Abstract
Chloroplasts and other members of the plastid organelle family contain a small genome of bacterial ancestry. Young chloroplasts contain hundreds of genome copies, but the functional significance of this high genome copy number has been unclear. We describe molecular phenotypes associated with mutations in a nuclear gene in maize (Zea mays), white2 (w2), encoding a predicted organellar DNA polymerase. Weak and strong mutant alleles cause a moderate (approximately 5-fold) and severe (approximately 100-fold) decrease in plastid DNA copy number, respectively, as assayed by quantitative PCR and Southern-blot hybridization of leaf DNA. Both alleles condition a decrease in most chloroplast RNAs, with the magnitude of the RNA deficiencies roughly paralleling that of the DNA deficiency. However, some RNAs are more sensitive to a decrease in genome copy number than others. The rpoB messenger RNA (mRNA) exhibited a unique response, accumulating to dramatically elevated levels in response to a moderate reduction in plastid DNA. Subunits of photosynthetic enzyme complexes were reduced more severely than were plastid mRNAs, possibly because of impaired translation resulting from limiting ribosomal RNA, transfer RNA, and ribosomal protein mRNA. These results indicate that chloroplast genome copy number is a limiting factor for the expression of a subset of chloroplast genes in maize. Whereas in Arabidopsis (Arabidopsis thaliana) a pair of orthologous genes function redundantly to catalyze DNA replication in both mitochondria and chloroplasts, the w2 gene is responsible for virtually all chloroplast DNA replication in maize. Mitochondrial DNA copy number was reduced approximately 2-fold in mutants harboring strong w2 alleles, suggesting that w2 also contributes to mitochondrial DNA replication.
Highlights
Chloroplasts and other members of the plastid organelle family contain a small genome of bacterial ancestry
Two hypotheses have been put forward regarding the functional significance of the high genome copy number in chloroplasts (Bendich, 1987): (1) The high copy number may compensate for stochastic DNA partitioning during plastid division, and (2) the high gene dosage may be necessary to provide an adequate supply of plastid ribosomal RNA to support the initial synthesis of the photosynthetic apparatus at the onset of chloroplast development
We explored the quantitative relationship between chloroplast DNA and chloroplast gene products by taking advantage of maize (Zea mays) mutants with insertions in the nuclear gene white2 (w2), which we show encodes a chloroplast DNA polymerase
Summary
Chloroplasts and other members of the plastid organelle family contain a small genome of bacterial ancestry. Two hypotheses have been put forward regarding the functional significance of the high genome copy number in chloroplasts (Bendich, 1987): (1) The high copy number may compensate for stochastic DNA partitioning during plastid division, and (2) the high gene dosage may be necessary to provide an adequate supply of plastid ribosomal RNA (rRNA) to support the initial synthesis of the photosynthetic apparatus at the onset of chloroplast development. In principle, these hypotheses could be addressed by experimentally manipulating plastid DNA levels. Maize Chloroplast DNA Polymerase Mutant mRNAs in C. reinhardtii are synthesized in excess of the amount that is needed to maintain the preassembled photosynthetic apparatus over the short time scale that was probed
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