O. N. Kulaeva

Cytokinins, abscisic acid and light affect accumulation of chloroplast proteins in Lupinus luteus cotyledons without notable effect on steady-state mRNA levels

Etiolated lupine (Lupinus luteus L.) cotyledons respond in a highly sensitive manner to phytohormones and light. The effects of cytokinin, abscisic acid, gibberellic acid (GA3) and indolylacetic acid (IAA) have been studied at the ultrastructural, steady-state mRNA and protein levels using 15 gene-specific probes for plastid proteins and corresponding antisera. No effect was noted with GA3 and IAA. As in other systems, N6-benzylaminopurine (BAP) and abscisic acid (ABA) operated antagonistically. In both instances, the steady-state mRNA levels remained relatively unaffected for plastid-encoded polypeptides, but not for those nuclear-encoded genes that could be tested. On the other hand, synthesis and accumulation of proteins of nuclear and plastid origin varied significantly. Cytokinin strongly promoted the accumulation of cytochrome b559 and subunit IV of the cytochrome b/f complex, while little effect was observed for cytochrome b6, the β subunit of the chloroplast ATP synthase or the large subunit of ribulose-1,5-bisphosphate carboxylase. In etiolated seedlings the level of chlorophyll-binding proteins (the 43-kDa chlorophyll a protein of photosystem II and subunits I a, b of photosystem I) was below the level of detectability. Their accumulation in light was promoted by cytokinin and inhibited by ABA though to different extents. Cytochrome b559 and the 33-kDa polypeptide of the water-oxidizing complex were not detectable in water-(control) and ABA-treated cotyledons. Cytokinin induced the synthesis of these proteins, even in darkness. These results indicate a protein-specific response to phytohormones, which can differ even for polypeptides belonging to the same membrane complex. They also suggest different modes of interaction between hormones and light, quite different phytohormone action in the two compartments, and demonstrate that phytohormones influence the biogenesis of the thylakoid membrane mainly posttranscriptionally.

Cytokinin Stimulates Chloroplast Transcription in Detached Barley Leaves

Chloroplasts are among the main targets of cytokinin action in the plant cell. We report here on the activation of transcription by cytokinin as detected by run-on assays with chloroplasts isolated from apical parts of first leaves detached from 9-d-old barley (Hordeum vulgare) seedlings and incubated for 3 h on a 2.2 × 10−5 m solution of benzyladenine (BA). Northern-blot analysis also detected a BA-induced increase in the accumulation of chloroplast mRNAs. A prerequisite for BA activation of chloroplast transcription was preincubation of leaves for 24 h on water in the light, resulting in a decreased chloroplast transcription and a drastic accumulation of abscisic acid. Cytokinin enhanced the transcription of several chloroplast genes above the initial level measured before BA treatment, and in the case of rrn16 and petD even before preincubation. Cytokinin effects on basal (youngest), middle, and apical (oldest) segments of primary leaves detached from plants of different ages revealed an age dependence of chloroplast gene response to BA. BA-induced stimulation of transcription of rrn16, rrn23, rps4, rps16, rbcL, atpB, and ndhC required light during the period of preincubation and was further enhanced by light during the incubation on BA, whereas activation of transcription of trnEY, rps14, rpl16, matK, petD, and petLG depended on light during both periods. Our data reveal positive and differential effects of cytokinin on the transcription of chloroplast genes that were dependent on light and on the age (developmental stage) of cells and leaves.

Cytokinin stimulates and abscisic acid inhibits greening of etiolated Lupinus luteus cotyledons by affecting the expression of the light-sensitive protochlorophyllide oxidoreductase.

Abstract Plastid biogenesis in etiolated lupine (Lupinus luteus L.) cotyledons is highly sensitive to cytokinins and abscisic acid. In the presence of the synthetic cytokinin N6-benzylaminopurine, greening and plastid biogenesis is substantially promoted as compared to untreated controls, whereas abscisic acid has an inhibitory effect. Faster greening in cytokinin-treated cotyledons is accompanied by a higher level and slower degradation of the light-sensitive protochlorophyllide-oxidoreductase (POR); while ABA has the opposite effect. The phytohormones appear to modulate POR gene expression, since the steady-state levels of POR mRNA, as well as transcripts of other nuclear genes for plastid proteins, are strongly increased by cytokinin and reduced by abscisic acid treatment. When etiolated lupine cotyledons were illuminated with far-red light prior to phytohormone application, the POR level substantially decreased; this was accompanied by the loss of the phytohormones effect on greening. Based on these findings it is concluded that the level of POR and the integrity of the prolamellar body is crucial for cytokinin- and abscisic acid-controlled greening following transfer of etiolated lupine cotyledons into the light.

The effect of ethylene and cytokinin on guanosine 5'-triphosphate binding and protein phosphorylation in leaves of Arabidopsis thaliana

Abstract. Binding of [α-32P]guanosine 5′-triphosphate ([α-32P]GTP) has been demonstrated in a Triton X-100-solubilised membrane fraction from leaves of Arabidopsis thaliana (L.) Heynh. Binding was stimulated by 1 h pre-treatment of leaves with ethylene and this effect was antagonised by the inclusion of N6-benzyladenine in the medium used for homogenisation. The ethylene-insensitive mutants eti5 and etr showed contrasting responses. In eti5 the constitutive level of GTP binding was higher than in the wild type whereas in etr the level was much lower. Neither ethylene nor cytokinin affected GTP binding in the mutants. The GTP-binding activity was localised in two bands at 22 and 25 kDa, both of which were immunoprecipitated by anti-pan-Ras antibodies, indicating that the activity is due to small GTP-binding proteins. In a similar membrane fraction, ethylene was shown to increase protein phosphorylation and benzyladenine antagonised this effect. In eti5 the constitutive level of protein phosphorylation was higher than in the wild type, but benzyladenine increased activity substantially while ethylene was without effect. In etr, protein phosphorylation was lower than in the wild type, ethylene was without effect, but cytokinin increased activity. A protein of Mr 17 kDa was detected on gels using antibodies to nucleoside diphosphate kinase. Phosphorylation of this protein was upregulated by ethylene but nucleoside diphosphate kinase activity was unaffected. The results are compared with the effect of the two hormones on the senescence of detached leaves and discussed in relation to pathways proposed for ethylene signal transduction.

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.

Abscisic acid represses the transcription of chloroplast genes

Numerous studies have shown effects of abscisic acid (ABA) on nuclear genes encoding chloroplast-localized proteins. ABA effects on the transcription of chloroplast genes, however, have not been investigated yet thoroughly. This work, therefore, studied the effects of ABA (75 μM) on transcription and steady-state levels of transcripts in chloroplasts of basal and apical segments of primary leaves of barley (Hordeum vulgare L.). Basal segments consist of young cells with developing chloroplasts, while apical segments contain the oldest cells with mature chloroplasts. Exogenous ABA reduced the chlorophyll content and caused changes of the endogenous concentrations not only of ABA but also of cytokinins to different extents in the basal and apical segments. It repressed transcription by the chloroplast phage-type and bacteria-type RNA polymerases and lowered transcript levels of most investigated chloroplast genes drastically. ABA did not repress the transcription of psbD and a few other genes and even increased psbD mRNA levels under certain conditions. The ABA effects on chloroplast transcription were more pronounced in basal vs. apical leaf segments and enhanced by light. Simultaneous application of cytokinin (22 μM 6-benzyladenine) minimized the ABA effects on chloroplast gene expression. These data demonstrate that ABA affects the expression of chloroplast genes differentially and points to a role of ABA in the regulation and coordination of the activities of nuclear and chloroplast genes coding for proteins with functions in photosynthesis.

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.

Recent advances and horizons of the cytokinin studying

Over the past two to three years, considerable progress has been achieved in the study of the cytokinin action mechanism. Genes encoding isopentenyltransferase, a key enzyme of the cytokinin synthesis in higher plants, were discovered. The membrane receptor for cytokinin was detected. The cytokinin primary response genes were found. Protein transcription factors involved in the control of these genes were isolated. Nuclear cytokinin-binding proteins, which control, in complex with cytokinin, transcription in nuclei, were studied. Fundamentally new results were obtained about cytokinin localization in cell organelles. Cytokinin-binding proteins, which are involved in the cytokinin-dependent regulation of chloroplast-genome transcription, were isolated from higher-plant chloroplasts. New evidence concerning the interaction between cytokinins and nitrogen metabolism was obtained. We surveyed the most important recent advances in the field of the study of cytokinins, analyzed the current state of the problem, and considered the possible pathways for further cytokinin investigation.

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.