N. N. Karavaiko

Isolation of a protein with cytokinin-receptor properties by means of anti-idiotype antibodies

A protein from cytosol of 10‐day‐old barley leaves with cytokinin‐receptor properties was isolated and 12 000‐fold purified by anti‐idiotype antibodies from anti‐BA serum. Cytokinin‐binding properties of this protein were demonstrated by its competition with Abba for immobilized BA in competitive ELISA. In the presence of BA the protein activates in vitro rRNA synthesis in the transcription elongation system containing chromatinbound RNA‐polymerase 1 from barley leaves. The protein with similar properties was isolated from barley leaf cytosol with BA‐Sepharose. The control proteins isolated with adenine‐ and ethanolamine‐Sepharose did not possess cytokinin‐binding properties and had no effect on RNA synthesis in vitro.

Chloroplasts affect the leaf response to cytokinin

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.

RECEPTOR OF TRANS-ZEATIN INVOLVED IN TRANSCRIPTION ACTIVATION BY CYTOKININ

Zeatin‐binding protein (67 ± 2 kDa) was isolated from the cytosol of the first leaf of 10‐day‐old barley plants. The protein fits to all requirements for a zeatin receptor: (i) it binds [3H]trans‐zeatin reversibly and specifically, (ii) it is recognized by anti‐idiotype antibodies from antiserum raised against trans‐zeatin, (iii) in concert with 10−8 M trans‐zeatin it activates rRNA synthesis in vitro in a transcription elongation system containing chromatin from barley leaves associated with RNA‐polymerase I. In the presence of trans‐zeatin, the protein activates also RNA synthesis directed by RNA‐polymerase I and RNA‐polymerase II in isolated nuclei from barley leaves.

Nuclear and chloroplast cytokinin-binding proteins from barley leaves participating in transcription regulation

The 67 cytokinin-binding protein (CBP) previously isolated from the cytosol of mature leaves of 10-day-old barley seedlings has now been purified from nuclei isolated from the same leaves. The procedure of CBP isolation included protein purification by Sephadex G-50, hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on zeatin-Sepharose. Interaction of trans-zeatin with the nuclear protein was demonstrated by the ELISA technique based on cytokinin competition with anti-idiotype antibodies (raised against antibodies to trans-zeatin) for complex formation with the protein immobilized on polystyrene microtiter plates. Nuclear 67 protein in concert with trans-zeatin activated transcription elongation in vitro in systems containing chromatin associated with RNA polymerase I or nuclei isolated from barley leaves. Nuclear 67 protein had no effect on the chloroplast transcription system. A 64 CBP was isolated from chloroplasts of barley leaves by the same procedure as that used for CBP isolation from nuclei. The cytokinin-binding properties of the chloroplast protein were demonstrated by competition of trans-zeatin with Aba-i in complex formation with the protein in ELISA. Chloroplast CBP activated RNA synthesis markedly in chloroplast lysate without any effect on transcription in the chromatin-containing system. Therefore both nuclear and chloroplast transcription machineries are regulated by cytokinins by means of special nuclear and chloroplast cytokinin-binding proteins which can be considered cytokinin receptors with the properties of transfactors regulating the elongation phase of transcription.

Cytokinin signalling systems

A role of endogenous cytokinins as the hormonal signal, by which the roots regulate leaf metabolism and prevent leaf senescence in particular, is discussed. Cytokinin signal perception and transduction in leaf cells were studied on fully expanded first leaves of 10–13-day-old barley plants (Hordeum vulgare L. cv. Viner). Their high sensitivity to exogenous cytokinins depends on dramatic decrease in endogenous cytokinin content during leaf growth. Cytokinin-binding protein of 28–30 kD was isolated from barley leaf cytosol by its affinity to synthetic cytokinin benzyladenine (BA). The 30-kD protein was involved in cytokinin-dependent activation of RNA synthesis in vitro in the system containing chromatin-bound RNA polymerase I from barley leaves. Multistage purification of the protein included affinity chromatography on trans-zeatin-Sepharose or zeatin riboside-Sepharose resulted in isolation of barley leaf cytosol 67-kD protein up to electrophoretic homogeneity. The protein was revealed as a single band in Western blot analysis developed with anti-idiotype antibodies from antiserum to zeatin. In concert with trans-zeatin, the 67-kD protein activated transcription elongation directed by RNA polymerase I (in the system containing chromatin-bound RNA polymerase I from barley leaves) and by RNA polymerase II (in nuclei isolated from barley leaves). The protein effect strongly depended on cytokinin concentration. The maximum activation was observed at trans-zeatin concentration of 10−8M. cis-Zeatin had no effect. These results together with data on reversible [3H]zeatin-binding moiety of the 67-kD protein [8] provide a definitive proof to consider this protein as one of the cytokinin receptors in barley leaf cells which is responsible for cytokinin activation of transcription elongation directed by both RNA polymerase I and RNA polymerase II.Cytokinin-sensitive protein kinase activity was detected in barley leaf chromatin. Stimulation of this protein kinase was specific for physiologically active cytokinin trans-zeatin and was not realized by its non-active isomer cis-zeatin. The protein kinase was co-isolated from chromatin with RNA polymerase I and phosphorylated its subunits. The results of this study are discussed in the context of the latest evidence on regulation of transcription elongation in eucariotic cells.The suggestion was put forward on the involvement of protein kinase and cytokinin-binding protein in cytokinin-dependent transcription regulation in leaves.

A NEW FAMILY OF CYTOKININ RECEPTORS FROM CEREALES

The highly specific recognition of a natural cytokinin, trans‐zeatin, by cytokinin‐binding protein (CBP) of 67 kDa from barley leaves was detected with an assay developed on the basis of cytokinin competition in ELISA with anti‐idiotype antibodies (raised against antibodies to zeatin) for complex formation with CBP. Monoclonal antibodies (mAbs) raised against 70 kDa CBP from etiolated maize seedlings cross‐reacted with barley 67 kDa CBP and prevented barley CBP and trans‐zeatin induced activation of transcription elongation directed by RNA polymerase I associated with barley chromatin. One mAb (Z‐6) had an agonistic effect. Maize CBP replaced barley CBP in activation of RNA synthesis with cytokinin in the barley transcription system. Hence, a new family of cytokinin receptors with common functions and immunodeterminants including maize and barley CBPs was found.

The Involvement of the Chloroplast Zeatin-Binding Protein in Hormone-Dependent Transcriptional Control of the Chloroplast Genome

Using a multistep system for protein purification, including affinity chromatography on zeatin riboside–Sepharose, we isolated a zeatin-binding protein from the chloroplasts of the first leaf of ten-day-old barley seedlings (ZBPChl). In combination with trans-zeatin, this protein enhanced the RNA synthesis in the chloroplast lysate from the same leaves. A hormonal specificity of ZBPChl-induced transcriptional stimulation was demonstrated. The protein activated the chloroplast but not nuclear genome. Age-dependent changes in the ZBPChl functional activity and in the response of the chloroplast transcription system to this protein and trans-zeatin were observed. In the chloroplast lysate from aging first leaves of 14-day-old barley plants, ZBPChl from the leaves of 3-, 10-, and 14-day-old barley plants activated transcription in the absence of the hormone, and trans-zeatin suppressed this activation. Thus, trans-zeatin played the role of the ZBPChl activator in chloroplast lysates from ten-day-old seedling and its inhibitor in lysates from 14-day-old seedlings. The combination of ZBPChl and trans-zeatin activated the RNA synthesis in lysates from lupine leaves, which indicates the absence of species-specificity in the ZBPChl action. It was shown that, in the heterologous transcription system, the principal factor of regulation was not a plant species, which was a source of the ZBPChl or chloroplast lysate, but the physiological state of plants used.

Cytokinin-binding protein (70 kDa) from etioplasts and amyloplasts of etiolated maize seedlings and chloroplasts of green plants and its putative function

Cytokinins regulate chloroplast differentiation and functioning, but their targets in plastids are not known. In this connection, the plastid localization of the 70 kDa cytokinin-binding protein (CBP70) was studied immunocytochemically in 4-d-old etiolated maize seedlings (Zea mays L., cv. Elbrus) using monoclonal antibodies (mAbs) against CBP70 recognizing this protein not only in nuclei and cytoplasm, but also in plastids. CBP70 was detected in the amyloplasts of the root cap and etioplasts of the mesocotyl, stem apex, and leaves encircling the stem axis in the node. Immunogold electron microscopy demonstrated CBP70 localization in amyloplasts outside starch grains and revealed a dependence of CBP70 content in etioplasts on the degree of their inner membrane differentiation: the low CBP70 amount in etioplasts at the early stages of membrane development, the high content in etioplasts with actively developing membranes, and a considerable decrease in plastids with the formed prolamellar body. This suggests that CBP70 is involved in etioplast structure development. CBP70 was also observed in chloroplasts of the bundle sheath of green maize leaves. CBP70 purified from etioplasts mediated trans-zeatin-dependent activation of transcription elongation in vitro in the transcription systems of maize etioplasts and barley chloroplasts, suggesting that CBP70 is a plastid transcription elongation factor or a modulator of plastid elongation factor activity. CBP70 involvement in the cytokinin-dependent regulation of plastid transcription elongation could be essential for the cytokinin control of the biogenesis of this organelle.

Cytokinin-binding protein from Arabidopsis thaliana leaves participating in transcription regulation

The 67 kDa cytokinin-binding protein (CBP) has been isolated from old rosette leaves of 9-week-oldArabidopsis thaliana plants. The procedure of CBP isolation included the protein puri?cation by SephadexG-50, hydrophobic chromatography on phenyl-Sepharose and a?nity chromatography on zeatin riboside-Sepharose. Nuclear localization in leaves of 67 kDa CBP was demonstrated. The recognition of a naturalcytokinin trans-zeatin by 67 kDa CBP from Arabidopsis leaves was detected by a very speci?c and sensitiveassay based on trans-zeatin competition in ELISA with anti-idiotype antibodies (Aba-i) for complex formationwith CBP. Aba-i were raised against antibodies against zeatin. trans-Zeatin competed with Aba-i in adose-dependent manner, demonstrating a high a?nity for the protein. In combination with trans-zeatin,67 kDa protein activated RNA synthesis in vitro in the transcription elongation systems containingchromatin-bound RNA polymerase I from barley leaves (heterologous system) or from Arabidopsis leaves(homologous system). These data permit a suggestion that the 67 kDa CBPs from monocots and dicotsful?ll similar functions. A comparison of functional activity of 67 kDa CBP from young, growing rosetteleaves of 3-week-old Arabidopsis plants, from mature leaves of 7-week-old plants and from senescent leavesof 9-week-old plants demonstrated age-dependent changes of the protein properties. The data are discussedin context of recent advances in the study of transcription regulation in eukaryotic cells.

Is a 67-kD Cytokinin-Binding Protein from Barley and Arabidopsis thaliana Leaves Involved in the Leaf Responses to Phenylurea Derivatives? (A Review)

Cytokinin-binding proteins (67-kD CBP) were isolated from barley first leaves of 8–10-day-old plants and Arabidopsis thaliana rosette leaves of 9-week-old plants. A capability of these proteins for cytokinin binding was assessed from trans-zeatin competition with antiidiotypic antibodies (ABa-i) for complex formation with immobilized 67-kD CBP under competitive ELISA conditions. ABa-i were obtained against anti-trans-zeatin antibody. 67-kD CBP from A. thaliana and barley leaves, in combination with trans-zeatin, activated transcriptional elongation in the in vitro system containing chromatin and RNA polymerase I from the same leaves. A phenylurea derivative demonstrating a high cytokinin activity, N-phenyl-N1-(2-chloro-4-pyridyl)urea (4-PU-30) could not displace ABa-i from its complex with 67-kD CBP from either barley or A. thaliana leaves, which indicates the absence of its interaction with CBP. In the presence of 67-kD CBP, 4-PU-30 did not activate transcription in the in vitro system containing chromatin and RNA polymerase I from A. thaliana or barley leaves. This means that, as distinct from trans-zeatin, 4-PU-30 did not use 67-kD CBP for transcription activation. A. thaliana leaf preincubation in the 4-PU-30 solution for 1 h enhanced RNA synthesis in the transcriptional system containing chromatin and RNA polymerase I from these pretreated leaves. Thus, leaves recognized the 4-PU-30 signal using another receptor. 4-PU-30 capacity to retard leaf senescence also supports this supposition. Another highly active phenylurea derivative thidiazuron also enhanced RNA synthesis in leaves and retarded their senescence.