Cynthia Perry and Dr. Brent Nielsen, MMBio

DNA replication is essential to all life on earth. Not only does replication occur in the nucleus, it also happens in organelles of the cell: the mitochondria and chloroplasts. In higher plants such as Arabidopsis thaliana, the genomes in mitochondria encode proteins which are responsible for DNA replication. These proteins also assist with DNA repair and recombination (Edmondson, AC et. al). One protein from the Arabidopsis thaliana mitochondrial genome is particularly of interest in our research: the single-stranded DNA-binding protein (SSB). The major function of this protein is to keep the two DNA strands separated while they are being replicated. SSB also seems to have additional functions such as mediating assembly of the Rec A complex on DNA (Edmondson, AC et. al). (The Rec A complex is a complex of proteins that functions in DNA recombination as well as repair.)

In Arabidopsis thaliana, two SSB proteins have been found: SSB1 and SSB2. Our research has mainly focused on SSB1. The SSB1 protein has not been completely characterized in the plant biology literature. It is assumed to have the function of a traditional SSB protein but it may have other unknown properties and functions. The goal of our research was to characterize this protein and its function by determining its localization in the cell and by using T-DNA insertion plants to disrupt the SSB1 gene and determine any phenotypic differences.

To determine where SSB1 was found in the cell, we used Western blot analysis. Fractionations of both mitochondria and chloroplasts from wild-type cells were made and then used in Western blots. We found that SSB1 protein was present in mitochondria but not in chloroplasts. We also did an additional Western blot that confirmed that SSB1 did not localize to chloroplasts. Also, GFP fusion protein analysis confirmed localization of the protein the mitochondria.

In order to elucidate the possible roles SSB1 plays in the cell, we used T-DNA insertion seedlines and compared them with wild type plants. T-DNA insertion seedlines are seedlines from plants who have had transfer DNA (T-DNA) from another organism (usually bacteria) inserted into their genome, disrupting a particular gene in the plant’s genome. Certain genes can be selected to be disrupted and we used seedlines that disrupted the SSB1 gene in Arabidopsis thaliana. Upon growing the mutant (T-DNA) seedlines, we observed distinct phenotypes, namely smaller plant size and slower growth compared to wild type plants of the same age. Also, some of the mutant plants were much smaller and had an albino phenotype. A Western blot analysis was used to confirm that the SSB1 protein had been disrupted in the mutant plants showing the different phenotypes.

We analyzed the plants from the mutant seedlines in a variety of ways, the first being a stress experiment. Both mutant plants and wild type plants were grown in a normal condition (20 degrees C) and a stress condition (25 degrees C). The mutant plants showed the albino phenotype more frequently in the stress condition than in the normal condition.

Secondly, quantitative PCR (QPCR) was used to analyze the amount of mitochondrial and nuclear DNA was found in the mutant and wild type plants. Because the SSB1 protein is involved in DNA replication and is found in the mitochondria, we hypothesized that there would be a considerable less amount of mitochondrial DNA in the plants without the SSB1 gene. DNA was isolated and used to establish baseline levels of nuclear and mitochondrial DNA. Q-PCR of the SSB1 mutant plants showed significant decrease in the amount of mitochondrial DNA versus nuclear DNA in the mutant plants as compared with wild-type plants.

Thirdly, tissue was isolated from wild-type plants on a weekly basis for six weeks. Western blots were performed to determine when the SSB1 protein was expressed most prominently in the plant. Weeks 2 and 3 showed the most expression of the SSB1 protein in wild-type plants. The same experiment will be performed on the mutant plant tissue at weeks 1-6.

Fourth, electron microscopy was performed on mutant plant samples. The mutant plants with the albino phenotype had the most unusual mitochondria morphology. Those plants with either homozygous or heterozygous for the T-DNA insertion had normal mitochondrial morphology but the mitochondria were noticeably smaller in size.

Lastly, a Southern blot was performed with the mutant plant tissue to determine if multiple T-DNA insertions were included in the mutant plant genome. If more than one insert is present, it’s possible that more than one gene has been disrupted. Thus, the phenotypes associated with the mutant plants (such as low mitochondrial DNA copy number) cannot be confidently attributed to the deletion of the gene of interest. Unfortunately, the results of the Southern blot were inconclusive. We also attempted to sequence part of the genome to determine the presence of multiple inserts but that was unsuccessful as well.

Further research is needed to complete the characterization of the SSB1 protein. Most importantly, the determination of single or multiple insertions needs to be completed. Our current plan is to begin back-crossing the mutant plants with wild-type plants while at the same time performing inverse PCR with mutant plant tissue to determine how many insertions are present. Also, we plan to confirm the preliminary results found with Q-PCR as well as determine the genotype of the mutant plants with the albino phenotype.

The results of our work were presented at the annual American Society for Plant Biologists conference in Minneapolis, Minnesota in August of 2011. We presented a poster of our work at the poster sessions of the conference.

References:

• Edmondson, AC, et al. “Characterization of a mitochondrially targeted single-stranded DNA-binding protein in Arabidopsis thaliana.” Mol Gen Genomics 273 (2005): 115-122.