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 x 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.

A functionally important region in the promoter of the spinach photosynthesis gene AtpC, which encodes the subunit γ of the chloroplast ATP synthase, is located immediately upstream of the CAAT-box. A single nucleotide exchange in this region (AAAATTCAAT → AAGATCAAT) uncouples the expression of an AtpCpromoter::uidA gene fusion from the regulation by light, cytokinin, and functional plastids and results in a high constitutive expression of the reporter gene. By screening anArabidopsis thaliana expression library with a double-stranded wild-type oligonucleotide from this promoter region, we have isolated cDNAs from Arabidopsis libraries that code for plant homologs of the CAAT-box binding factor (CBF)-C. Binding occurs only in the presence of nuclear extracts, consistent with reports from metazoa CBFs that the subunits A and B in addition to C are required for the formation of the CBF-DNA complex. At least eight genes with homologies to CBF-C are present in theArabidopsis genome; one of them exhibits striking similarities to the gene for the human global transcriptional repressor Drap1. In gel mobility shift assays, low binding activity of CBF to the wild-type AtpC promoter sequence was observed with nuclear extracts from tissue with low AtpC expression levels,i.e. extracts from etiolated and photobleached seedlings, whereas high binding activity was detectable with extracts from tissues with high AtpC expression levels, i.e. extracts from light-grown seedlings and etiolated seedlings treated with cytokinin. Binding to the mutant sequence, which directs constitutive high level uidA expression in vivo, is significantly stronger than to the wild-type sequence. The data are consistent with the idea that the assembly of CBF at theAtpC promoter is regulated in response to light and cytokinin and that the low level of expression in etiolated and photobleached material is caused by an inhibitory effect. The structure/function relationships of the Arabidopsis CBFs are discussed in relation to their regulatory function inAtpC gene expression.

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.