Research
RESEARCH PROJECTS
Evolution of Cytonuclear Interactions
The basic functionality of the eukaryotic cell depends on coordinated interactions between two or more genomes. In the case of plants, three distinct genomic compartments—the nucleus, mitochondria, and plastids—all coexist within cells but experience very distinct conditions with respect to DNA replication, recombination, damage, repair, and inheritance. What effects do these contrasting genetic environments have on co-evolution between the genomic compartments? Because of the striking differences in mutation rates between mitochondrial and nuclear DNA in animals, it has long been hypothesized that differential mutation pressure may shape the interactions between organelle genomes. For example, high mutation rates in animal mitochondrial genes may create selection for compensatory mutations in the nuclear genome because mitochondrially encoded gene products experience direct protein-protein interactions with nuclear-encoded gene products. Theory suggests that frequent mutations and lack of recombination in mtDNA create selection for transfer of genes from the mitochondria to the nucleus to avoid the deleterious consequences of mutation accumulation. The natural mitochondrial mutation rate variation found in the angiosperm genus Silene (see below) presents an opportunity for us to test these hypotheses and better understand key mechanisms of cellular function (and dysfunction), using a combination of comparative genomic analysis and manipulative experiments that employ genome-editing techniques. We are also investigating the evolutionary dynamics associated with interactions between tRNAs and the aminoacyl-tRNA synthetase enzymes that are responsible for charging them with the correct amino acid. We have ongoing projects in Silene and in parasitic plants to investigate how these systems respond when the relationships between organelle-encoded tRNAs and nuclear-encoded enzymes are perturbed.
Protein structures of pairs of subunits within the mitochondrial ATP synthase complex (left) and OXPHOS complex III (right), highlighting the fact that enzyme function involves direct interactions between mitochondrial-encoded (green) and nuclear-encoded (yellow) subunits. Adapted from Havird et al. 2015 Evolution.
Mechanisms of Mitochondrial Mutation and DNA Damage
Although we typically think of mitochondrial genomes as enduring a much higher rate of point mutations than the nucleus, it is not clear how general this pattern is outside of animals, and it is most certainly not the case in plants, in which the rates of nucleotide substitution in mtDNA are exceptionally slow. The high mutation rates in human mitochondrial genomes are of great biomedical importance, making it valuable to understand how some other eukaryotic lineages like plants have evolved such low rates. Using special modifications to high throughput DNA sequencing techniques, we are detecting de novo mutations and DNA damage at low frequencies within plant tissues. Applying these approaches in mutant backgrounds of the model angiosperm Arabidopsis thaliana with disruptions in some key components of mitochondrial DNA replication and recombination is helping us elucidate the mechanisms the determine rates of mutation and DNA damage. Our work has identified the gene MSH1 as a key player in suppressing mitochondrial and plastid mutation rates and shaping the dynamics of heteroplasmic sorting -- the process by which a new mutation either proliferates or is eliminated among the many cytoplasmic genome copies that exist within the cell. Ongoing efforts are aimed at better understanding the mechanisms by which MSH1 affects these processes.
Summary of high-fidelity DNA sequencing methods used to detect de novo mutations present at ultra-low frequencies within tissue samples. From Sloan et al. 2018 Trends in Biotechnology.
Hypothesized model of MSH1 function in suppressing plant mitochondrial and plastid mutation rates via introducing double-strand breaks in response to mismatched bases and initiating homologous recombinational repair. From Broz et al. 2022 PNAS.
Intracellular Gene Transfer and Functional Replacement
We are interested in the long-term evolutionary process by which mitochondria and plastids (chloroplasts) have become genetically integrated into the eukaryotic cell. This process has involved functional transfer of genes from the organelle genomes to the nucleus, as well as the functional replacement of organelle genes by existing nuclear genes. We have collaborative projects examining the evolutionary mechanisms that shape both of these processes, which include the functional constraints that limit effective gene transfer and the coevolutionary consequences of "swapping" anciently divergent genes. Current work in this area is addressing the evolutionary dynamics of losing mitochondrial tRNA genes and replacing them by importing nuclear-encoded tRNAs from the cytosol. We are taking advantage of the fact that such changes are still ongoing in some Silene species.
A summary of tRNA gene transfer and functional replacement in plant mitochondria. From Warren and Sloan 2020 Mitochondrion.
The Evolution of Plant Organelle Genome Architecture
We and others have identified a number of flowering plant groups with dramatically elevated mitochondrial mutation rates. Mutation rates in these atypical plant lineages appear to be as fast or even faster than those normally seen in animals. We work on one of these groups, the genus Silene (Caryophyllaceae), in which accelerations in mutation rate appear to have occurred in just the past few million years, making it an excellent system to investigate the consequences of mitochondrial mutation rate variation.
Recent accelerations in the rate of mitochondrial sequence evolution within Silene and other angiosperm lineages. Adapted from Sloan 2015 New Phyt.
By sequencing complete mitochondrial genomes from a number of species, we found that increases in mitochondrial mutation rates in Silene have been associated with extreme changes in genome size and structure as well as reductions in the frequency of RNA editing. Remarkably, the fast-evolving Silene mtDNAs include the largest organelle genomes ever identified (up to 11.3 Mb in size) and have been fragmented into dozens of circular-mapping chromosomes. However, in many cases, the observed changes in these species run counter to existing theories about the relationship between mutation rates and genome architecture. Our current efforts are aimed at understanding the origins of this mitochondrial genomic diversity and identifying the mechanisms by which the enormous, multichromosomal genomes in Silene are replicated and inherited.
Summary of structural changes and whole-chromosome loss between mitochondrial genomes from two populations of the same angiosperm species (Silene noctiflora). Adapted from Wu et al. 2015 PNAS.
Evolution of Plastid Clp Protease Complex
Genes found within the plastid (chloroplast) genome are typically highly conserved. A glaring exception is the clpP1 gene, which has undergone extreme accelerations in rates of sequence evolution in manly lineages of flowering plants. This gene encodes a protein subunit that assembles with many nuclear-encoded proteins to form an essential plastid protease. We have identified evidence of intense selection pressures and coevolutionary dynamics between these plastid- and nuclear-encoded subunits, but the functional causes and consequences of rapid clpP1 evolution are still mysterious. In collaboration with the labs of Pal Maliga and Klaas van Wijk, we are testing the hypothesis that these unusual patterns of molecular evolution reflect a form of selfish conflict between plastid and nuclear genomes, using a combination of genome editing, proteomics, and comparative phylogenomics.
Extreme variation in rates of ClpP1 protein evolution across the green plant phylogeny compared to the more conserved protein PsaA. Long branches indicate lineages with elevated amino-acid substitution rates. From Williams et al. 2019 Plant Journal.
The Effects of Whole Genome Duplication (Polyploidy) on Cytonuclear Interactions
Whole genome duplication events have had a fundamental effect in shaping the evolution of eukaryotes, especially plant lineages. Although the consequences of polyploidy on genetic interactions within the nucleus have been the subject of extensive research, we know very little about how doubling the nuclear genome perturbs interactions with mitochondrial and plastid genomes. In collaboration with Jonathan Wendel's lab at Iowa St., we are investigating how plant cells respond to these stoichiometric changes. In addition, because whole genome duplication often coincides with interspecific hybridization events (allopolyploidy), we are examining the evolutionary consequences of pairing mitochondrial and plastid genomes from one species with paternal subgenomes from another.
Summary of potential stoichiometric responses to maintain cytonuclear interactions in the wake of whole genome duplication in the nucleus. From Sharbrough et al. 2017 Am J Bot.