Abstract
Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles.
Highlights
Through the process of endosymbiosis, ancient bacteria were engulfed by precursors of eukaryotic cells, and over time most of the genes required for organelle function from these ancestral bacteria have been moved into the nucleus
We have examined the effects of mutations in the A. thaliana organellar DNA polymerases on chloroplast DNA (ctDNA) replication by quantitative PCR analysis of organelle DNA levels
At 21 days, there is a slightly greater reduction in the PolIB mutant (∼60% decrease) compared to the PolIA mutant (∼50% decrease). These results indicate that both DNA polymerases affect ctDNA copy number, in contrast with the finding in maize that a single DNA polymerase is responsible for ctDNA replication (Udy et al, 2012)
Summary
Through the process of endosymbiosis, ancient bacteria were engulfed by precursors of eukaryotic cells, and over time most of the genes required for organelle function from these ancestral bacteria have been moved into the nucleus. This raises the question, if most genes have migrated to the nucleus, why not all of them? John Allen (2015) proposes, supported by significant evidence from the literature, that redox regulation of gene expression is required within the membranebound compartment. Evidence for regulation of chloroplast DNA (ctDNA) by the redox state of cells has been reported in Chlamydomonas reinhardtii (Kabeya and Miyagishima, 2013), and for yeast mitochondrial DNA (mtDNA; Hori et al, 2009)
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