. 2A). Theroot elongation zone contained 10 times less CBP70 thanthe meristem, but significantly more than the zones of rootcell differentiation and lateral root formation (Fig. 2A).The lowest level of CBP70 was in mesocotyl. In shoots,the highest amount of CBP70 was in the node containingdividing cells of shoot meristem and leaflets. Thus, the

Fig. 1. Western immunoblot detection of CBP70 in crude proteinextracts from various parts of 4-d-old etiolated maize seedlings. Crudeprotein extracts were analysed by SDS-PAGE. The samples loadedwere equalized in the amounts of CBP70 on the basis of sandwichELISA data. Blots were probed with mouse anti-CBP70 mAb, and theantibody was detected using the secondary anti-mouse antibodyconjugated with alkaline phosphatase. Lane 1, root meristem; lane 2,root elongation zone; lane 3, root differentiation zone; lane 4, zone oflateral root formation; lane 5, first leaf; lane 6, node; lane 7, coleoptile;lane 8, mesocotyl; lane 9, molecular weight standards.

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highest amount of CBP70 in roots and shoots was in thezones of cell division.Since the sizes of cells from various tissues are very

different, it was important to calculate the content ofCBP70 not only per fresh weight (FW) basis, but also percell. This was achieved by counting the number of thecells per 1 g FW (Table 1) and calculating the relativeamount of CBP70 per cell in various tissues (Fig. 2B).The total protein was also measured in various parts ofseedlings, and the accumulation of CBP70 and totalprotein in cells of these tissues were compared (Fig. 2B, C).The average cell in the elongation zone was 8-fold

larger and contained three times more total protein than anaverage cell in the meristem, but the content of CPP70 inthe cells was half that in the meristem cells (Fig. 2B), indi-cating partial degradation of CBP70 during cell elongation.The amount of CBP70 in various parts of seedlings per

milligram of protein was also calculated. The dataobtained (Fig. 2D, E) suggested that CBP70 constituteda considerably larger fraction of total protein in meris-tematic cells than in other root tissues. It also revealeda significant CBP70 accumulation in the node, whichcontained dividing cells of the shoot apex and youngleaflets (Fig. 2E). No correlation was observed betweenthe content of total protein and CBP70 in different parts ofthe seedling (cf. Fig. 2B, C). Thus, CBP70 was pre-dominantly located in the root and shoot zones containingdividing cells, particularly in the root meristem.

Distribution of endogenous cytokinins in seedlingroot zones

Analysis of endogenous cytokinins in the root zonesdemonstrated that the meristem contained much more ofall cytokinin forms than other root zones (Fig. 3). Thecontent of zeatin, a physiologically active form ofcytokinin, decreased in the elongation zone more sharplythan that of other cytokinin forms. At the same time,cytokinin content in the elongation zone was much higher

Fig. 2. Relative contents of CBP70 and total protein in various parts of 4-d-old-etiolated maize seedling. CBP70 content in crude protein extractswas estimated by sandwich ELISA using a pair of anti-CBP70 mAbs. Biotinylated mAbs were recognized by a streptavidin–peroxidase conjugate.Peroxidase reaction was recorded at 492 nm. Mean values 6SE are presented. 1, Root meristem and root cap (0–2 mm from the root tip); 2,elongation zone (2–7 mm from the root tip); 3, 10 mm segment of the differentiation zone (7–17 mm from the root tip); 4, 10 mm segment from themiddle part of the lateral root formation zone; 5, mesocotyl; 6, node; 7, coleoptile; 8, first leaf. (A) Relative content of CBP70 on a fresh weight basis(A492 g

�1 FW). (B) Relative content of CBP70 on a cell basis (A492 per 106 cells). (C) Total protein content on a cell basis (mg per 106 cells). (D)

Relative content of CBP70 per total protein unit (A492 mg�1 total protein). (E: insert) Enlarged fragment of D.

Table 1. The number of cells in various tissues of 4-d-oldetiolated maize seedlings

The root meristem, the elongation zone, a 10 mm long segment of thedifferentiation zone, and a 10 mm segment from the middle part of thelateral root formation zone were excised from primary roots of 4-d-oldseedlings. Mesocotyls, nodes, coleoptiles, and first leaves were excisedfrom seedlings. Three independent experiments were carried out withfive replications each. Mean values 6SE are presented.

Root zone and shoot organ No. of cells3106

g�1 FW

Root meristem 129.168.4Root elongation zone 15.962.6Root differentiation zone 9.860.9Zone of lateral root formation 12.060.4Mesocotyl 9.861.1Node 8.061.9Coleoptile 14.261.8Leaf 243.765.3



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than in the zones of differentiation and lateral rootformation, i.e. a progressive decrease in all cytokinin formswas observed from the meristem to other root zones. Thesedata correspond fully to a CBP70 distribution pattern in theroot zones of etiolated maize seedlings.

CBP70 localization within the cells of theetiolated maize seedlings

CBP70 localization within the cells of the 4-d-oldetiolated maize seedlings was examined immunocytochem-ically, using a mouse mAb against CBP70 (Zagranichnayaet al., 1997). Localization of the antibody was detectedwith anti-mouse biotinylated antibody and streptavidinconjugated with alkaline phosphatase. CBP70 was notdetected on control longitudinal sections of the rootmeristem, which were not treated with mAb againstCBP70 (Fig. 4B, F). Root cells treated, instead of withmAb against CBP70, with non-immune serum (Fig. 4G)or with mouse mAbs raised against a protein which isabsent in plants (anti-Myc antibody, Fig. 4H), were alsonot stained. These data indicate a high specificity ofCBP70 detection using anti-CBP70 antibodies.The highest CBP70 concentration was observed in the

nuclei of the meristem cells (Fig. 4A, C, D, blackarrowheads) and especially in their nucleoli (Fig. 4C, D,black double arrowhead). CBP70 was also present in thecytoplasm of meristematic cells (Fig. 4C, D, whitearrowhead). CBP70 was unevenly distributed within themeristem. The cells of the quiescent centre (QC) con-tained much less CBP70 than meristematic cells (Fig. 4A,C). Stem cells adjoining the QC, which are characterizedby a slow division rate (Laux, 2003), contained lessCBP70 in both the cytoplasm and nuclei than their rapidlyproliferating daughter cells (Fig. 4A, C). A high contentof CBP70 was found in the nuclei of root cap cells (Fig.4A, rc). A longitudinal section of the root primary cortex

in the region of provascular tissue development demon-strates a high CBP70 concentration in the cytoplasm ofthe protoderm and epidermis cells (Fig. 4D, whitearrowhead). In the cells of the elongation zone, CBP70was detected in nuclei (Fig. 4E, black arrowhead) andespecially in nucleoli (Fig. 4E, black double arrowhead).CBP70 was also discerned in the cytoplasmic strands inelongating cells (Fig. 4E, black arrow). However, the totalcontent of CBP70 in the cells of the elongation zone wasconsiderably lower than in the meristem cells (Fig. 4E).

Fig. 3. Cytokinin content in roots of 4-d-old etiolated maize seedlings.Root zones: 1, meristem; 2, elongation zone; 3, zone of differentiation;4, zone of lateral root formation. Mean values 6SE (n ¼ 9).

Fig. 4. Immunolocalization of CBP70 on longitudinal sections of theroot tip of etiolated maize seedlings. LR White-embedded tips (0–2mm) of the main root from dark-grown 4-d-old maize seedlings wereused. (A, C–E) Sections were incubated with mouse anti-CBP70 mAbs,biotinylated anti-mouse antibodies, and streptavidin–alkaline phospha-tase conjugate without counterstaining. (B, F) A negative control:sections incubated without primary antibody. Root cells treated withnon-immune serum (G) or with mouse monoclonal antibodies raisedagainst a protein which is absent in plants (anti-Myc antibody; H),instead of with mAb against CBP70, were also not stained. (A) CBP70was detected in the nuclei (black arrowhead) and the cytoplasm ofmeristematic cells and in nuclei of the root cap (rc) cells. (C) A 3-foldmagnified view of the lower part of (A). Nuclei and cytoplasm of thequiescent centre (QC) cells contain much less CBP70, as compared withmeristematic cells. CBP70 accumulated in nuclei (black arrowhead),with the highest concentration in nucleoli (black double arrowhead). (D)The root cortex in the region of provascular tissue development. Thehighest accumulation of CBP70 was detected in nuclei (blackarrowhead), mainly in nucleoli (black double arrowhead). The CBP70concentration in the cytoplasm is especially high in proepidermal andepidermal cells (white arrowhead). (E) Root central cylinder, elongationzone. CBP70 is localized in nuclei, mainly in nucleoli (black doublearrowhead) and in cytoplasmic strands (black arrow) of elongating cells.Scale bars are 75 mm (A, B) and 25 mm (C–H).

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These data confirmed the results of biochemical experi-ments (Fig. 2) and emphasized that the CBP70 concentra-tion was significantly higher in zones of cell division.Transverse sections of mesocotyl, node, and coleoptile

of 4-d-old etiolated maize seedlings demonstrated CBP70localized in shoot cells (Fig. 5). Control sections, whichwere not treated with mAb against CBP70, were notstained (Fig. 5A, F, G). Also not stained were controlsections treated, instead of with mAb against CBP70, withmouse non-immune serum or with mouse antibodiesagainst a protein which is absent from plants (anti-Mycantibody) (data not shown). These three negative controlsdemonstrated that CBP70 detection in shoot cells wasspecific. In the mesocotyl, characterized by the lowestcontent of CBP70 (Fig. 2), this protein was detectedmainly in the cells of the bundle sheath (Fig. 5B). CBP70was also present in large parenchymal cells (Fig. 5C). Inthese tissues, as well as in the cells of the coleoptileparenchyma (Fig. 5H) and vascular tissue (Fig. 5I), cellwalls/peripheral cytoplasm were strongly stained and itwas impossible to discriminate whether CBP70 waspresent in the cell walls or in the peripheral cytoplasm.However, immunogold electron microscopy clearly dem-onstrated that CBP70 was absent in the cell wall butabundant in the cytoplasm adjoining the cell wall. Thissuggests a similar CBP70 distribution in the cellspresented in Fig. 5. CBP70 was much more abundant inthe node (Fig. 5D, E) than in the mesocotyl and coleoptile(Fig. 5B, C, H, I). The highest CBP70 concentration wasobserved in the shoot apex and actively dividing leaf cells(Fig. 5D, white double arrowheads). An enlarged frag-ment of the stem apex section demonstrates a predominantCBP70 localization in the nuclei (Fig. 5E, black arrow-heads) and especially in the nucleoli (Fig. 5E, blackdouble arrowhead). The protein was also present in thecytoplasmic strands of growing cells (Fig. 5E, blackarrow). Again, the highest CBP70 content was observedin the sites of cell division, where it occurred in both thenucleus and cytoplasm (Fig. 5E, white arrowheads). In thelarge parenchymal cells of the mesocotyl and coleoptile,CBP70 was detected in the nuclei pressed by the vacuoleagainst the cell wall (Fig. 5B, C, H, I, black arrowheads).Because there was insufficient resolution, it was difficultto be sure that stained structures are really nuclei.However, when tissue sections were treated with safraninO according the method described by Sylvester and Ruzin(1994), these CBP70-containing structures were stainedbright red, which is characteristic of nuclei (data notshown). This evidence proves that these structures arereally nuclei. The coleoptile vascular tissue containedmuch more CBP70 than the parenchyma (Fig. 5I).Thus, the results of the immunocytochemical analysis of

CBP70 localization in shoot organs presented in Fig. 5 arecompletely consistent with the results of biochemicalanalyses considered in the previous sections.

Fig. 5. Immunolocalization of CBP70 on cross-sections of mesocotyl(A–C), node (D–F), and coleoptile (G–I) of etiolated maize seedlings.LR White-embedded tissues from dark-grown 4-d-old maize seedlings.(B–E, H, I) Sections were incubated with mouse anti-CBP70 mAbs,biotinylated anti-mouse antibodies, and streptavidin–alkaline phospha-tase conjugate. (A, F, G) A negative control: sections incubated withoutprimary antibody. (B) Vascular bundle of the mesocotyl. CBP70 is seenin nuclei (black arrowhead) of phloem cells. (C) Parenchyma of themesocotyl. CBP70 is localized in the nuclei (black arrowheads), mainlyin nucleoli of parenchymal cells. (D) Stem apex region of the node. Thehighest accumulation of CBP70 is detected in actively dividing cells ofthe leaf blade edges (white double arrowheads). (E) Detail of (D): stemapex. The cytoplasm and nuclei of small cells at the sites of celldivision are well stained (white arrowheads). In largely vacuolatedparenchyma cells, nuclei (black arrowhead), especially nucleoli, andcytoplasmic strands (black arrow) are stained. (H) Cross-section of thecoleoptile parenchyma. CBP70 is localized in nuclei adjacent to the cellwall (black arrowheads) and in nucleoli. (I) Cross-section of thecoleoptile vascular tissue. CBP70 is localized in nuclei (black arrow-heads), mainly in nucleoli (black double arrowhead). Scale bars are50 mm (A–C, G–I), 200 mm (D), and 25 mm (E, F).



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Immunogold electron microscopic detection of CBP70in cells of the root meristem and leaves of 4-d-oldetiolated maize seedlings

Subcellular localization of CBP70 and the ultrastructure ofcells of the root meristem and leaves of 4-d-old etiolatedmaize seedlings were studied by immunogold electronmicroscopy. Ultrathin sections prepared from the rootmeristem and leaves were successively treated with mousemAb against CBP70 (or with non-immune serum ascontrol), biotinylated anti-mouse antibody, and streptavi-din–gold. The labelled sections were post-stained inuranyl acetate and Reynold’s lead citrate. Figure 6illustrates a representative image of the root meristem(Fig. 6A, C) and leaf cells (Fig. 6B, D). Figure 6C and Ddemonstrates control sections treated with mouse non-immune serum instead of anti-CBP70 antibody. Goldparticles indicate CBP70 localization in the cells (Fig.6A, B). The highest CBP70 concentration was found inthe nucleolus (Fig. 6A, B, black arrowhead). CBP70 wasalso present in the nucleoplasm (Fig. 6A, B, whitearrowhead). The protein concentration in the nucleoplasmwas one half of that in the nucleolus (Table 2). Theprotein distribution in the nucleoplasm was not uniform,demonstrating the regions with predominant CBP70localization. CBP70 was also well defined in the cyto-plasm (Fig. 6A, B). Its concentration in the cytoplasm wassubstantially lower than in the nucleoplasm and nucleoli(Fig. 6A, B; Table 2). The protein was practically absentfrom the vacuole and cell wall (Fig. 6A, B). Thus,electron microscopy using the immunogold techniquerevealed CBP70 localization in the cytoplasm, nucleo-plasm, and nucleolus, with predominant accumulation ofthe protein in the nucleolus, which corresponds to thecytochemistry data (Figs 4, 5).

CBP70 from etiolated maize seedlings mediatescytokinin-dependent activation of transcript elongationin the model system

The high concentration of CBP70 in nuclei and especiallyin nucleoli of all seedling cells suggested that CBP70could be involved in the control of transcription. To verifythis assumption, a system of transcript elongation in vitrodirected by RNA polymerase I, which is tightly associatedwith the chromatin isolated from barley leaves, was used.RNA polymerase II was lost during chromatin purification(Selivankina et al., 2004). Previously, this system wasused to demonstrate CBP67 (isolated from barley leaves)involvement in the cytokinin-dependent transcriptionactivation. CBP70 was purified from etiolated maizeseedlings and used for the transcription assay (Fig. 7).In the presence of tZ, CBP70 activated elongation oftranscripts in vitro by RNA polymerase I (Fig. 7). CBP70or tZ alone had no effects on transcription, indicatingthat CBP70 mediated a cytokinin-dependent regulation

of transcript elongation. A specific inhibitor of RNApolymerase II, a-amanitin, had no significant effect ontranscription (Fig. 7); thus, under the experimentalconditions used, transcript elongation was mediatedby RNA polymerase I only. The mAb raised againstCBP70 blocked the protein activity in transcription regu-lation (Fig. 7). Taken together, the data suggest that thepresence of functionally active CBP70 is essential for thecytokinin control of rRNA synthesis. CBP70 activatedtranscription elongation not only in complex with tZ,but also in complex with other physiologically activenatural and synthetic cytokinins: isopentenyladenine, 6-benzylaminopurine, and kinetin (Table 3). In contrastto this, inactive analogues of cytokinin and its glucosidehad no such effects (Table 3). Therefore, CBP70 in-teraction with cytokinins is very specific and results ineffective complex formation only with functionally activecytokinins.

Discussion

Predominant localization of CBP70 in the zones of celldivision of etiolated maize seedlings, its accumulationin nuclei with enrichment in nucleoli

The use of ELISA and western blotting (Shepelyakovskayaet al., 2002) allowed demonstration of the CBP70 dis-tribution pattern in etiolated maize seedlings. CBP70 wasdetected in all seedling parts and tissues (Figs 1, 2),suggesting its vital importance in plant life.It is the first information on localization in the whole

plant of the protein that belongs to the family of CBPs

Fig. 6. Subcellular localization of CBP70. Ultrathin sections obtainedfrom the root meristem (A, C) and leaf cells (B, D) of 4-d-old etiolatedmaize seedlings were treated with mouse anti-CBP mAbs (A, B) or withnon-immune serum (C, D), biotinylated anti-mouse antibodies, andstreptavidin–gold (15 nm). CBP70 is localized in the cytoplasm (blackarrows), nucleoplasm (white arrowheads), and nucleolus (black arrow-heads). N, nucleus; n, nucleolus; m, mitochondria; c, cytoplasm; a,amyloplast; v, vacuole; w, cell wall. Scale bars are 1 lm.

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mediating a cytokinin-dependent regulation of transcriptelongation. It was done for CBP70 as the first example ofthis type of proteins.Predominant CBP70 localization was established in the

zones of cell division of etiolated maize seedlings,especially in the root meristem, where the CBP70concentration was significantly higher than in other plantorgans and tissues (Fig. 2A, B, D). Predominant localiza-tion of CBP70 in the zones of active cell proliferationsuggested its involvement in the processes related to celldivision. CBP70 is distributed in the root meristem non-uniformly. Cells in different regions of a meristem havedistinct fates (Laux, 2003). Mitotically inactive cells ofthe QC did not contain a detectable amount of CBP70(Fig. 4A, C). Divisions of stem cells adjoining the QC areless frequent than divisions of their daughter cells (Laux,2003). Corresponding to their lower mitotic activity, stemcells contained less CBP70 than daughter cells (Fig. 4A,

C). The data on CBP70 distribution in the root meristemgive an additional and a very strong argument for therelationship of the protein to cell division. Activelydividing cells of the root cap (Esau, 1977) were alsocharacterized by a high content of CBP70 in their nuclei(Fig. 4A). It is especially interesting in the context ofthe data published by Aloni et al. (2005) demonstratingthe highest content of active endogenous cytokinins in theroot cap of A. thaliana. This mysterious accumulation ofCBP70 and cytokinin in the root cap remains to beunderstood. The present data on cytokinin localization inroot zones (Fig. 3) were also correlated with thedistribution pattern of CBP70 (Fig. 2), supporting the ideathat CBP70 is involved in the cytokinin-dependentregulation of cell division.In the shoot, as well as in the root, CBP70 was localized

mainly in the zone of cell divisions. In the mesocotyl andcoleoptile, the zones of cell division are represented bydeveloping vascular tissues (Fig. 5B, I), whose proportionis low relative to extended parenchymal cells (Fig. 5C, H).This explains why a low amount of CBP70 was found inthese parts of seedlings by biochemical methods (Fig. 2).A high content of CBP70 in the node detected bybiochemical methods (Fig. 2) is consistent with cytologi-cal data indicating CBP70 accumulation in the sites of celldivision in the shoot apex and leaflets (Fig. 5D). Thuscytochemical data (Figs 4, 5) are in full agreement withbiochemical results (Fig. 2) revealing CBP70 accumula-tion in the zones of cell proliferation. CBP70 was alsopresent in the root elongation zone, although in consider-ably lower amounts than in the meristem (Figs 2A, B, D,4E). At the same time, the content of CBP70 in this zonewas higher than in many other analysed tissues, and it wasdecreased further in the zones of differentiation and lateralroot formation (Fig. 2A). These results correspond to the

Table 2. Gold particle distribution in the root meristem andleaf cells of 4-d-old etiolated maize seedlings (per lm2)

Part of seedling mAb againstCBP70

Cytoplasm Nucleoplasm Nucleolus

Root meristem + 5.060.5 11.065.0 21.560.8Root meristem – 0.5060.08 0.5160.09 0.3560.06Leaf + 6.060.4 10.064.0 21.061.0Leaf – 0.5360.10 0.3960.08 0.2060.06

Fig. 7. Cytokinin-dependent activation of transcript elongation in vitroby CBP70 from etiolated maize seedlings. The effects of CBP andtrans-zeatin (tZ) on RNA synthesis were studied in the transcriptionelongation system containing RNA polymerase I associated withchromatin isolated from barley leaves. The effects of the followingsubstances added to the reaction medium (100 ll) were studied:CBP70 (20 lg), mAb (20 lg) raised against CBP70, AM, a-amanitin(4 lg ml�1), and tZ (10�7 M),

Table 3. Effects of CBP70 from etiolated maize seedlings,natural and synthetic cytokinins, and their inactive analogueson RNA synthesis in vitro in the transcription elongation systemcontaining chromatin-bound RNA polymerase I from maturebarley leaves

Compound Concentration(M)

[3H]UMPincorporationinto RNA

cpm per 20lg of DNA

%

Control (water) – 10 7316397 100Trans-zeatin 10�7 31 92061309 297Isopentenyladenine 10�7 25 14261055 2346-Benzylaminopurine 10�7 28 93161128 270Kinetin 10�7 22 2616 957 207Adenine 10�7–10�5 No effect –Trans-zeatin-O-glucoside

10�7–10�5 No effect –

6-Benzylthiopurine 10�7–10�5 No effect –



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pattern of active cytokinin distribution in the root zones ofA. thaliana (Aloni et al., 2005) and to the data onisopentenyltransferase gene expression in the roots of A.thaliana (Takei et al., 2004).Cytochemical analysis using the mAb against CBP70

showed the presence of this protein in the cytoplasm ofmeristematic cells and its accumulation in nuclei andespecially in nucleoli (Fig. 4A, C, D). Nuclear localizationof CBP70 in meristematic cells corresponded to thepresence of zeatin in nuclei of the cells of the vegetativeshoot apex detected in tobacco plants by Dewitte et al.(1999). CBP70 was clearly revealed not only in the cellsfrom dividing zones but also in the nuclei and nucleoli ofnon-dividing cells, for instance cells of the root elongationzone and mature parenchymal cells of the mesocotyl andcoleoptile (Figs 4E, 5C, H). Cytochemical data on CBP70accumulation in the nucleolus were confirmed by electronmicroscopy with the use of immunogold labelling (Fig. 6;Table 3). The highest concentration of gold particles in theroot meristem and leaf cells was found in the nucleoli,where it was 3–4 times higher than in the cytoplasm andtwice as high as in the nucleoplasm (Table 3). CBP70was not detected in the vacuole and cell wall. As far aswe know, this is the first evidence of such a type of CBPlocalization in plant cells with predominant accumulationin nucleoli.The data on CBP70 accumulation in nuclei and nucleoli

of cells in all seedling tissues suggest the involvement ofthe protein in the control of transcription.

CBP70-mediated cytokinin-dependent activation oftranscript elongation in vitro

The suggestion of the involvement of CBP70 in transcrip-tion regulation was confirmed in experiments on transcriptelongation in vitro in a model system containing RNApolymerase I tightly associated with chromatin isolatedfrom barley leaves (Selivankina et al., 2004). In thissystem, new transcripts are not initiated; the system onlymaintains the elongation of transcripts initiated in vivo.Previously, using this system, a cytokinin-dependentactivation of transcript elongation in vitro by barleyCBP67 was demonstrated (Selivankina et al., 2001). Inthe present study, it was shown that maize CBP70 alsoactivated transcript elongation by RNA polymerase I inthe presence of tZ (Fig. 7).The inhibition of this effect by mAbs against CBP70

emphasized the requirement for the functionally activeprotein for the cytokinin-dependent regulation of tran-scription (Fig. 7). Other natural and synthetic physiolog-ically active cytokinins replaced tZ in formation ofa complex with CBP70, which enhanced transcriptelongation in the model system (Table 3), whereasadenine, an analogue of cytokinins but lacking theirhormonal activity, and other inactive analogues ofcytokinin had no such effects (Table 3). Hence, CBP70

interaction with cytokinins in the regulation of transcriptelongation is very specific and is achieved only bybioactive cytokinins. As mentioned above, transcriptelongation in the model system in the present experimentswas directed by RNA polymerase I synthesizing rRNAprecursors. This process is localized in nucleoli. Immuno-cytochemistry (Figs 4, 5) and immunogold electronmicroscopy (Fig. 6) demonstrated that CBP70 accumu-lated in seedling cells in nucleoli. Co-localization ofCBP70 and RNA polymerase I in nucleoli, together withresults of the in vitro experiments are very important newarguments for the protein’s involvement in the cytokinin-dependent regulation of rRNA precursor synthesis. Thisconclusion is in agreement with cytokinin-induced activa-tion of rRNA synthesis in vivo, which was establishedpreviously (Gaudino and Pikaard, 1997). Thus, the resultsobtained in the model system used in the present experi-ments adequately reflect the effect of the cytokinin in thecell. All these data suggest that maize CBP70 belongs tothe class of transcript elongation factors or proteinscontrolling elongation factor activity.The present results demonstrated CBP70 activity only in

cytokinin-dependent regulation of transcript elongation byRNA polymerase I. There are no data on the effect ofCBP70 on RNA polymerase II, which was lost duringchromatin purification (Selivankina et al., 2004), butbarley CBP67 (related to CBP70) activated transcriptelongation directed not only by RNA polymerase I, butalso by RNA polymerase II in nuclei isolated from barleyleaves (Selivankina et al., 2001). These results areconsistent with the discovery of the common mechanismcontrolling transcript elongation in yeast by RNA poly-merase II and RNA polymerase I (Fath et al., 2004). Thepresence of CBP70 in maize seedling cells not only innucleoli but also in the nucleoplasm (Figs 4C, D, E, 6)demonstrated the importance of studying further theparticipation of this protein in the regulation of RNApolymerase II activity.

Possible involvement of CBP70 in the cytokinincontrol of cell proliferation

The significance of cytokinins for cell division is wellknown (Miller et al., 1955; Mok and Mok, 2001).Cytokinins participate in the regulation of the expressionof D-cyclins and other genes involved in the cell cycle(Frank and Schmulling, 1999; Riou-Khamlichi et al.,1999; Fountain et al., 2003; Hartig and Beck, 2005).As discussed above, a predominant localization of

CBP70 in the root meristem and other zones of celldivision suggests that the protein could be involved incytokinin control of cell proliferation. At the same time, itis known that receptor histidine kinases CRE1/AHK4/WOL, AHK2, and AHK3 participate in the regulation ofcell division by cytokinin (Inoue et al., 2001; Ueguchiet al., 2001; Higuchi et al., 2004; Nishimura et al., 2004).

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The functions of the three AHKs partially overlap, but allof them are necessary for the proper response of culturedcells to cytokinin. All three AHKs are required for themaintenance of cell division in both stem and rootmeristems (Higuchi et al., 2004; Nishimura et al., 2004).The analysis of b-glucuronidase (GUS) gene expressionunder the control of AHK promoters in the roots oftransformed A. thaliana plants demonstrated that thegenes of all three AHKs were expressed in the rootmeristem (Nishimura et al., 2004), but CBP70 localizationcorresponded more accurately to the zones of cell division(Fig. 4A, C) than GUS gene expression under the controlof AHK promoters. In intact A. thaliana plants, triplemutants displaying damage to all three AHKs hadseverely reduced meristems of the shoot and root, whichindicates a suppression of cell division and the systemsmaintaining cells in the state of division and retardingtheir transit to growth and differentiation. Nevertheless,some reduced mitotic activity was maintained in triplemutants. They develop embryos, seedlings, and dwarfplantlets. Based on these facts, it was concluded that themechanism of cytokinin action may involve, together withthe receptor histidine kinases and phosphate cascade,some other unknown system of hormonal signal percep-tion and transduction (Higuchi et al., 2004; Nishimuraet al., 2004). In this connection, maize CBP70, studied inthis work, might also be involved in transduction of thecytokinin signal. As discussed above, CBP70 belongs tothe transcript elongation factors or proteins controllingelongation factor activity. Recently, numerous eukaryoticelongation factors facilitating transcription directed byeither RNA polymerase I or II were identified (Bjorklundet al., 1999; Yik et al., 2005). A complex system involvedin the control of elongation factor activity was elucidatedrecently, and some participating proteins were isolated(Fath et al., 2005; Yik et al., 2005). It was found that thecontrol of transcript elongation is a very important stepfor the regulation of gene expression in eukaryotes(Bjorklund et al., 1999) and that transcript elongationfactors are involved in the regulation of cell proliferationnot only in animals but also in plants (Grasser, 2005;Nelissen et al., 2005). Further studies of the mechanism ofcytokinin-dependent regulation of transcript elongation byCBP70 could be a key to understanding the protein’sinvolvement in the processes related to cytokinin controlof cell division.

Acknowledgements

We thank Professors Nella L Klyachko, Vasily M Studitsky, andRichard M Napier for critical reading and helpful discussion of thismanuscript, and Dr Elena A Burkhanova for counting the cellnumbers and protein estimation. This work was partly supported bythe Russian Foundation for Basic Research, project nos 05-04-

48289 and 05-04-49411, and by a grant from the President of theRussian Federation for the Support of Leading Scientific Schools,no. NSh-3692.2006.4

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