An Ace in the Hole Explosive Seed Discharge may be Facilitated by Declining Stomatal Density Dylan Ziegler, Lyn Baldwin, Nancy Flood, Cynthia Ross Friedman INTRODUCTION RESULTS  Dwarf mistletoes parasitize conifers by invading the host’s vasculature, stealing water and nutrients1  Floral structures (the tepals and stigma) persist through second year of maturation (Figure 1)  Fruit recurves downwards as it approaches discharge in September  Arceuthobium americanum uses explosive discharge of the seed from the fruit as a dispersal mechanism2  A. americanum possesses transverse stomata  Stomata only observed on the stems, bracts, pedicels, and tepals of the plant; stomata were never observed on the fruit  A. americanum infection causes timber losses estimated at 3.8 million m³ in western Canada and 11.3 million m³ in the western United States3  Fruit length and diameter was found to increase over the growing season in all five trees sampled  Stomata are small pores in the plant epidermis involved in gas exchange and water movement  Integral to understanding the accumulation of hydrostatic pressure prior to discharge  Objective: To determine if stomatal density changes as the fruit matures METHODS  Tepal whorl surface area increased in all trees  Stomatal density declined in all trees (Figure 2) Figure 1. Second year DM fruit SEM micrographs taken in A) April B) May C) June D) July E) August and F) September. Scale bars represent 400 µm in each micrograph. Images taken at 56 Pa of extended pressure by Zeiss LS Evo Scanning Electron Microscope. Micrographs have been artificially colourized, showing the fruit in green, the tepal whorl in beige, and highlighting the stomata in orange. DISCUSSION  Floral organs may provide necessary transpiration  Fruit diameter increases leading up to discharge; presumably due to water accumulation in the fruit Collect A. americanum fruits from Stake Lake (once per week) from five randomly selected trees from AprilSeptember  Cuticle development likely functions in water retention  The observed decline in stomatal density any of the following three non-exclusive mechanisms: 1. Stomata number remains constant, surface area increases due to growth, consequently reducing stomatal density Examine fruits under Environmental Scanning Electron Microscope at a partial pressure of 56 Pa Image the whole fruit and tepal whorl, take measurements of the fruit length, diameter and stomatal density using the image analysis software, ImageJ 2. Stomatal density decline may be a mechanism of water retention. A reduction in stomata reduces transpiration rate; allows the fruit to accumulate water pressure 3. A reduction in transpiration heats the plant up. Given the plant relies on thermogenesis to explode, this could be an additional mechanism to trigger the process2 Figure 2. Stomatal density (stomata/mm²) change over the growing season. Curve generated by averaging all 5 trees sampled. Measurements taken using ImageJ. Acknowledgements I would like to thank the numerous supervisors and committees that enabled me to conduct this project. I would like to thank Joanna Urban for her input regarding mistletoe physiology and development from a genetic perspective. I would also like to thank Mark Paetkau for his assistance in helping me design my data analysis methods. Additionally, Dave Pow’s technical assistance on the scanning electron microscope was indispensible in this project. In preparation for this poster, Amanda Ziegler graciously lent her time to colourize the electron micrographs, for which I am grateful for. Lastly, I am very thankful to NSERC for funding my project through Summer 2015.  Future research: Investigate transpiration rates in the fruit and explore the otherwise undocumented genome Literature Cited 1. Hawksworth F.G, Wiens D. 1996. Dwarf Mistletoes: Biology, Pathology and Systematics. United States Department of Agriculture Forest Service Agricultural Handbook 709. 2. deBruyn, R. A. J., Paetkau, M., Ross, K. A., Godfrey, D. V. & Friedman, C. R. Thermogenesis-triggered seed dispersal in dwarf mistletoe. Nat Commun 6, 6262 (2015). 3. Shamoun S.F., Ramsfield T.R, van der Kamp B.J. 2003. Biological control approach for management of dwarf mistletoes. New Zealand Journal of Forestry Science. 33(3): 373-384.