Variations in sweetgum tree (Variations in sweetgum tree (Liquidambar styracifluaLiquidambar styraciflua L.) leaf ultra-structure after exposure to L.) leaf ultra-structure after exposure to elevated COelevated CO22

April D NesbitApril D Nesbit1*1*, Johnna D Sholtis, Johnna D Sholtis11, David T Tissue, David T Tissue11

1 1 Department of Biology, Texas Tech UniversityDepartment of Biology, Texas Tech University

Sweetgum trees were exposed to ambient CO2 (37/44 Pa daytime/nighttime) and elevated CO2 (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 CO2 treatment.

ABSTRACTABSTRACTThe physiological and growth responses of trees to elevated CO2 are well documented, but considerably less is known about the effects of elevated CO2 on leaf ultra-structure. Recent studies with sweetgum trees have indicated that elevated CO2 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 CO2. Therefore, we measured the depth of different cell layers (epidermis, palisade parenchyma, mesophyll) within leaves of established sweetgum trees growing in ambient CO2 (~ 364 ppm) and elevated CO2 (~ 553 ppm) at the Oak Ridge National Lab Free-Air CO2 Enrichment (FACE) facility in eastern Tennessee. Preliminary data in the second year of CO2 treatment indicated that the number of palisade parenchyma cell layers increased in trees grown in elevated CO2. A more comprehensive study in the third year of CO2 treatment, in which leaf samples were collected in early- (May-June) and mid-growing season (July), indicated that neither CO2 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 CO2 treatment.

SUMMARYSUMMARY• Elevated CO2 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 CO2 treatment.

METHODSMETHODS• Sweetgum trees were exposed for three years to ambient (~364 ppm) or elevated (~553 ppm) CO2 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.

ACKNOWLEDGEMENTSACKNOWLEDGEMENTSFinancial 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.

INTRODUCTIONINTRODUCTION

The partial pressure of atmospheric CO2 is increasing at unprecedented rates. Substantial effects of elevated CO2 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 CO2 (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 CO2. We used a Free-Air CO2 Enrichment (FACE) facility at Oak Ridge National Laboratory in Tennessee to expose sweetgum trees to ambient (364 ppm) and elevated (553 ppm) CO2 treatments to determine whether long-term growth in elevated CO2 would affect leaf cell ultra-structure.

VARIATION OF PALISADE CELL NUMBERSVARIATION OF PALISADE CELL NUMBERS

RELATIONSHIPSRELATIONSHIPS

SINGLE VARIABLE EFFECTSSINGLE VARIABLE EFFECTS

Leaf position significantly affected the thickness of the palisade layer, spongy mesophyll, and total leaf (p≤0.0001).

Elevated CO2 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 CO2 treatment and leaf position.

No significant relationship was observed between CO2 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.

REFERENCESREFERENCESGriffin, K. L., et al. 2001. Plant growth in elevated CO2 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 CO2 enrichment. Plant, Cell and Environment. 25:379-393.Thomas, J. F. and C.N. Harvey. 1983. Leaf anatomy of four species grown under

continuous CO2 enrichment. Bot. Gaz. 144:303-309.

Ambient COAmbient CO22-Base (July)-Base (July) Ambient COAmbient CO22-Sylleptic (July)-Sylleptic (July) Ambient COAmbient CO22-Tip (July)-Tip (July)

Palisade Cells/Unit Area versus Each Treatment

0.0

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250.0NS NSNS NSSig.

Date versus CO2 versus Thickness

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Palisade Total Leaf TotalEpider.

LowerEpider.

UpperEpider.

Spongy

JunxAmbJulxAmbJunxElvJulxElv

NS

NS

NSNS NS

NS

Leaf Position versus CO2 versus Thickness

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Palisade Total Leaf TotalEpider.

LowerEpider.

UpperEpider.

Spongy

BxAmbSxAmbTxAmbBxElvSxElvTxElv

NS

NS

NSNS NS

NS

Date versus Thickness

0.0000

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Palisade Total Leaf TotalEpider.

LowerEpider.

UpperEpider.

Spongy

early summermid-summer

NS

NS

NS

Sig. Sig.

Sig.

CO2 Treatment versus Thickness

0.0000

0.0500

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Palisade Total Leaf Total Epider. LowerEpider.

UpperEpider.

Spongy

Thickness (mm)

AmbientElevated

NS

NS NS

NSNS NS

Leaf Position versus Leaf Thickness

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Palisade Total Leaf Total Epider. LowerEpider.

UpperEpider.

Spongy

Thickness (mm)

BaseSyllepticTip

Sig.

Sig.Sig.

NSNS NS

Ambient COAmbient CO22 (June) (June) Elevated COElevated CO22 (June) (June)

Elevated COElevated CO22- Base (July)- Base (July) Elevated COElevated CO22-Sylleptic (July)-Sylleptic (July) Elevated COElevated CO22-Tip (July)-Tip (July)

LowerEpidermis

Spongymesophyll

UpperEpidermis

Palisade