1. Variations in sweetgum tree (Liquidambar styraciflua L.) leaf ultra-structure after exposure to elevated CO 2 April D Nesbit 1*, Johnna D Sholtis 1, David T Tissue 1 1 Department of Biology, Texas Tech University Sweetgum trees were exposed to ambient CO 2 (37/44 Pa daytime/nighttime) and elevated CO 2 (57/65 Pa) in a Free Air Carbon dioxide Enrichment (FACE) facility in a tree plantation at Oak Ridge National Lab in Tennessee, USA. Trees were 11-12 years old and approximately 15 m tall in a closed canopy with the live canopy beginning at 8 m from the ground. There were two 25-m diameter rings per CO 2 treatment. ABSTRACT The physiological and growth responses of trees to elevated CO 2 are well documented, but considerably less is known about the effects of elevated CO 2 on leaf ultra-structure. Recent studies with sweetgum trees have indicated that elevated CO 2 may produce significant structural changes in cellular organelles, such as increased numbers of mitochondria per unit cell area and a greater proportion of stroma to grana thylakoids. These changes in cellular structure may reflect a shift in plant metabolism that partially explains enhanced plant growth in elevated CO 2. Therefore, we measured the depth of different cell layers (epidermis, palisade parenchyma, mesophyll) within leaves of established sweetgum trees growing in ambient CO 2 (~ 364 ppm) and elevated CO 2 (~ 553 ppm) at the Oak Ridge National Lab Free-Air CO 2 Enrichment (FACE) facility in eastern Tennessee. Preliminary data in the second year of CO 2 treatment indicated that the number of palisade parenchyma cell layers increased in trees grown in elevated CO 2. A more comprehensive study in the third year of CO 2 treatment, in which leaf samples were collected in early- (May- June) and mid-growing season (July), indicated that neither CO 2 treatment nor time of season affected the depth of different cell layers. However, leaf position within a branch significantly affected leaf ultra-structure. Leaves at the base of a branch had thicker palisade parenchyma cells (70%), spongy mesophyll cells (28%), and greater total leaf thickness (41%), as well as greater numbers of palisade cells per unit leaf area (17%), than did leaves at the tip of a branch. These results suggest that leaf position may play a larger role in controlling leaf ultrastructure than CO 2 treatment. SUMMARY Elevated CO 2 treatment did not have a significant effect on leaf ultra- structure. Leaf position had a significant effect on leaf cell ultra-structure such that the palisade layer, spongy mesophyll layer, total leaf thickness, and palisade cells/unit area were smallest for leaves at the tip of the primary branch and largest for leaves at the base of the primary branch. There were significant seasonal effects on leaf ultra-structure resulting in thinner epidermal layers and a thicker spongy mesophyll layer in mid-season compared with early-season. These results suggest that leaf position may play a larger role in controlling leaf ultra-structure than CO 2 treatment. METHODS Sweetgum trees were exposed for three years to ambient (~364 ppm) or elevated (~553 ppm) CO 2 using FACE technology. Sixteen leaves were harvested from each treatment during early summer and eight leaves were harvested from each treatment in mid-summer. Leaves were preserved in 5 mL Formalin, 5 mL Glacial Acetic Acid, and 90 mL Ethyl Alcohol (FAA) mixture. Dehydration and embedding of samples was done using the Tissue-Tek II Histological Tissue Processor and Embedding Center. Samples were then embedded in Paraplast® X-tra Tissue Embedding Medium, and an American Optical® 820 Microtome was used to slice the tissue 8 microns thick. Slides were dyed with Safranin O and Fast Green and analyzed using light microscopy at 20x. Pictures were taken using E-6 film in an Olympus camera attached to the microscope. ACKNOWLEDGEMENTS Financial support was provided by DOE Program for Ecosystem Research. I would like to thank Dr. Larry Blanton and the Howard Hughes Medical Institute (HHMI) for additional financial support for this research project. I would also like to thank Dr. Jim Carr, Amy Russell, Mark Grimson, Dr. Marilyn Houck, and Laura Morris-Olson for technical and scientific guidance. INTRODUCTION The partial pressure of atmospheric CO 2 is increasing at unprecedented rates. Substantial effects of elevated CO 2 on plant physiology and growth have been documented, but relatively little research has been conducted on the potential effects on cell ultra-structure. Some experiments have shown a marked increase in cellular components, such as increased mitochondria per unit cell area and a greater proportion of stroma to grana thylakoids, and physiological testing of our site has shown increases in light-saturated photosynthetic rates and decreases in stomatal conductance due to elevated CO 2 (Griffin et al. 2001;Gunderson et al. 2002). Therefore, the overall ultra-structure of the leaf may have also been altered. Although the thickness of certain cell layers - epidermis, palisade, and spongy mesophyll - are controlled in part by genetics, Thomas and Harvey (1983) showed significant increases in palisade layer and total leaf thickness due to growth in elevated CO 2. We used a Free-Air CO 2 Enrichment (FACE) facility at Oak Ridge National Laboratory in Tennessee to expose sweetgum trees to ambient (364 ppm) and elevated (553 ppm) CO 2 treatments to determine whether long-term growth in elevated CO 2 would affect leaf cell ultra-structure. VARIATION OF PALISADE CELL NUMBERS RELATIONSHIPS SINGLE VARIABLE EFFECTS Leaf position significantly affected the thickness of the palisade layer, spongy mesophyll, and total leaf (p≤0.0001). Elevated CO 2 had no significant effects on any of the cell layers. Thickness of the lower epidermis, upper epidermis and spongy mesophyll varied significantly due to time of season. No significant relationship was observed between CO 2 treatment and leaf position. No significant relationship was observed between CO 2 treatment and time of year. The only significant change in palisade cells per unit area was in the leaf position (p=0.0023). This was calculated by counting the number of palisade cells on the monitor at a set magnification. REFERENCES Griffin, K. L., et al. 2001. Plant growth in elevated CO 2 alters mitochondrial number and chloroplast fine strucutre. PNAS. 98:2473-2478. Gunderson, C. A., et al. 2002. Environmental and stomatal control of photosynthetic enhancement in the cnaopy of a sweetgum (Liquidambar styraciflua L.) plantation during 3 years of CO 2 enrichment. Plant, Cell and Environment. 25:379-393. Thomas, J. F. and C.N. Harvey. 1983. Leaf anatomy of four species grown under continuous CO 2 enrichment. Bot. Gaz. 144:303-309. Ambient CO 2 -Base (July) Ambient CO 2 -Sylleptic (July) Ambient CO 2 -Tip (July) Ambient CO 2 (June) Elevated CO 2 (June) Elevated CO 2 - Base (July) Elevated CO 2 -Sylleptic (July) Elevated CO 2 -Tip (July) Lower Epidermis Spongy mesophyll Upper Epidermis Palisade