Plastid Transcription Activity and DNA Copy Number Increase Early in Barley Chloroplast Development1

Plastid transcription activity and DNA copy number were quantified during chloroplast development in the first foliage leaf in dark-grown and illuminated barley (Hordeum vulgare L.) seedlings. Primary foliage leaves of seedlings given continuous illumination from 2 days post-imbibition reached a final mean length of 15 centimeters at 6.5 days, whereas primary leaves of darkgrown seedlings required 7 days to reach a similar length. Dividing cells were observed in the basal 0.5 to 1 centimeter of primary leaves until 5.5 days post-imbibition. Plastids isolated from cells located in the basal meristem of 4-day-old seedlings were small (-2 micrometers in diameter), exhibited low transcription activity and contained approximately 130 copies of plastid DNA per organelle. Cell size increased from 18 to 60 micrometers in a 1 to 3 centimeter region located adjacent to the leaf basal meristem. In this region, transcriptional activity per plastid increased 10fold and DNA copy number increased from 130 to 210. Plastid transcriptional activity declined rapidly in illuminated plants with increasing leaf cell age and plastid DNA copy number also declined but with a slower time course. In dark-grown seedlings, plastid transcriptional activity declined more slowly than in illuminated plants while DNA copy number remained constant with increasing cell age. These data show that plastid transcriptional activity and DNA copy number increase early in chloroplast development and that transcriptional activity per DNA template varies up to 5-fold during barley leaf biogenesis. In monocots such as barley or wheat, leaf cells are produced primarily by a meristem located in the leaf base (5). The meristematic cells of the leaf base contain small (1-2 ,um diameter) undifferentiated prochloroplasts (26). In contrast, mesophyll cells located in mature portions of barley leaves contain up to 60 chloroplasts which are 6 to 8 Am in diameter (12, 26). The increase in plastid size during leaf cell development is accompanied by an accumulation of plastid proteins, many of which are encoded by plastid genes (12, 29). The plastid genes are located on a circular DNA which is 120 to 180 kbp2 in size. This DNA contains up to 137 genes which encode tRNAs, rRNA (16S, 23S, 4.5S, and 5S), and numerous proteins. The proteins encoded by plastid DNA are involved in transcription, translation, and photosynthesis. Therefore, 'This research was supported by the Texas Agricultural Experiment Station and by National Science Foundation grant DCB 8616156 'Abbreviations: kbp, kilobase pair; rbcl, gene encoding the large subunit of ribulose bisphosphate carboxylase. the biogenesis of chloroplasts requires activation of plastid gene expression (reviewed in Ref. 21). Transcription activity in plastids varies during chloroplast development (10, 22). This could be due to changes in RNA polymerase levels or activity (2, 30). In addition, variation in plastid DNA copy number might influence transcription activity, particularly if plastid transcription is template limited. The amount ofDNA in plastids varies considerably (4-7, 15, 17, 20). For example, in wheat, plastid DNA copy number has been reported to increase up to 7.5-fold during leaf development (20). Based in part on this observation, Bendich (3) proposed that increases in plastid DNA could be required for the build-up of chloroplast ribosomes during chloroplast biogenesis. He further suggested that plastid transcription activity and DNA content may be tightly coupled during chloroplast development. As a first step toward addressing these possibilities, we have measured plastid transcription activity and DNA copy number during plastid development in dark-grown and illuminated barley seedlings. MATERIALS AND METHODS