Elevation of Pollen Mitochondrial DNA Copy Number by WHIRLY2: Altered Respiration and Pollen Tube Growth in Arabidopsis

In plants, the copy number of the mitochondrial DNA (mtDNA) can be much lower than the number of mitochondria. The biological significance and regulatory mechanisms of this phenomenon remain poorly understood. Here, using the pollen vegetative cell, we examined the role of the Arabidopsis (Arabidopsis thaliana) mtDNA-binding protein WHIRLY2 (AtWHY2). AtWHY2 decreases during pollen development, in parallel with the rapid degradation of mtDNA; to examine the importance of this decrease, we used the pollen vegetative cell-specific promoter Lat52 to express AtWHY2. The transgenic plants (LWHY2) had very high mtDNA levels in pollen, more than 10 times more than in the wild type (ecotype Columbia-0). LWHY2 plants were fertile, morphologically normal, and set seeds; however, reciprocal crosses with heterozygous plants showed reduced transmission of LWHY2-1 through the male and slower growth of LWHY2-1 pollen tubes. We found that LWHY2-1 pollen had significantly more reactive oxygen species and less ATP compared with the wild type, indicating an effect on mitochondrial respiration. These findings reveal that AtWHY2 affects mtDNA copy number in pollen and suggest that low mtDNA copy numbers might be the normal means by which plant cells maintain mitochondrial genetic information.Reflecting their endosymbiotic origin, mitochondria contain DNA genomes (mtDNA) encoding several key proteins for oxidative phosphorylation. As most genes identified in the mitochondrial genome are indispensable for mitochondrial function, it is generally believed that each mitochondrion must possess at least one full copy of the genome. Indeed, this seems to be the case in animals. For example, although the number of mitochondria per cell varies in human, mouse, rabbit, and rat cell lines, the mtDNA copy number per mitochondrion remains constant at 2.6 ± 0.3 (Robin and Wong, 1988). Also, in mouse egg cells, each mitochondrion contains an estimated one to two copies of the mtDNA (Pikó and Matsumoto, 1976).Plant cells, however, have very few copies of the mtDNA compared with the number of mitochondria. For example, in the Cucurbitaceae, cells containing 110 to 140 copies of the mtDNA have 360 to 1,100 mitochondria (Bendich and Gauriloff, 1984). In Arabidopsis (Arabidopsis thaliana), leaf cells each contain approximately 670 mitochondria (Sheahan et al., 2005) and approximately 50 copies of the mtDNA (Draper and Hays, 2000). Thus, in plant cells, each mitochondrion does not possess one complete copy of the mtDNA, a phenomenon that occurs commonly in somatic cells of plants (Preuten et al., 2010). In addition, work in Arabidopsis, barley (Hordeum vulgare), and tobacco (Nicotiana tabacum) showed that cells in leaves, stems, and roots contain few copies of the mtDNA (40–160), whereas cells in root tips contain more copies (300–450; Preuten et al., 2010). This is consistent with the mitochondrial nucleoid diminishment previously observed in developing root and shoot tips (Fujie et al., 1993, 1994), which suggests that the low copy numbers in plant cells result from a decrease in the mtDNA copy number in nondividing cells during development.One question raised by these findings is whether some mitochondria have complete mtDNAs while others have no mtDNA or whether mitochondria have partial mtDNAs. Using techniques for the direct visualization of small amounts of DNA, our group revealed that up to two-thirds of mitochondria in Arabidopsis mesophyll cells totally lack mtDNA and the remaining one-third of mitochondria possess mtDNA of about 100 kb on average (Wang et al., 2010). This agrees well with a previously reported value for mtDNA copy number (about 50 copies per cell; Draper and Hays, 2000) and is consistent with the idea that plant mitochondrial genomes exist as submolecules smaller than the total genomic sizes (Satoh et al., 1993; Kubo and Newton, 2008). Among plant cells possessing low mtDNA copy numbers, the vegetative cell in the pollen grains is an extreme case; a mature pollen grain of Antirrhinum majus, containing many more mitochondria than a somatic cell, possesses only 16 copies of the mtDNA (Wang et al., 2010). Similar to the changes observed in somatic cells, this extremely low level of mtDNA in pollen vegetative cells results from a rapid decrease in mtDNA copy number during pollen development (Sodmergen et al., 1991; Nagata et al., 1999). In A. majus, the vegetative cell in its initial developmental stage has 482.7 copies of the mtDNA per cell, indicating a 30-fold decrease (482.7/16) during development (Wang et al., 2010). These results from both somatic and reproductive cells led to the intriguing idea that the mtDNA copy number in plants decreases in parallel with cell differentiation, to a very low value, and thus that several mitochondria must share the genetic information carried on a single copy of the mtDNA. Plant cell mitochondria undergo frequent and coupled fusions and divisions, which may explain how mitochondria share this information (Arimura et al., 2004). However, the biological significance of why plant cells lose their mtDNA, and how this benefits these cells, remains unknown. Given that pollen germination, pollen tube elongation, and sperm cell delivery all require energy conversion, the extremely low mtDNA copy numbers, such as in pollen vegetative cells, must not compromise mitochondrial function.The mtDNA copy numbers remain constant in various tissues, however, indicating that cellular mechanisms accurately regulate the levels of mtDNA in relation to cell type (Robin and Wong, 1988; Preuten et al., 2010). In yeast and animals, this regulation involves the core enzymes of mtDNA replication, such as DNA polymerase-γ (Sharief et al., 1999), RNA polymerase (Wanrooij et al., 2008), and mitochondrial helicase (Liu et al., 2009), as well as a group of DNA-binding proteins such as ARS-binding factor2 protein in yeast (Saccharomyces cerevisiae; Newman et al., 1996), MITOCHONDRIAL TRANSCRIPTION FACTOR A (TFAM) in human (Alam et al., 2003), and mitochondrial single-stranded DNA binding protein in Drosophila spp. (Maier et al., 2001). Overexpression of TFAM causes an increase in the mtDNA copy number, and RNA interference of TFAM decreases the mtDNA copy number (Ekstrand et al., 2004; Kanki et al., 2004). Also, the homozygous knockout of TFAM in mouse results in embryos that lack mtDNA and thus fail to survive (Larsson et al., 1998). Clearly, protein factors within mitochondrial nucleoids play a crucial role in the regulation of mtDNA copy number.Recent investigation in Arabidopsis revealed that, similar to the case in yeast and animal cells, DNA polymerase, the core enzyme of mtDNA replication, functions to maintain mtDNA levels in plants. Mutation of Arabidopsis PolIA or PolIB (homologs of bacterial DNA polymerase I) causes a reduction in mtDNA copy number, and double mutation of these proteins is lethal (Parent et al., 2011). Also, an Mg²⁺-dependent exonuclease, DEFECTIVE IN POLLEN ORGANELLE DNA DEGRADATION1 (DPD1), degrades organelle DNA, helping to produce the proper amounts of mtDNA in pollen cells (Matsushima et al., 2011; Tang et al., 2012). These results provide insights into the molecular control of mtDNA levels in plants, via both mtDNA replication and mtDNA degradation. Except for these enzymes, however, other protein factors (such as TFAM in animals) have not been identified in plants. The DNA-binding proteins, such as MutS Homolog1 (MSH1), Organellar Single-Strand DNA Binding Protein1 (OSB1), Recombinase A1 (RecA1), RecA3, and WHIRLY2 (WHY2), identified so far in plant mitochondria likely participate in genomic maintenance by affecting substoichiometric shifting (Abdelnoor et al., 2003), stoichiometric transmission (Zaegel et al., 2006), genomic stability (Shedge et al., 2007; Odahara et al., 2009), and DNA repair (Cappadocia et al., 2010). None of these plant nucleoid factors (DNA-binding proteins) has been implicated in the control of mtDNA copy number; thus, the mechanisms by which nonenzyme protein factors regulate mtDNA copy number in plants remain obscure.To test whether nucleoid DNA-binding proteins can affect mtDNA copy number, we examined the effect of producing Arabidopsis WHY2, a single-stranded DNA-binding protein (Cappadocia et al., 2010), in the pollen vegetative cell, which generally does not express WHY2 (Honys and Twell, 2004). We found that expression of WHY2 resulted in a 10-fold increase in mtDNA copy number in the pollen vegetative cell. This increase affected mitochondrial respiration, mitochondrial size, and pollen tube growth. Thus, our results uncover a novel function for WHY2, a member of the plant Whirly protein family, in regulating mtDNA amounts and indicate that, in plants, low mtDNA copy number does not compromise mitochondrial function but rather promotes proper mitochondrial function.