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.

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.

A cytokinin-binding protein (CBP) was isolated from etiolated maize (Zea mays L.) seedlings by two protocols. Ammonium sulfate fractionation, hydrophobic chromatography on Toyopearl HW-60, and affinity chromatography on immobilized trans-zeatin were used for CBP isolation according to the first protocol. The second protocol included a combination of ion-exchange chromatography and gel filtration followed by affinity chromatography on zeatin riboside-Toyopearl as in the first protocol. In order to remove proteins that could be nonspecifically bound to the affinity matrix, chromatography on adenosine-Toyopearl preceded chromatography on zeatin riboside-Toyopearl. Following these procedures, a CBP with a molecular weight of 70 kD was isolated. This protein specifically and reversibly bound dihydrozeatin and, in the presence of trans-zeatin, activated in vitro RNA synthesis in a system containing chromatin-bound RNA polymerase I from barley leaves. The protein cross-reacted with anti-idiotypic antibodies isolated from antizeatin serum, and, therefore, these antibodies could be considered antibodies to zeatin-binding protein. The data thus obtained indicate that the isolated CBP is a cytokinin receptor.

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(-8) M. cis-Zeatin had no effect. These results together with data on reversible [H-3]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 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 10-8 M. 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 procedure for isolating a protein with high affinity for the naturally occurring cytokinin trans-zeatin from the barley leaf cytosol was developed. Along with gel filtration and hydrophobic chromatography, this procedure involved affinity chromatography using trans-zeatin immobilized on Epoxy-activated Sepharose. The high affinity for zeatin in protein with mol wt of 67 +/- 2 kD was demonstrated by its competition with antibodies against trans-zeatin in the competitive-binding ELISA system when trans-zeatin was immobilized on polystyrene microtiter plates. When assayed in direct-binding ELISA and by Western-blot analysis, the protein cross-reacted with anti-idiotypic antibodies isolated from anti-zeatin serum. In the in vitro system containing barley chromatin and RNA polymerase I, the isolated protein enhanced RNA synthesis elongation in the presence of trans-zeatin. Data obtained permitted us to consider the isolated protein as a receptor for the naturally occurring cytokinin trans-zeatin, which mediated transcription activation with this phytohormone.

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.

The effects of cis- and trans-zeatin on the activity of the protein kinase associated with barley leaf chromatin were studied. Substances tested were added directly into the incubation medium for enzyme activity estimation. Only trans-zeatin activated the chromatin-associated protein kinase. Maximum activation was detected at a trans-zeatin concentration of 10−9 m. cis-Zeatin had no activity in a range of concentrations from 10−10 to 10−5 m. Comparison of the R-(-)-N 6-1-(1-naphthyl)ethyl-1H-purine-6-amine (R-NEPA) and S-(+)-N 6-(1-naphthyl)ethyl-1H-purine-6-amine (S-NEPA) effects on the enzyme activity showed that only S-NEPA activated the protein kinase from barley leaf chromatin, whereas R-NEPA had no such effect. The data on the effect of other synthetic analogues of cytokinin on the protein kinase activity are also presented. The results are discussed in terms of the specificity and sensitivity of the in vitro response of the chromatin-associated protein kinase from barley leaves to cytokinins. The advantages and limitations of this in vitro assay to test cytokinin activity are also considered.

A preparation of calcium- and phospholipid-dependent protein kinase was isolated from barley (Hordeum vulgare L.) leaf cytosol by ion-exchange chromatography on DEAE-cellulose followed by affinity chromatography on adenine-linked Sepharose. The protein kinase revealed a cross-reactivity with monoclonal antibodies against the rat brain protein kinase C. Polypeptides from barley leaf cytosol and histone H1 were the substrates for the isolated protein kinase. Its activity was stimulated by 1,2-sn-diacylglycerol. With regard to its specificity for substrates and activators, the enzyme resembled C-type protein kinases. From barley leaf cytosol, a calcium- and phospholipid-independent protein kinase with a mol wt of 48 +/- 2 kD was also isolated. This enzyme might be a catalytically active fragment of C-type protein kinase.

Protein kinase activity was detected in preparations of RNA-polymerase solubilized from chloroplast thylakoid membranes. Investigation of its substrate specificity indicated that casein suppressed protein kinase activity, and histone H1 could be used as a phosphorylation substrate only to a slight extent. The detected protein kinase activity depended on the presence of Mg2+ in the reagent medium and was Dot stimulated by cAMP or polyamines. Action of 6-benzylaminopurine and abscissic acid in vitro on protein kinase activity associated with solubilized chloroplast RNA polymerase is shown to be possible. Partial purification of solubilized RNA polymerase by means of centrifugation in a glycerin centrifugation gradient and chromatography on a column containing phosphocellulose P-11 did not lead to complete detachment of protein kinase activity from RNA polymerase activity, which enables us to postulate either the presence of common subunits in these enzymes or a fairly strong bond between them that is not broken by the purification methods used.

We studied changes of RNA polymerase and protein kinase activities in preparations of chromatin nuclei, and solubilized RNA polymerase of chloroplasts from leaves of bean plants (Vicia faba L.) grown at different light intensities (10, 50, 80, and 125 W.m-2 PAR) and during variation of illumination conditions. Chromatin-associated RNA polymerase I was most sensitive to changes of light intensity. Such changes affected the activities of RNA polymerase II and solubilized chloroplast RNA polymerase to a lesser extent. Changes of protein kinase activity in preparations of chromatin, nuclei, and solubilized chloroplast RNA polymerase were for the most part correlated with changes of their RNA polymerase activity. Such unidirectional changes of RNA polymerase and protein kinase activities were observed both during prolonged growth under light of different intensities and during variation of illumination conditions. Possible involvement of phosphorylation in light regulation of nuclear and chloroplast RNA polymerase activity is discussed in the paper.

We investigated the effects of blue and red light on protein kinase activity of thylakoid membranes in chloroplasts isolated from barley and pea leaves. It is demonstrated that blue light activates protein kinase of thylakoid membranes in vitro more significantly than red light does. This was observed both without the addition of exogenous phosphorylation substrates and after the addition of histone H1 as a substrate. Addition of casein to the reagent mixture under any illumination conditions and in the dark lowered the protein kinase activity of thylakoid membranes. Results obtained in experiments with thylakoids from normal and mutant pea plants employing an inhibitor of PSII at the level of plastoquinone indicate that reduced plastoquinone is not the only mediator of action exerted by light of different quality on activity of protein kinases bound with chloroplast thylakoids.

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.

A protein fraction containing protein kinase similar in properties to animal protein kinase C has been isolated by chromatography on adenine-Sepharose from the cytosol of barley leaves (Hordeum vulgare L.). Protein kinase activity was stimulated by Ca2+ ions, phosphatidylserine, diacylglycerol, and physiologically active phorbol ester (an inactive phorbol ester analog did not possess such a property). A series of polypeptides of the barley leaf cytosol and histones isolated from barley leaf chromatin served as substrate for the protein kinase. On the basis of the indicated properties taken altogether, the studied protein kinase can be relegated to the category of protein kinases of the C type. Cytokinin (6-benzylaminopurine) stimulated in vitro activity of the enzyme in the presence of Ca2+ ions, phosphatidylserine, and diacylglycerol, but exerted no influence in their absence. The authors discuss the possible involvement of C-type protein kinase in the response of plant cells to cytokinin.