Cytokinins are crucial endogenous regulators of chloroplast biogenesis [1]. They activate the synthesis of chloroplast proteins and photosynthetic pigments [2], enhance expression of genes encoding enzymes of the chlorophyll biosynthesis [3], accelerate differentiation of etioplasts and chloroplasts [1,2]. The effects of cytokinins on chloroplasts are mostly related to the involvement of phytohormones in the control of expression of nuclear and chloroplast genes encoding plastid proteins. The control of nuclear genes was shown to occur mainly at the level of transcription [2,4]. We demonstrated that, in greening lupine cotyledons, cytokinins evidently regulated expression of the chloroplast genes mainly at the posttranscriptional and/or translational levels [2]. A considerable progress achieved during recent years in the studying of the mechanisms of chloroplast gene's transcriptional regulation [5] makes sense to perform further investigations of cytokinin effect on transcription. Such investigations seem promising in connection with our isolation of a chloroplastic cytokinin-binding protein (chlCBP), which, in the presence of cytokinin, activated the synthesis of total RNA in vitro in the chloroplast transcription system [6,7]. In this connection, the objective of this work was to study the regulation of particular chloroplast genes by cytokinin. Since chlCBP was isolated from completely expanded first barley leaves [6], experiments were performed on these leaves. The task was complicated by the fact that cytokinins are endogenous regulators of chloroplast biogenesis, which are present in plant leaves [8], in chloroplasts in particular [9]. Therefore, in preliminary experiments, we choose the conditions permitting us to detect the effects of exogenous cytokinin on transcription in chloroplasts. To this end, we attempted to reduce the level of endogenous cytokinins in barley seedlings, limiting their nutrient supply and illuminance (from 4200 to 1000 lx). In both cases, this resulted in plant weakening and a decrease in the total transcription rate but did not allow a detection of the exogenous cytokinin influence. Keeping of detached leaves on moistened filter paper was more efficient in the exhausting of endogenous cytokinin pools in them because cytokinins are transported into leaves mainly from roots. The best result was obtained after leaf incubation on water for 24 h. We also compared the responses to cytokinin of chloroplasts isolated from growing three-day-old and mature nine-day-old leaves. Only chloroplasts from mature leaves manifested a clear response to exogenous cytokinin. Earlier, we have demonstrated that a 30-min leaf treatment with cytokinin resulted in the activation of transcription of the nuclear gene encoding 18S rRNA [10]. A longer leaf preincubation with cytokinin was required for the activation of chloroplast transcription.This preliminary work determined the following condition for experiments. Barley ( Hordeum vulgare L., cv. Luch) plants were grown in the phytotron chamber under illum...
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