DNA Biosynthesis in Chloroplasts: I. CHARACTERIZATION OF AND DEVELOPMENTAL VARIATION IN BOTH LIGHT- AND ATP-DRIVEN THYMIDINE INCORPORATION INTO DNA IN ISOLATED INTACT CHLOROPLASTS

Intact chloroplasts from young pea leaves were able to incorporate[³H]thymidine into DNA at relatively high rates (50 pmol mg^–1chlorophyll h^–1 or more), using light as the sole energysource. The intact plastids were also able to synthesize DNAin darkness, but only if ATP and MgCl₂ (MgATP) were both present.The rates of MgATP-driven assimilation in darkness were equalto or greater than light-driven activity. Neither light nordithiothreitol pretreatments enhanced thymidine incorporationin darkness, suggesting that enzymes of chloroplast DNA (ctDNA)biosynthesis are not regulated via a thioredoxin-type system.Although exogenous nucleosides (other than [³H]thymidine) werenot an absolute requirement, dramatically elevated rates ofincorporation (over 300 pmol mg^–1 chlorophyll h^–1)were seen when adenosine, cytidine, guanosine and thymidinewere supplied in combination (500 mmol m^–3 each). RadiolabelledDNA synthesized by the isolated chloroplasts was prepared usinga new heat extraction method. After digestion by restrictionendonucleases, ctDNA synthesized in organello was found to givetypical autoradiography patterns for chloroplast DNA. ExonucleaseIII studies suggested that 5% to 15% of the newly synthesizedDNA might be in a closed circular form. MgATP-driven synthesisin darkness was highly age-dependent. Chloroplasts from young(6 to 8-d-old) plants, or alternatively the youngest leavesof more mature plants, were 4–10 times more active thanthose from older tissues. Although these data do not establishconclusively that replication-type synthesis was occurring inthe isolated chloroplasts, they are consistent with this suggestion. Key words: Chloroplast DNA replication, isolated chloroplasts, chloroplast DNA synthesis