V. V. Kusnetsov

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.

Chloroplast RNA polymerases: Role in chloroplast biogenesis.

Plastid genes are transcribed by two types of RNA polymerase in angiosperms: the bacterial type plastid-encoded RNA polymerase (PEP) and one (RPOTp in monocots) or two (RPOTp and RPOTmp in dicots) nuclear-encoded RNA polymerase(s) (NEP). PEP is a bacterial-type multisubunit enzyme composed of core subunits (coded for by the plastid rpoA, rpoB, rpoC1 and rpoC2 genes) and additional protein factors (sigma factors and polymerase associated protein, PAPs) encoded in the nuclear genome. Sigma factors are required by PEP for promoter recognition. Six different sigma factors are used by PEP in Arabidopsis plastids. NEP activity is represented by phage-type RNA polymerases. Only one NEP subunit has been identified, which bears the catalytic activity. NEP and PEP use different promoters. Many plastid genes have both PEP and NEP promoters. PEP dominates in the transcription of photosynthesis genes. Intriguingly, rpoB belongs to the few genes transcribed exclusively by NEP. Both NEP and PEP are active in non-green plastids and in chloroplasts at all stages of development. The transcriptional activity of NEP and PEP is affected by endogenous and exogenous factors. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

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.

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.

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.

Expression of the ribosomal proteins S14, S16, L13a and L30 is regulated by cytokinin and abscisic acid: Implication of the involvement of phytohormones in translational processes

By employing the differential display method we have isolated a cDNA clone for rps14 from lupine, which encodes the ribosomal protein (r-protein) S14. The polypeptide consists of 150 amino acid residues with a molecular mass of 18.8 kDa [Bot. Acta 111 (1998) 287]. Northern analysis revealed that the mRNA level of rps14 responds rapidly, i.e. within 5 h, to phytohormone treatments: the level is stimulated by benzylaminopurine (BA), a synthetic analog of natural cytokinin, and repressed by abscisic acid (ABA). A comparable regulation was observed for the homologous gene in Arabidopsis. In contrast, hormone effects on the S14 protein level could only be detected after 48 h. Western analyses discovered two fractions of the S14 protein in lupine cotyledons: a BA-independent membrane-associated pool and a BA-stimulated soluble pool. S14 is predominantly found in young leaves and hypocotyls. Comparable hormone regulation was found for the mRNA of the ribosomal proteins S16 of the small (40S) subunit as well as for L13a and L30 of the large (60S) subunit of the cytosolic ribosomes. The phytohormone effects on the expression of r-proteins will be discussed in the context of their possible functions in protein synthesis.

Cytokinin and abscisic acid control plastid gene transcription during barley seedling de-etiolation

The regulatory role of cytokinins and abscisic acid (ABA) during de-etiolation of monocotyledonous plants is not sufficiently studied. The effects of cytokinin—6-benzyladenine (BA) and ABA on leaf greening and chloroplast gene transcription were studied on leaves detached from etiolated barley (Hordeum vulgare L.) seedlings. The influence of both phytohormones was age-dependent. Cytokinin practically had no effect on the youngest (3-day-old) leaves, increased chlorophyll accumulation in 6-day-old leaves, and this effect decreased in 9-day-old leaves. ABA reduced chlorophyll accumulation in the youngest leaves, and this effect was blocked by cytokinin. ABA alone had practically no effect on greening of leaves detached from 6- and 9-day-old seedlings, but fully inhibited activation of this process by cytokinin. Thus, antagonism between ABA and cytokinin was observed for seedlings of all ages. The rate of transcription of chloroplast genes was studied by run-on transcription in isolated chloroplasts in the course of etiolated seedling growth in darkness, and age-dependent decrease was demonstrated. It was established that cytokinin activated transcription of 11 chloroplast genes among 30 studied, and ABA suppressed transcription of 12 plastid genes. Such differential effect of cytokinin and ABA on chloroplast gene transcription in monocot seedlings during etiolated leaf greening was not known before.

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.

Application of run-on transcription method for studying the regulation of plastid genome expression

This paper describes in detail the main stages of the run-on transcription assay, which permits studying the regulation of the transcription rates for nuclear, chloroplast, and mitochondrial genes. This is very important for understanding the action mechanisms of phytohormones, light, temperature, and other factors. This method includes isolation of DNA-containing organelles, in vitro transcription in their lysates in the presence of labeled 32P-precursors of RNA, isolation of newly synthesized transcripts, and their hybridization with gene-specific DNA fragments fixed on a special membrane. We discuss some possible difficulties in the assay performing and some occasionally met inaccuracies in the result interpretation. In spite of a great potential of this method, it is not essentially applied by Russian researchers, in particular those studying plant biology. The objective of this work was to facilitate a rapid introduction of the run-on transcription method in the practice of scientific research of Russian plant physiologists.