1. The Use of Portable Gas Exchange Systems to Measure Plant Leaf Photosynthesis: Comparing Different Methods to Control Humidity Bailey Kramer  Dr. Tali Lee (Mentor)  Biology Department  University of Wisconsin-Eau Claire Introduction Methods Results This study was performed on the C3 species, coleus (Solenostemon scutellarioides), and the C4 species, amaranth (Amaranthus caudatus), growing in the greenhouse at the University of Wisconsin-Eau Claire. Two separate plants of each species underwent measurements, for a total of two leaves per plant (n=4), using a LI- COR 6400 when water was controlled for in three ways (FLOW, RH, H2OS). FLOW was set at a constant rate of 400 mol s -1. The LI-6400 was switched to holding H 2 OS constant and measurements were auto-logged every 30s until gas exchange stabilized. This step was repeated holding FLOW constant and then RH constant. The sequence was reversed until all the leaves were measured, always allowing the leaf to stabilize before the measurements were taken. Resources: Fanjul, L., Jones, H.G. 1982. Planta. 154: 135-138. Long, S.P., Bernacchi. C.J. 2003. Journal of Experimental Botany. 54: 2393-2401. Long. S.P., Farage. P.K., Garcia. R.L. 1996. Journal of Experimental Botany.47: 1629-1642. Resources: Fanjul, L., Jones, H.G. 1982. Planta. 154: 135-138. Long, S.P., Bernacchi. C.J. 2003. Journal of Experimental Botany. 54: 2393-2401. Long. S.P., Farage. P.K., Garcia. R.L. 1996. Journal of Experimental Botany.47: 1629-1642. Figure 3. Coleus (Solenostemon scutellarioides) Figure 2. Amaranth (Amaranthus caudatus) Photosynthesis is often measured during field and laboratory experiments as an important indicator of plant health and function. Today over 95% of photosynthetic CO 2 uptake measurements listed in journals use commercially available portable infrared gas analyzers like the LI-6400 (Long and Bernacchi, 2003, Fig. 1). The LI-6400 can control for the water within the chamber three different ways by setting constant: the rate of airflow across the leaf (FLOW), the relative humidity (RH), or the absolute amount of moisture in the chamber (H 2 OS). It is not known if the environmental fluctuations that result from using these different measurement techniques differentially affect leaf responses, which could impact the speed and accuracy of measurements. LI-6400 The overall trend shows holding FLOW constant to be the fastest measurement technique for both species, which does not support my hypothesis that FLOW would increase time to stabilization for C3 vs. no affect on C4. One similar study found that there were significant changes to leaf stomatal conductance shortly after humidity was changed when measuring apple tree leaves (Fanjul and Jones, 1982). Using the LI-6400, when FLOW is held constant humidity fluctuates. If the stomatal conductance in leaves is very responsive to these fluctuations then this could explain why the rate of stabilization varied across species and technique used to control atmospheric water. Another study assessed the ability of stomatal function to decrease the photosynthetic rate of a leaf (Long and Bernacchi, 2003). Due to the dependence of C3 photosynthesis on stomata that readily fluctuate in response to changes in atmospheric water this could explain why coleus did not stabilize faster under any of the measurement methods. The C4 species have a CO2 concentrating mechanism which allows for greater independence from stomatal fluctuations and might be the reason the C4 species was able to stabilize faster under the constant FLOW conditions. In conclusion, these results suggest that when using the LI-6400 to measure photosynthesis and stomatal conductance, the method used to control atmospheric water will not impact measured rates, but for some species the method chosen could reduce the time it takes for rates to stabilize. Discussion The technique used to control atmospheric water did not significantly affect the time to stabilization of photosynthetic or stomatal conductance rates in coleus (ANOVA, P=0.124, Fig. 5A; P=0.3192, Fig. 5B), however time to stabilization in amaranth did depend on measurement technique used (ANOVA,P=0.0011, Fig. 5A; P=0.0186, Fig. 5B). Photosynthesis in amaranth stabilized 88% faster, on average, when FLOW compared to H 2 OS was held constant and 85% faster compared to when holding RH constant (Fig. 5A). Similarly, stomatal conductance in amaranth stabilized 76% faster, on average, when FLOW compared to H 2 OS_mml was held constant, however, there was not a significant effect on time to stabilization when holding FLOW vs. RH constant (Fig. 5B). Figure 4. Gas Exchange Rates. A.) net leaf photosynthesis (µmol CO 2 m -2 s -1 ) and B.) leaf stomatal conductance to water vapor (mol H2O m -2 s -1 ) of coleus and amaranth for each water control method (FLOW, H 2 OS_mml, RH). Each bar represents the mean +/- SE of 4 replicates. Means with differing letters differ significantly at P<0.05, Tukey HSD within species Figure 5. Time to Stabilization (s) of A.) photosynthesis and B.) stomatal conductance to water vapor of coleus and amaranth for each water control method (FLOW, H 2 OS_mml, RH). Each bar represents the mean +/- SE of 4 replicates. Means with differing letters differ significantly at P<0.05, Tukey HSD within species. Objective The objective of this study was to determine if changing the LI-6400’s method of controlling atmospheric water affects leaf photosynthesis and stomatal conductance (gas exchange) rates, the time it takes for these rates to stabilize, and whether these differ in plants with contrasting photosynthetic pathways (C3,C4). I hypothesized that there would be no significant difference in the gas exchange rates when either FLOW, RH, H2OS were held constant on the LI-6400. I further hypothesized that the C3 species gas exchange rates would stabilize faster under constant RH and H2OS conditions when compared to FLOW, but that the method of controlling water would have no effect on the C4 species. The LI-6400 (LI-COR, INC., Lincoln, NE) portable infrared gas analyzer consists of a console and a leaf chamber/IRGA (Infrared Gas Analyzer). Once an attached leaf is in the chamber, LI-6400 takes measurements of the gases going into and out of the chamber. Using these measurements the machine calculates net photosynthesis and stomatal conductance, based on the equations derived by Caemmerer and Farquhar (1981). Among the variables processed by the LI- 6400 are: flow rate, pressure, temperature, humidity, and leaf area (Long, et al., 1996). All of these variables are interdependent and a change in one can cause the others to fluctuate. This creates a system where it is only possible to hold certain variables constant while the others fluctuate such as FLOW, RH, and H 2 OS. Figure 1. LI-6400 portable infrared gas analyzer. Acknowledgements: Lynn Young-Janik for supplying the plants & Learning and Technology Services (LTS) for printing this poster. Acknowledgements: Lynn Young-Janik for supplying the plants & Learning and Technology Services (LTS) for printing this poster. A. The technique used to control atmospheric water did not affect leaf net photosynthesis or stomatal conductance to water vapor in the C3 or C4 species (ANOVA, P=0.9784, Fig. 4A; P=0.9273, Fig. 4B). A A A A A A A A A A A A AA A B B B B B B B B AB A. B.