The response of leaf nitrogen metabolism to seasonal changes in ammonium and nitrate for competing invasive Phalarisarundinacea and native CarexstrictaElizabeth F. Waring and A. Scott HoladayTexas Tech University, Department of Biological Sciences, Lubbock, TX, USA Methods-Analysis Leaf area for specific leaf area (SLA) (cm2 g-1) was calculated using a LI-3100 Leaf Area Meter the tissues were then samples were dried at 65 ºC for 24 hrs and weighted. The dried samples were ground for analysis for leaf nitrogen which was analyzed using a CE Elantech NCS 2500 elemental analyzer. Leaf tissue used for measuring nitrate reductase (NR) activity was measured spectrophotometerically at A540 by using the Griessreaction to measure nitrite formation (Granger et al. 1996). Soil samples were analyzed by Waters Agricultural Laboratories, Inc. (Owensboro, KY) for total ammonium and nitrate content. Statistical analysis of leaf tissue data were done using a random effects model followed by an ANOVA. This was done using the nlme package in R (Pinhero et al. 2009). The random effect was the repeated individuals from season to season. The soil data was statistically analyzed by using a liner regression using time as a continuous variable and an ANOVA was then run on the results of the linear regression using R. • Conclusions • Phalaris appears to be sensitive to changes in the soil nitrogen in which it grows. Site 1 is dominated by Phalaris and contains more total nitrogen and specifically more nitrate than the other site (Fig. 2). This difference in soil nitrogen has contributed to higher leaf nitrogen and SLA in site 1 for Phalaris (Figs. 3 and 5). Conversely, Carex does not change its leaf nitrogen metabolism strategy depending on how much soil nitrogen in present. Despite the higher amounts of nitrogen in site 1, the amount of leaf nitrogen did not vary between site nor did NR activity or SLA for Carex (Figs. 3-5). • Although soil nitrate increased seasonally, NR activity decreased seasonally in leaves of Phalaris (Fig. 5). It is possible that Phalaris down-regulates NR activity in its leaves seasonally while up-regulating NR activity in its roots. The high nitrate reductase activity combined with the higher levels of soil nitrate in the site dominated by Phalaris (site 1) suggests that Phalaris responds better to high nitrate than does Carex. In 2011, we did not conduct NR assays on root tissue. In 2012, roots are being collected for NR activity analysis. • These data indicate that Carex slows its incorporation of nitrogen into its leaves earlier in the year than Phalaris (Fig. 3), potentially giving it an advantage to invade more space from the summer into the autumn. It is possible that Phalaris continues incorporating nitrogen into its leaves until it is too cold for the above-ground tissue to survive. The earlier senescence of Carex leaves compared to Phalaris could assist Phalaris in its ability to invade Carex dominated wetlands. • This research is being continued in 2012 at the same sites and includes photosynthetic data as well as root enzyme assays to fully explore how seasonally changes effect both carbon and nitrogen metabolism. • Introduction • The invasion of Phalaris arundinacea is an ideal model system to study the physiological effects of eutrophication on invasion (Lavergne and Molofsky 2004). Phalaris often invades sedge meadows containing Carex stricta. Previous work has determined that Phalaris exhibits higher physiological performances across a broad range of temperatures compared to Carex in nitrogen enriched areas (He et al. 2011). What is unknown is the role of nitrogen assimilation efficiency and soil nitrogen preference play in determining where each species is competitive. As well as how seasonal variations in nitrogen metabolism and availability affect each species. Present research is addressing the following questions: (1) How do seasonal changes affect leaf traits and nitrogen assimilatory processes of each species under the current climatic conditions?; (2) Does soil nitrogen vary seasonally?; (3) Does either nitrate or ammonium affect the performance of either species? 1a 1b 1d 1c Figure 2 (left): Changes in soil nitrate and ammonium over time for both sites. Significant differences over time for ammonium and significant differences in date and site for nitrate (p<0.05) Figure 3 (right): Changes in leaf nitrogen over time for both species as well as between sites. Significant differences in species and site (p<0.05) Acknowledgements Funding for this project was provided by a grant through The Wetland Foundation to EFW and the department of biological sciences at Texas Tech University. The authors thank Dylan Schwilk for assistance with statistics, HasithaGuvvala and Yuanhua Wang for their assistance in the field and Nastasja van Gestel for assistance with analysis of leaf tissues. 1e 1f Literature Cited Granger, DL, Taintor, RR, Boockvar, KS, Hibbs, JB. 1996. Measurement of nitrate and nitrite in biological samples using nitrate reductase and Griess reaction. Nitric Oxide, Pt a - Sources and Detection of No; No Synthase. San Diego: Academic Press Inc. He, Z, Bentley, JP, Holaday, AS. 2011. Greater seasonal carbon gain across a broad temperature range contributes to the invasive potential of Phalaris arundinacea L. (Poaceae, reed canary grass) over the native sedge Carex Stricta LAM. (Cyperaceae). American Journal of Botany. 98:20-30. Lavergne, S, Molofsky, J. 2004. Reed canary grass (Phalaris arundinacea) as a biological model in the study of plant invasions. Critical Reviews in Plant Sciences 23: 415-429. Pinheiro, J, Bates, D, DebRoy, S, Sarkar , D and the R Development Core Team . 2011. nlme: Linear and Nonlinear Mixed Effects Models. R package version 3.1-102 R Development Core Team (2011). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL Wickham, H. ggplot2: elegant graphics for data analysis. Springer New York, 2009. Figure 4 (left): Changes in NR activity over time for both Phalaris and Carex and between sites. Significant differences in species, date, and site (p<0.05) Figure 5 (right): Changes in SLA over time for both Phalaris and Carex and between sites. Significant differences in species, date, and site (p<0.05) Figures 1a-f: Site variations by seasons. Site 1 is Phalaris dominated and appears the same through spring (1a), summer (1c), and autumn (1e). Site 2 is Carex dominated changes seasonally between spring (1b), summer(1d), and autumn (1f) • Results • While not significantly different, there was substantially more ammonium in Site 2 (Carex dominated) compared to Site 1 (Phalaris dominated). Soil ammonium decreased significantly over time (p<0.05) (Figure 2) • Significantly more nitrate the soil samples in Site 1 compared to Site 2 (p<0.001) and nitrate increased significantly over time in both sites (p<0.001) (Figure 2) • Significantly more leaf nitrogen in Phalaris compared to Carex (p<0.0001). Overall leaf nitrogen increased in Phalaris over time while decreasing in Carex. There were no significant differences between date or site. There was a significant interaction of species and date. (Figure 3) • Significantly greater NR activity in Phalaris compared to Carex (p<0.0001), significantly less activity over time (p<0.05) and was a significant difference between sites (p<0.05) (Figure 4) • Significantly greater SLA in Phalaris compared to Carex (p<0.0001), SLA decreased significantly over time in Carex (p<0.001), and Site 1 had higher SLAs in both species overall (p<0.05). There was also a significant interaction of Species and Date (p<0.0001) (Figure 5) • Methods-Data Collection • Data were collected from a Phalaris-dominated site former maize field (Site 1) (Figs 1a,c,e) and an adjacent Carex-dominated sedge meadow (Site 2) (Figs 1b,d,f) in north-central Indiana during spring, summer, and autumn in 2011. Both species were present at both sites. Data for the Spring were collected 18 May 2011 (Julian Date 138), Summer on 18 July 2011 (Julian Date 208), and Autumn on 13 October 2011(Julian date 285). Four plants were sampled from each site in each season. Soil samples were also collected seasonally from each site (n=4).