DNA replication is a process that occurs in all cells of all organisms to create new cells with the same DNA composition. DNA re-replication, however, creates numerous copies of the DNA within single cells instead of creating numerous new cells. This process occurs in many types of organisms but there is little knowledge of the potential ecological or evolutionary benefits of re-replicating DNA for the organism. This project will provide a whole-genome perspective of the effects of DNA re-replication on gene regulation and will relate those expression differences to plants under the stress of herbivory, ultimately determining if DNA re-replication provides beneficial gene regulatory effects to aid the plants? regrowth, survival, and enhanced seed production following damage.
The genetic effects of DNA re-replication may be important for organisms in combating a wide range of environmental stresses, and this study will provide the genetic underpinning of the adaptive potential of DNA re-replication generally. Investigating under-researched mechanisms influencing plant regrowth following damage is particularly important with the increased production of repeatedly-harvested biofuel and resprouting crops. This project will provide opportunities for undergraduates to gain experience in cutting-edge gene expression techniques and serve as material for discussion in K-12 classrooms via Skype and online web forums.
A broad, consequential issue in the ecology and evolution of plant-animal interactions regards the ways in which plants respond to herbivore-induced damage. Plants have evolved a wide variety of responses to damage by animal herbivores, including resistance strategies that aim to prevent damage and tolerance strategies that aim to prevent the reduction in seed production upon sustaining damage. Resistance traits often entail preventing damage via structural impediments or by decreasing the palatability of the plant tissue, and may include the production of defensive compounds that serve as toxins. Tolerance generally involves increased stem production and/or branching following damage that results in increases in photosynthetic output and plant growth rates. Resistance and tolerance strategies have traditionally been considered alternative forms of plant defense of herbivory with plants primarily exhibiting either defense or tolerance due to the necessity of allocating limited nutrient resources to toxin production or regrowth, respectively. Our recent research into the genetic underpinnings of tolerance provides evidence that tolerance and defense may not be alternative strategies to defend against damage. The oxidative pentose phosphate pathway (OPPP) is a metabolic pathway that provides the intermediate molecules necessary for biosynthesis of a wide range of compounds. Our past studies demonstrate that the OPPP is up-regulated following damage in the Arabidopsis thaliana genotype Columbia-4, and that Columbia-4 overcompensates following damage (i.e. damaged plants have greater seed yield than undamaged plants). The A. thaliana genotype Landsberg erecta, in contrast, has lower OPPP activity following damage and undercompensates (i.e. damaged plants have lower seed yield than undamaged plants). The metabolic activity of the OPPP not only appears to influence the tolerance abilities of these genotypes, however, but the intermediate molecules produced by the OPPP can also be used for the production of toxins. Theoretically, the OPPP may therefore not only influence the abilities of these plants to tolerate damage but potentially also provide a mechanism for the concurrent production of compounds that promote resistance. To assess the ability of the OPPP to promote both tolerance and resistance, we conducted gene expression analyses on damaged and undamaged plants of A. thaliana Columbia-4, Landberg erecta, and a genotype produced from their crossing (CS1936). Specifically, we performed whole-transcriptome RNA-sequencing of five plants of each genotype before being damaged and then again after being damaged to compare gene expression changes induced by damage for individual plants. This was achieved by clipping the primary stem of each plant with scissors during its growth, simulating natural mammalian herbivory. The expression of all genes in the removed tissue was measured and reflects each plantâ€™s gene regulation during its normal growth. When the regrowing stems reached approximately the same size as the initial primary stem, we removed the stem tissue of each plant again and measured total gene expression, which reflects the gene regulation of each plant during regrowth. Concurrently, we grew additional plants of each genotype, clipped half of the plants during their growth to simulate natural damage, and left the other half of the plants unclipped for comparison. For these plants, we measured biomass and seed yield at the end of life and compared these measures for clipped versus unclipped plants for each genotype, providing measures of tolerance. Our analyses indicate that tolerance and resistance are positively correlated in these plants. Columbia-4 equally compensated (i.e. tolerated) the damage, with no difference in biomass or seed yield between clipped and unclipped plants. Columbia-4 also significantly increased the expression of genes involved in the production of two classes of resistance compounds following damage, including glucosinolates which are considered the primary anti-herbivore toxins in this species. Landsberg erecta and CS1936 both suffered decreased biomass and seed yield when damaged relative to when undamaged and experienced significant decreases in the expression of genes involved in glucosinolate production. These results collectively suggest that tolerance and resistance are not necessarily alternative forms of plant defense to damage, but rather may be employed together or not at all depending on the genotype. A metabolic pathway involved in both, like the OPPP, could be a central factor in determining the fate of plants following damage. This research addresses a fundamental issue in the study of plant-animal interactions while providing opportunities and insights in a broader context. In total, four students were trained in plant growth, care, maintenance, clipping protocols, data collection (biomass and seed yield), statistical analysis, laboratory procedures (RNA extraction, purification, quantitation), genomic data analysis, and dissemination of results in scientific venues (both via manuscripts and presentations). Our results should be of particular importance to agriculture where increases in biomass, seed yield, the ability to tolerate damage, and natural plant resistance are highly desirable traits. For example, overcompensation of certain soybean cultivars following damage have produced world record soybean yields in recent years. Additionally, pest resistant, repeatedly-harvestable biomass crops (like Miscanthus) could be developed from our findings.