Arabidopsis thaliana organellar DNA polymerase IB mutants exhibit reduced mtDNA levels with a decrease in mitochondrial area density

Abstract

Plant organelle genomes are complex and the mechanisms for their replication and maintenance remain unclear. Arabidopsis thaliana has two DNA polymerase genes, DNA polymerase IA (polIA) and polIB, that are dual targeted to mitochondria and chloroplasts and are differentially expressed in primary plant tissues. PolIB gene expression occurs at higher levels in tissues not primary for photosynthesis. Arabidopsis T-DNA polIB mutants have a 30% reduction in relative mitochondrial DNA (mtDNA) levels, but also exhibit a 70% increase in polIA gene expression. The polIB mutant shows an increase in mitochondrial numbers but a significant decrease in mitochondrial area density within the hypocotyl epidermis, shoot apex and root tips. Chloroplast numbers are not significantly different in mesophyll protoplasts. These mutants do not have a significant difference in total dark mitorespiration levels but exhibit a difference in light respiration levels and photosynthesis capacity. Organelle-encoded genes for components of respiration and photosynthesis are upregulated in polIB mutants. The mutants exhibited slow growth in conjunction with a decreased rate of cell expansion and other secondary phenotypic effects. Evidence suggests that early plastid development and DNA levels are directly affected by a polIB mutation but are resolved to wild-type levels over time. However, mitochondria numbers and DNA levels never reach wild-type levels in the polIB mutant. We propose that both polIA and polIB are required for mtDNA replication. The results suggest that polIB mutants undergo an adjustment in cell homeostasis, enabling them to maintain functional mitochondria at the cost of normal cell expansion and plant growth.

Plastid Genome Instability Leads to Reactive Oxygen Species Production and Plastid-to-Nucleus Retrograde Signaling in Arabidopsis

“…Interestingly, a similar high-light tolerance was reported in msh1, but the exact cause of this adaptation remains elusive, as both chloroplast and mitochondria exhibit genomic instability in this mutant (Xu et al, 2011). Similarly to Msh1, PolIB is known to act in both organelles (Parent et al, 2011;Cupp and Nielsen, 2012), and thus it is possible that alterations in DNA metabolism from both chloroplast and mitochondria contribute to the nuclear genetic reprogramming observed in why1why3polIb-1. However, the synergistic increase in ptDNA rearrangements observed when plastid-localized Whirly proteins and PolIB are affected suggests that chloroplast defects are the main factor at the origin of changes in the nuclear gene expression.…”

“…In Arabidopsis (Arabidopsis thaliana), PolIA and PolIB, two type-I DNA polymerases, are proposed to perform chloroplast DNA replication (Mori et al, 2005;Parent et al, 2011). The mutation of either of the genes encoding these proteins is enough to cause replication stress, and PolIB has been demonstrated to have additional roles in DNA repair (Parent et al, 2011) and mitochondrial homeostasis (Cupp and Nielsen, 2012). In addition to PolIB, plant organelles possess Whirly proteins that act as safeguards of plastid genome stability by binding and stabilizing single-stranded DNA, thereby limiting the occurrence of illegitimate recombination (Maréchal et al, 2009;Cappadocia et al, 2010).…”

Enzymes involved in organellar DNA replication in photosynthetic eukaryotes

“…The identified DNA polymerase was first isolated from plastids of rice, and its localization was confirmed by immunoblot analysis of isolated plastids (Kimura et al, 2002). Subsequent studies using GFP-fusion proteins and/or immunoblotting demonstrated that the polymerases, which were named PolI-like, PolI or Polγ, are localized to both plastids and mitochondria in Arabidopsis thaliana and tobacco (Christensen et al, 2005; Mori et al, 2005; Ono et al, 2007; Parent et al, 2011; Cupp and Nielsen, 2013). We also identified this type of DNA polymerase in algae and ciliates (Moriyama et al, 2008, 2011, 2014).…”

“…The spatial expression patterns of POPs were analyzed in A. thaliana and rice by in situ hybridization, which revealed that POP genes are strongly expressed in the apical meristems of roots and shoots, leading to high POP protein levels in these tissues (Kimura et al, 2002; Mori et al, 2005). In A. thaliana , the expression of two POPs, AtPOP1 (At1g80840) and AtPOP2 (At3g20540), were compared by quantitative RT-PCR (Cupp and Nielsen, 2013). The analysis demonstrated that AtPOP1 is mainly expressed in rosette leaves, whereas AtPOP2 is predominantly found in the meristems of roots and shoots.…”

“…POP mutants of A. thaliana have been characterized by two research groups (Parent et al, 2011; Cupp and Nielsen, 2013). The A. thaliana genome encodes two POPs, AtPOP1 and AtPOP2, which are both localized to plastids and mitochondria.…”