Effects of Reduced Chloroplast Gene Copy Number on Chloroplast Gene Expression in Maize    

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.

Chloroplast DNA in Mature and Senescing Leaves: A Reappraisal

“…However, in the case of maize, for which total genomic sequence data are available, our analyses show that a few NUPTs (versus more than 1000 authentic ptDNA copies) have no significant influence on the estimated copy numbers per cell. Recent findings about the role of the maize white2 gene mutation on plastid genome copy numbers, which showed that the fraction of the total signal in real-time qPCR measurements that could arise from NUPTs is in the order of 1% (Udy et al, 2012), corroborate our findings. In Arabidopsis, the impact of coamplification of NUPTs is expected to be even lower because of the significantly lower abundance of NUPTs in the genome (Smith et al, 2011) and the possibility of selecting primers (on the basis of complete genome sequence information) that will not coamplify nuclear DNA sequences (Zoschke et al, 2007).…”

“…By contrast, data about plastid DNA (ptDNA) amounts in chloroplasts and cells of mature, ageing, and senescent tissue differ and are highly controversial. Basically two patterns have been described: the maintenance of more or less constant amounts of ptDNA per cell and/or organelle (Li et al, 2006;Zoschke et al, 2007;Rauwolf et al, 2010;Udy et al, 2012) or a significant decrease in copy number brought about by either continued organelle and cell division without ptDNA replication (Lamppa and Bendich, 1979;Scott and Possingham, 1980;Tymms et al, 1983) or by ptDNA degradation (Baumgartner et al, 1989;Sodmergen et al, 1991). In a series of communications, Bendich and coworkers recently reported that ptDNA levels decline drastically before leaf maturation in several plant species.…”

“…However, since contradictory data were reported for the same species that were grown under comparable, if not identical, conditions (Rowan et al, 2004(Rowan et al, , 2009Li et al, 2006;Oldenburg et al, 2006;Shaver et al, 2006;Zoschke et al, 2007;Evans et al, 2010;Udy et al, 2012), it is apparent that some of them must reflect methodological insufficiencies of the experimental approaches employed.…”

Chloroplast DNA Replication Is Regulated by the Redox State Independently of Chloroplast Division in Chlamydomonas reinhardtii

“…Mutation in the organellar DNA polymerase in maize (Zea mays) reduces the chloroplast DNA copy number as well as the chloroplast-encoded transcripts and proteins, suggesting that the DNA copy number is a limiting factor for the expression of chloroplastencoded genes (Udy et al, 2012). Other recent studies revealed a reduction in the chloroplast DNA copy number under a phosphate-limited condition in C. reinhardtii (Yehudai-Resheff et al, 2007) and an increase in the copy number under a phosphate-rich condition in the green alga Nannochloris bacillaris (Sumiya et al, 2008).…”

“…[See online article for color version of this figure. ] the replication of chloroplast DNA is performed by the plant organellar DNA polymerase (POP; Ono et al, 2007;Moriyama et al, 2008;Parent et al, 2011;Udy et al, 2012), which is similar to bacterial DNA polymerase I and is targeted to both chloroplasts and mitochondria. We performed BLAST searches in the C. reinhardtii database in an effort to identify POP orthologs.…”