Chloroplasts affect the leaf response to cytokinin

J. Plant Physiol. 159. 1309 – 1316 (2002)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

Chloroplasts affect the leaf response to cytokinin Olga N. Kulaeva1, Emilia A. Burkhanova1, Natalia N. Karavaiko1, Svetlana Yu. Selivankina1, Svetlana A. Porfirova1, Galina G. Maslova1, Yana V. Zemlyachenko1, Thomas Börner2 * 1

Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia

2

Institute of Biology (Genetics), Humboldt-University Berlin, Chausseestr. 117, D-10115 Berlin, Germany

Received February 15, 2002 · Accepted July 1, 2002

Summary The effect of cytokinin on green and white leaves of the mutant line albostrians of barley was studied. Chloroplast development is completely blocked in white leaves of albostrians barley due to a nuclear mutation causing a lack of plastid ribosomes. We found white leaves to contain distinctly less abscisic acid than green leaves. In contrast, cytokinin (zeatin, zeatinriboside) content was higher in white vs. green leaves. Both white and green leaves contain a cytokinin-binding protein (CBP) of 67 kDa. CBP from white and green leaves, together with trans-zeatin (10 –7 mol/L), activated RNA synthesis in an in vitro transcription elongation system containing chromatin associated with RNA polymerase I isolated from wild-type barley leaves. In spite of the high cytokinin content and the presence of a protein showing properties of a cytokinin receptor, white leaves differed markedly from green ones in their response to added cytokinin. Cytokinin promoted stomatal opening in both types of leaves, though white leaves proved to be less sensitive. During senescence of detached leaf segments, protein degradation in white leaves occurred much more rapidly and was less retarded by cytokinin than in green leaves. Cytokinin enhanced the incorporation of methionine into protein in green leaves, but did so to a much lower degree in white leaves. Therefore, we conclude that unimpaired chloroplast development and/or chloroplast gene expression is required for normal leaf responses to cytokinin. Key words: albostrians mutant – abscisic acid – cytokinin – cytokinin-binding protein – Hordeum vulgare – senescence – transcription Abbreviations: ABA = abscisic acid. – Aba – i = anti-idiotype antibodies. – Abz = antibodies against zeatin. – BA = benzyladenine. – CBP = cytokinin-binding protein. – FC = fusicoccin. – Z = trans-zeatin. – ZR = trans-zeatin riboside. – ZR-Sepharose = trans-zeatinriboside Sepharose

Introduction Although cytokinin plays a central role in plant development including cell division, cell differentiation, leaf senescence, * E-mail corresponding author: [email protected]

and apical dominance, our knowledge of the distribution, perception and signal transduction of cytokinin is limited (for reviews see Schmülling et al. 1999, Schmülling 2001, Mok and Mok 2001). Recently it has been shown that a substantial part of leaf cytokinins are localised to chloroplasts (Benkova et al. 1999). Plastids and their development are among the 0176-1617/02/159/12-1309 $ 15.00/0

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major targets of cytokinin action in the leaf cell (Parthier 1979). Cytokinins activate etioplast differentiation in darkness and chloroplast development in the light (e.g. Khokhlova et al. 1971, Chory et al. 1991). There is abundant evidence to indicate that cytokinin activates the synthesis of chloroplast proteins encoded by both nuclear and plastid genomes, enhances photosynthetic pigment production, and stimulates chloroplast structural differentiation (e.g. Buschmann and Lichtenthaler 1977, Lerbs et al. 1984, Axelos et al. 1987, Reski et al. 1991, Kasten et al. 1992, 1997, Kusnetsov et al. 1994, 1998, 1999, Kubo and Kakimoto 2000). However, virtually nothing is known about the influence of chloroplasts on the cytokinin signal perception and transduction in leaf cells. Particularly, no data exist that describe the potential importance of the development state of chloroplasts and chloroplast gene expression for the cell response to cytokinin. To study this problem, we have investigated the effect of cytokinin on green and white leaves of the mutant line of barley, albostrians (Hagemann and Scholz 1962). Chloroplast development is completely blocked in white leaves of albostrians barley due to a nuclear mutation causing a lack of plastid ribosomes and thus of all plastid-encoded proteins (Hess et al.1993 a, b). The block in chloroplast development has been shown to affect other compartments in white leaves of the albostrians mutant as well, including the transcription of certain nuclear and mitochondrial genes indicating an influence of the developmental state of plastids on inter-organellar signal transduction (Hedtke et al. 1999, Hess et al. 1994, 1997). In this study, we compared the effects of cytokinin on the stomatal aperture (Jewer and Incoll 1980) and senescence (Mothes et al. 1959, Kulaeva et al. 1996) of primary leaves of white and green seedlings of the albostrians mutant. The data provide the first evidence for a role of chloroplast development and/or chloroplast gene expression in the response of leaf cells to cytokinin.

Materials and Methods Albostrians barley (Hordeum vulgare L. cv. Haisa) plants were grown in soil in a growth chamber under the following constant conditions: irradiance of 50 W m – 2, a 16-h photoperiod, a day temperature of 22 – 23 ˚C, a night temperature of 18 ˚C, and a humidity of 80 %. The primary leaves of entirely white or green eight to ten day old seedlings were collected separately and used for stomata aperture measuring in senescence studies and for CBP and chromatin isolation. For stomata aperture measuring, the upper 5 cm of the leaves were placed in Petri dishes onto filter paper moistened with water (control) or one of the following solutions: BA (10 – 6, 10 – 5, 10 – 4 mol/L), ABA (2 × 10 – 5 mol/L), BA (10 – 5 mol/L) + ABA (2 × 10 – 5 mol/L), and FC (5 × 10 – 5 mol/L). Petri dishes with leaf pieces were kept in a growth chamber at 23 ˚C and illumination of 50 W m – 2 for 15 h. Thereafter, the epidermal strips were peeled from the abaxial surface and immediately fixed with 2.5 % glutaraldehyde in 0.1 mol/L Na-phosphate buffer (pH 7.2) for 30 min. Epidermal strips were examined under a light microscope. The stomata aperture was measured with an ocular mi-

crometer AZ/K-15x (Carl Zeiss, Jena) at a magnification of 16 × 15 or 40 × 15. In a preliminary experiment, we demonstrated that glutaraldehyde fixation of the epidermal strips did not significantly change the stomatal aperture. Leaf senescence was tested with 2-cm-long segments from the middle part of fully expanded green and white primary leaves of 9 days old plants. The leaf segments were incubated in the dark at 22 ˚C on water or BA solution (10 – 6 –10 – 5 mol/L) for 2 – 4 days. Total soluble protein was estimated in leaf segments according to Lowry et al. (1951). Protein biosynthesis was studied in plants grown in axenic culture. Seeds were sterilised with 1 % sodium hypochlorite. Plants were grown axenically on agar-solidified Murashige and Skoog (1962) medium in a growth chamber under the same conditions as the plants grown in soil. 2-cm-long leaf segments of 7-day-old plants pre-incubated on water or BA solution were incubated during the last 3 h with 35S-methionine (3.7 MBq/mL, with a specific activity of 11 PBq/mmol) at 22 ˚C in the light. Then leaf segments were washed with cold methionine, frozen in liquid nitrogen, and kept at –70 ˚C. Protein extraction, estimation of 35S-methionine uptake by leaf segments, and its incorporation into proteins were performed by routine techniques as described by Burkhanova et al. (1991). CBP isolation was performed according to Karavaiko et al. (1995). All procedures were carried out at 2 – 4 ˚C. Barley leaves were homogenised in 3 – 4 volumes of buffer A (50 mmol/L Tris-HCl, pH 7.7; 100 mmol/L MgCl2; 250 mmol/L sucrose; and 5 mmol/L β-mercaptoethanol) and centrifuged at 160,000 g for 2 h. The supernatant was passed through a Sephadex G-50 column equilibrated with buffer B containing 20 mmol/L Tris-HCl, pH 7.7; 10 mmol/L MgCl2; 0.5 mol/L NaCl, and 5 mmol/L β-mercaptoethanol followed by hydrophobic chromatography on phenyl-Sepharose. During all steps of isolation, CBP was identified by its interaction with Aba – i in direct ELISA. The phenyl-Sepharose column was washed with 20 mmol/L Tris-HCl buffer, pH 7.7. Proteins were eluted with distilled water. Further purification of CBP was carried out by affinity chromatography using ZR-Sepharose. The protein fraction was applied to affinity matrix in 20 mmol/L Tris-HCl buffer, pH 7.7, with 200 mmol/L NaCl. CBP was eluted from ZR-Sepharose with 0.2 N NaOH after the main protein peak. CBP isolated by affinity sorbent was dialysed against 20 mmol/L Tris-HCl buffer, pH 7.7, and used for analysis. Protein content in this experiment was determined according to Bradford (1976). Cytokinin-binding properties of the isolated proteins were tested by their interaction with Aba – i in direct ELISA (Kulaeva et al. 1995). The proteins to be tested were immobilised on microtitration plates and then treated with Aba – i. Aba – i associated with immobilised protein were estimated using second anti-rabbit antibodies conjugated with horseradish peroxidase; o-phenylenediamine was used as a chromogen. The reaction product was measured at 490 nm (Multiskan MS, Kabsystems, Finland). Aba – i were isolated from antiserum raised against monospecific Abz by immunoaffinity chromatography on Abz-Sepharose (Kulaeva et al. 1995). PAGE of CBP was performed by the Laemmli (1970) procedure. Chromatin was isolated according to the protocol described previously (Selivankina et al. 1982). All procedures were carried out at 4 ˚C. Leaves were homogenised in 100 mmol/L Tris-HCl, pH 8.0, containing 250 mmol/L sucrose, 100 mmol/L MgCl2, and 20 mmol/L β-mercaptoethanol. The homogenate was filtered through several layers of gauze and then through a layer of Miracloth and centrifuged at 1000 g

Chloroplasts affect cytokinin for 10 min. The chromatin containing pellet was resuspended in 100 mmol/L Tris-HCl, pH 8.0, containing 350 mmol/L sucrose, 10 mmol/ L β-mercaptoethanol, and 2 % Triton X-100 and centrifuged at 7,500 g for 5 min. The procedure was repeated six times. The pellet was then washed twice with the same buffer, but without Triton X-100. Chromatin was resuspended in a buffer containing 50 mmol/L Tris-HCl, pH 8.0, 20 mmol/L MgCl, 1mmol/L β-mercaptoethanol, and 10 % glycerol, and used for transcription assays. The effects of CBP and cytokinin on RNA synthesis were studied in the transcription elongation system containing RNA polymerase I from barley leaves associated with chromatin as described by Selivankina et al. (1979, 1982). RNA polymerase activity was measured in the reaction medium (100 µL) containing (in µmoles): Tris-HCl buffer, pH 8.0 – 5; MgCl2-1; β-mercaptoethanol-1; CTP, UTP, CTP, and ATP – 0.2 of each, α-33P-ATP, (74 PBq/mol) and chromatin amount corresponding to 10 – 50 µg of chromatin DNA. Reaction was carried out at 32 ˚C for 20 min and stopped by the addition of an equal volume of cold 0.9 % sodium pyrophosphate in 10 % trichloroacetic acid. Cytokinins (Z + ZR) and ABA were extracted from leaves by routine techniques using 80 % ethanol containing 200 µg of diethyldithiocarbamate (25 mL for 1 g fresh leaf material) and purified according to Yokota et al. (1980). The extract was passed through SepPak C18 (Waters), ethanol was evaporated, the residue was acidified to pH 2.5 and passed through Dowex 50Wx8. ABA was not retained by the column. It was extracted from the liquid passed through the column by ethyl ether. After ether evaporation, ABA was dissolved in 20 mmol/L Tris-HCl buffer, pH 7.2, with 200 mmol/L NaCl. Cytokinins were eluted from Dowex 50Wx8 by 4 mol/L NH4OH. After ammonium evaporation they were dissolved in the same buffer as for ABA. Specific polyclonal antisera with high titres in ELISA (1: 30,000 for Z + ZR and 1: 60,000 for ABA) were obtained according to Weiler (1984). Z + ZR and ABA were determined by a competitive version of ELISA (Weiler 1982). All experiments were repeated at least three times. Figures represent means from three independent experiments and their standard errors.

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Figure 1. Effect of benzyladenine, abscisic acid and fusicoccin on stomatal aperture in green (䊏) and white (䊐) albostrians mutant leaves. Leaf segments were incubated on: 1 – water, 2 – BA (10 – 6 mol/ L); 3 – BA (10 – 5 mol/L); 4 – BA (10 – 4 mol/L); 5 – ABA (2 × 10 – 5 mol/L); 6 – BA (10 – 5 mol/L) + ABA (2 × 10 – 5 mol/L); 7 – FC (5 × 10 – 5 mol/L).

Table 1. Abscisic acid (ABA) and zeatin + zeatin riboside (Z + ZR) content in primary leaves of 9 days old green and white albostrians seedlings. Phytohormone

ABA Z + ZR

pmol/leaf White

Green

13.1 ± 2.1 13.8 ± 2.3

28.6 ± 2.4 8.2 ± 1.8

Results Cytokinin promotion of stomata opening in green and white leaves To test the sensitivity of leaves to cytokinins, we analysed the effect of BA (10 – 6 –10 – 4 mol/L) on stomata opening in green and white leaves. The enhancement of stomata aperture by BA (10 – 6 –10 – 5 mol/L) demonstrates a sensitivity of stomata cells of white leaves to cytokinin (Fig. 1). However, stomata cells of white leaves were clearly less sensitive to cytokinin, because BA at a concentration of 10 – 6 mol/L induced in green leaves the same effect as 10 – 5 mol/L BA in white leaves. Moreover, white leaves did not respond to higher BA concentrations: BA at a concentration of 10 – 4 mol/L increased stomata aperture in green leaves, but had no effect on white leaves (Fig. 1). To check whether the mechanism of stomata opening in white leaves was impaired, we analysed the effects of FC (5 × 10 – 5 mol/L), which is known to induce maximal opening of stomata (Marre 1979). FC dramatically enhanced stomata

opening of green as well as of white leaves (Fig. 1). Final stomata aperture of both leaf types was virtually the same (4.76 ± 0.34). Hence, the mechanism of stomata opening was not defective in white leaves. The stomata of white leaves were highly sensitive to ABA. This phytohormone, at a concentration of 2 × 10 – 5 mol/L, induced the closing of stomata in white leaves, but had no significant influence on stomata in green leaves. Cytokinin completely abolished the ABA effect on stomata aperture in white leaves and enhanced stomata opening two-fold in comparison with untreated control leaves. Conversely, ABA decreased the cytokinin effect on stomata aperture of these leaves. ABA and BA at equimolar concentrations, 2 × 10 – 5 mol/L and 10 – 5 mol/L, respectively, completely counter-acted each other in green leaves and their joint action had no effect on stomata aperture in these leaves. Thus, stomata cells of white leaves were sensitive to cytokinin and ABA, but their sensitivity to both phytohormones differed significantly from that of green leaves. Therefore, we determined the endoge-

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Figure 2. Protein content in green and white albostrians mutant leaf segments incubated in the dark on water (䊉) or 10 – 5 mol/L BA (䊊).

Table 2. Effect of BA (10 – 6 mol/L) on 35S-methionine accumulation in leaf segments, its incorporation into protein and percent of labeled methionine incorporation into protein of its total accumulation in leaf tissue of green and white albostrians leaves. 35S-methionine accumulation in leaf segments and its incorporation to protein are calculated in cpm/10 µg protein x 10 – 3. Treatment

Water BA Water BA

Incubation time [h]

24 24 48 48

Green leaves 35

35

S-methionine accumulation

S-methionine incorporation into protein

286 ± 14 215 ± 6.0 549 ± 9.0 484 ± 10

20 ± 0.3 33 ± 1.0 35 ± 0.5 44 ± 1.5

White leaves % of label incorporation into protein of its accumulation in leaf segments

nous levels of ABA and cytokinins (Z + ZR) in green and white leaves by ELISA (Table 1). Cytokinins and ABA were present in both types of leaves, though at strikingly different levels. Whilst white leaves were found to contain distinctly more cytokinins than green leaves (drastically more if related to the protein content, Fig. 2), the ABA content was much less in white than in green leaves.

Cytokinin effect on senescence of green and white leaves Senescence of green and white leaves was tested by assaying the loss of protein in leaf segments incubated on water or 10 – 5 mol/L BA solution in the dark. Figure 2 shows that protein

7 15 6 9

35

35

S-methionine accumulation

S-methionine incorporation into protein

449 ± 13 430 ± 19 479 ± 0.5 505 ± 10

58 ± 0.7 72 ± 2 65 ± 4 80 ± 0.9

% of label incorporation into protein of its accumulation in leaf segments 13 16 14 16

content in white leaves was significantly lower than in green ones due to the absence of all proteins synthesised within the chloroplasts and also of the nuclear encoded proteins involved in photosynthesis (Hess et al. 1993 a, 1994). As evident from the changes in protein content, senescence of white leaves occurred much more rapidly in comparison with green leaves. If incubated on water, the protein content of green leaves started to decrease only after 3 days, whereas a rapid decrease was already observed after 1 day in white leaves. Thus, the higher internal content of cytokinin could not prevent a fast degradation of proteins in white leaves. Moreover, externally supplied BA could only slightly retard the decrease of protein content in white leaves, whilst segments of green leaves showed even a slight increase in their protein content under identical conditions.

Chloroplasts affect cytokinin Responses of green and white leaves to cytokinin were compared by studying the BA effect on 35S-methionine incorporation into leaf proteins as well. These experiments were carried out axenically to exclude bacterial contamination. Segments of green and white leaves were incubated in the dark on water or BA solution. During the last 3 h, incubation was carried out in the presence of 35S-methionine. Total label accumulation in leaf segments and its incorporation into protein were measured. Label accumulation was much higher in white than in green leaves (Table 2). To compare the cytokinin effect on protein synthesis in white and green leaves, the percentage of 35S-methionine incorporation into protein of its total accumulation in the tissue of both types of leaves was calculated. Cytokinin activated protein synthesis not only in green, but also in white leaves, although the cytokinin effect on methionine incorporation in green leaves was much more pronounced. Interestingly, protein synthesis (in the cytoplasm) seemed to be more active in white vs. green leaves, since the share of radioactive methionine incorporated into protein of its total accumulation in tissue was significantly higher in white than in green leaves under most of the experimental conditions (Table 2).

Cytokinin-binding proteins from green and white leaves The observed differences in cytokinin response of green and white leaves prompted us to search for a cytokinin binding protein (CBP) that had previously been reported to be involved in the cytokinin-dependent stimulation of transcription in nuclei isolated from barley leaves (Kulaeva et al. 1995). The primary leaves of 9 days old, entirely white or green seedlings were used for CBP isolation. CBP purification revealed a 67 ± 2 kDa polypeptide and a minor band of 64 kDa in eluates from green as well as from white leaves (Fig. 3). The 67-kDa proteins from both white and green barley leaves reacted with Aba – i (raised against Abz) in direct ELISA, demonstrating zeatin-binding properties of the protein (Fig. 4). Pre-immune se-

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Figure 4. Interaction of Aba – i (raised against Abz) with CBP of 67 kDa from green (A) and white (B) primary leaves of albostrians barley. Open columns – pre-immune serum, closed columns – Aba – i, shaded columns – Abz. Reaction products were determined photometrically at 490 nm.

Table 3. Effect of trans-zeatin (10 –7 mol/L) and CBP from white and green leaves on RNA synthesis in an in vitro system containing chromatin-bound RNA polymerase I from leaves of barley wild-type leaves. Source of CBP

– – White leaves White leaves Green leaves Green leaves

transzeatin

– + – + – +

[α-33 P]AMP incorporation into RNA Cpm/50 g DNA

%

50 822 ± 4190 49 705 ± 3920 48 356 ± 4135 130 925 ± 8460 52 620 ± 4210 151 495 ± 7320

100 98 95 258 103 298

rum and Abz used as control for nonspecific binding did not interact with the protein. Functional activity of 67-kDa CBPs from white and green leaves were tested in an in vitro transcription elongation system containing RNA polymerase I associated with chromatin isolated from wild-type barley leaves. 67-kDa CBPs isolated from green and white leaves, respectively, enhanced RNA synthesis in vitro in the presence of 10 –7 mol/L trans-zeatin (Table 3). CBPs alone as well as transzeatin alone were not effective, thus indicating the activation to be dependent on the cytokinin-CBP complex.

Discussion Figure 3. SDS-PAG electrophoresis of soluble proteins isolated from barley leaves. The proteins were purified by a procedure including a ZR-Sepharose column, separated by SDS-electrophoresis in 10 % PAG and stained with Coomassie R-250. Samples were made from green leaves (lane 1) and white albostrians leaves (lane 2). Numbers indicate molecular mass of marker proteins (kDa).

We studied the effects of cytokinin on green and white leaves of the albostrians mutant to answer the question as to whether the developmental status of the plastid and/or plastid protein synthesis are of importance for the response of leaf cells to cytokinin. Cytokinins are known to enhance stomata opening (Jewer and Incoll 1980), an effect that can be used to study

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the sensitivity of leaves to cytokinin. Therefore, we compared BA effects on stomata aperture in green and white leaves of the albostrians mutant. BA promoted stomata opening in both types of leaves, although low concentrations of BA were less effective on white vs. green leaves. Cytokinin effects on white leaves were especially pronounced in the presence of ABA. ABA induced complete stomata closing in white leaves, and BA overcame this ABA action. Hence, stomata cells of white leaves are sensitive to cytokinin (as well as to ABA) and the cytokinin perception and transduction systems were functional in this type of cells, although stomata of white leaves were clearly less sensitive to low BA concentrations. We detected cytokinins and ABA (Table 1) in white and green leaves by ELISA. White leaves contained two times less ABA than green leaves. Plants have two pathways for ABA biosynthesis. One of the pathways is stress induced, localised in chloroplasts and branches from carotenoid synthesis (Parry and Horgan 1992). Quarrie and Lister (1984) observed that white leaves of albostrians barley did not respond to stress by enhanced ABA accumulation. The reason for the low ABA amount and lack of stress-induced ABA synthesis is probably the impaired carotenoid synthesis in the mutant plastids (Börner and Meister 1980). The low endogenous amount of ABA in white leaves could explain their extremely high sensitivity to exogenous ABA that leads to fully closed stomata apertures in these leaves and their low resistance to high (10 – 4 mol/L) BA concentration. There are also two pathways leading to cytokinins (Astot et al. 2000). The genes encoding enzymes of cytokinin biosynthesis in plants were identified recently (Takei et al. 2001, Kakimoto 2001). It remains to be seen why white leaves of albostrians barley contain a surprisingly higher amount of cytokinin than green leaves. To test the response of mesophyll cells of white leaves to cytokinin, we examined the effect of BA on senescence by measuring the amount and synthesis of proteins. In spite of their higher content of cytokinins, the process of senescence in white leaves, as detected by the loss of protein, proceeded much faster as compared to green leaves (Fig. 2). This might be the result of carbohydrate starvation in detached white leaves. In contrast, protein synthesis seems to be more active in the white leaves, as indicated by a higher incorporation of labelled methionine into proteins (related to total methionine accumulation) in comparison with green leaves (Table 2). Taking into account the fast protein degradation in white leaves, these data demonstrate a higher protein turnover in white vs. green leaves. Exogenous BA retarded protein loss, not only in green, but also in white leaves. However, this response was drastically reduced in white leaves as compared to green leaves (Fig. 2). Moreover, there was only a low BA-induced stimulation of 35S-methionine incorporation into protein in white leaves (Table 2). Thus, not the synthesis of cytokinins, but cytokinin perception and/or cytokinin-induced signal transduction is impaired in barley leaves with undifferentiated plastids. These results prompted us to study cytokinin perception in white leaves by

searching for a putative cytokinin receptor. A 67 kDa protein (CBP) was isolated previously from fully expanded primary leaves of wild-type barley cv. Viner (Kulaeva et al. 1995) that fulfilled the requirements of a cytokinin receptor: it specifically recognised only physiologically active cytokinins (Kulaeva et al. 1998) and, in complex with them, activated RNA synthesis in an in vitro system containing chromatin or isolated nuclei (Kulaeva et al. 1995). It is well known that cytokinin activates RNA synthesis in barley leaves (Kulaeva et al. 1996). Hence the 67 kDa CBP, in concert with trans-zeatin, reproduced in vitro a typical in vivo effect of cytokinins. Furthermore, the 67 kDa CBP was localised to nuclei of barley leaves (Kulaeva et al. 2000). Using the previously developed procedure, we isolated a 67 kDa protein from both white and green albostrians mutant leaves. SDS-PAGE revealed an additional minor band of 64 kDa, similar to that observed with CBP from leaves of wildtype barley (Karavaiko et al. 1995). The 67 kDa proteins from white and green leaves of albostrians mutant reacted with Aba – i in ELISA (Fig. 4), i.e. represented cytokinin-binding proteins. The complexes of trans-zeatin with the CBPs from green and white leaves activated RNA synthesis in an in vitro system containing chromatin-bound RNA polymerase I isolated from wild-type green barley leaves, cv. Viner or cv. Haisa (Table 3). Thus, a functionally active cytokinin-binding protein is present in both white and green mutant leaves. The system of cytokinin signal perception and transduction evidently involves a number of other components. Membrane cytokinin receptors with properties of sensor histidine kinases participating in cytokinin signal perception and transduction were discovered in Arabidopsis thaliana (Inoue et al. 2001, Yamada et al. 2001). Several genes encoding components of two-component regulatory systems were found to be cytokinin-inducible and should participate in primary cell responses to cytokinin (Brandstatter and Kieber 1998, Sakakibara et al. 1998, Taniguchi et al. 1998, D’Agostino et al. 2000). Similar proteins are expected to exist in barley. It is, therefore, quite possible that proteins involved in cytokinin perception or signal transduction other than the 67 kDa protein are responsible for the observed altered responses of white leaves to cytokinin. The data presented in this report demonstrate that the inability of white seedlings of the barley albostrians mutant to develop photosynthetically active green chloroplasts does not correlate with an impaired synthesis and accumulation of cytokinins; white leaves were found to contain high amounts of cytokinins (zeatin and zeatinriboside). However, our data on the effects of cytokinin on stomata cells and senescence, features that are not directly related to plastids, indicate that functional chloroplasts are required for the normal response of leaf cells to cytokinin. Probably chloroplasts possess their own system for cytokinin signal perception and transduction. Chloroplasts of tobacco and wheat leaves contain the whole spectrum of natural cytokinins (Benkova et al. 1999). A cytokinin-binding protein of 64 kDa participating in cytokinin-dependent activation of chloroplast tran-

Chloroplasts affect cytokinin scription was found in chloroplasts of barley leaves (Selivankina et al. 1997, Kulaeva et al. 2000). Therefore, it will be of great interest to find out to what extent direct effects of cytokinin on chloroplasts are affected by the albostrians mutation. Acknowledgements. The work was partially supported by the Russian Foundation for Fundamental Research, Grants 02-04-49367 and 00-15-97899, and by the Deutsche Forschungsgemeinschaft (436RUS-17/32).

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