1. PROJECT SUMMARY This study will determine the relationship between roadside car pollution, and a tomato crop’s natural defenses against a predator. Although endogenous substances are known to affect gene expression, the role of exogenous substances are relatively unknown. The chemical to be studied is pin II, a proteinase inhibitor, that is produced by plants upon attack. When ingested by insects, it will block the insect’s ability to break down proteins. The experiment will use a tomato field that is at the intersection of two roadways. Plants will be damaged with tobacco hornworms and pin II activity will be measured after 24 hours. Throughout the growing season, pollutant levels will be measured. A bioassay will also be conducted to see if tobacco hornworms are affected by the various levels of pin II. The results will yield a correlation between pollutants and concentration of pin II produced. Results of this research will establish a relationship between proximity to roadside pollution & damage to crops by insect pests. The Effect of Car Pollutants on the Expression of the Pin II Gene in Tomato Plants. Daniel Kani York College of P.A. Dept of Biological Sciences INTRODUCTION Plants use many natural chemicals to protect themselves against attack Proteinase Inhibitor Two (pin II) blocks an insect’s ability to break down proteins they have ingested, thus making the plant an undesirable food source We know that endogenous substances, such as, abscisic acid, and jasmonic acid will effect pin II expression, but we know very little about exogenous substances It is known that certain car pollutants will increases a plant’s susceptibility to insect attack In order for pin II to be produced, the plant must go through a pathway to reach actual expression of pin II. If one or more steps are interrupted, pin II will not be produced REVIEW OF LITERATURE Atmospheric SO2 was found to positively effect insect feeding preference. (Hughes, et al. 1981) Higher concentrations of atmospheric SO2 and NO2 positively correlated to insect growth and performance. (Bell, et al. 1984) Insects seem to prefer roadway plants because of higher levels of NOx, which the insects are deficient in. (Port and Thompson 1980) Abscisic acid (ABA) and Jasmonic Acid (JA) can initiate production, (therefore are part of the pathway). (Pena- Cortes et al. 1995) (Fig 1) Electrical current will also cause pin II production. (Pena-Cortes et al. 1995 & Davies and Stankovic 1996) Oligogalacturonides (pathogen), and chitosan (cell walls of fungus) will start the pathway. (Doares, et al 1995) (Fig. 1) Figure 1. Figure 1. Flowchart showing all the steps leading up to pin II production, from attack, to expression. (Pena-Cortes et al. 1995) HYPOTHESIS There is a negative correlation between car pollution and pin2 expression Tobacco Hornworms will prefer to feed upon, and will thrive on plants that have been exposed to high levels of car pollution RESEARCH DESIGN AND METHODS A tomato plant field, after a full growing season, will be split up into sectors, and random plants will be damaged by a tobacco hornworm. Plant samples will be collected after 24 hours, and frozen in liquid nitrogen (Figure 2) During the growing season, the pollutants CO, NO, and NO 2, will be measured weekly in each sector, using gas analysis tubes Each plant sample will then be analyzed, first with RNA isolation, followed by PCR, to isolate and amplify the desired gene Then each sample will be placed on a electrophoresis gel, and banding patterns will be measured by computer, giving a density of each band Separate from this experiment, plant samples will be fed to 2 nd instar tobacco hornworms. Feeding habits, as well as weight gain will be measured. A sample size of 10 will be used for each plant sample from each sector Figure 2 Figure 2. This is a pictorial map of the tomato field I will be studying. Each symbol is representative of a different sector. EXPECTED RESULTS Plants will show a lower density of pin II in sectors where pollutant levels are higher Tobacco hornworms will prefer plants with lower levels of pin2, and higher levels of pollutants LITERATURE CITED Bell, J. N. B., Dohmen, S., and McNeill, S., 1984. Air pollution increases Aphis fabae pest potential. Nature 307: 52-53 Doares, S.H., Syrovets, T., Weiler, E.W., and Ryan, C.A. 1995. Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway. Proceedings of the National Academy of Sciences USA 92: 4095-4098. Hughes, P.R., Potter, J.E., and Weinstein, L.H. 1981. Effects of air pollutants on plant-insect interactions: reactions of the mexican bean beetle to SO2-fumigated pinto beans. Environmental Entomology 10: 741- 744. Peña-Cortés, H., Fisahn, J., and Willimitzer, L. 1995. Signals involved in wound-induced proteinase inhibitor II gene expression in tomato and potato plants. Proceedings of the National Academy of Sciences USA 92: 4106-4113. Port, G.R., and Thompson, J.R. 1980. Outbreaks of insect herbivores on plants along motorways in the united kingdom. Journal of Applied Ecology 17: 649-656. Acknowledgements Karl Kleiner, PhD, YCP Research Mentor Bradley Rehnberg, PhD Jeffery Thompson, PhD