Regulation of chloroplast transcription and DNA replication during light-induced pea leaf development

Abstract

The development of the pea primary leaf was characterized in plants grown in darkness or continuous white or far-red light. Leaf growth in continuous far-red light was a high-irradiance response most likely mediated by PhyA. White light, but not farred light, was able to increase cell size and plastid number per cell during development from 8 to 12 days post-imbibition (dpi). Increases in DNA copy number and DNA synthesis rates appear to be activated by growth in far- red light. At 6 dpi, these levels were twice that observed in white-light grown plants. The role of plastid transcription in modulating chloroplast gene expression was also examined. Increases in plastid transcription are activated by growth in far-red light. At 7 dpi, transcription levels in far-red light-grown peas were twice that observed in white light-grown plants. Plastid RNAs accumulated in illuminated plants from 5 to 7 dpi. A overall decline of RNA levels was observed for white-light grown pea chloroplasts between 7 to 14 dpi as compared to far-red light, which may be due to a general decrease in RNA stability. Transcription of genes encoding factors of the transcriptional and translational complexes increased relative to genes encoding photosynthetic proteins from 4 to 5 dpi and declined from 5 to 9 dpi. The data show that differential transcription and modulation of mRNA stability regulate chloroplast gene expression in light-grown peas. A highly purified DNA polymerase fraction was obtained from pea chloroplasts to elucidate the mechanism of light-activated chloroplast DNA replication. This purification resulted in the appearance of two bands with molecular masses between 40 to 45 kDa. Although these polypeptides co-elute with DNA polymerase activity during chromatography, they do not possess DNA synthesis activity. However, a 90 kDa protein was directly observed to possess DNA polymerase activity. Experiments suggest that at least one polypeptide between 40 to 45 kDa stimulates the activity of the chloroplast DNA polymerase. The expression of the DNA polymerase is constitutive. The 40/45 kDa protein accumulates in far-red but not dark-grown plastids. The light induced accumulation of the 40/45 kDa protein may be one mechanism chloroplasts utilize to regulate DNA replication.