Carbon Dynamics - Part 2

Question:  How do organisms acquire energy?

Humans usually cultivate crop plants, such as maize and soybeans, in single-species stands (monocultures).  In most non-agricultural settings, however, plants occur in evolutionarily diverse communities of many species.  How and why does the photosynthetic and respiratory exchange of carbon (i.e. carbon dynamics) vary among coexisting species in a community?  In this week's lab you will address this question with plant species of the tallgrass prairie, the plant community that, before U.S. settlement, covered about 80% of Iowa's landscape.  You will apply biochemical and physiological concepts and methods you learned last week as you describe and explain variation in the carbon dynamics of leaves.  Some of your data and analysis will be used next semester in Biology 252 in studies of the interactions between leaf-level carbon dynamics, plant growth rate, competition between species, and carbon cycling in tallgrass-prairie ecosystems.

Photosynthesis and Respiration of Prairie Plants

Each group will be provided one or more plants of a species commonly found in the prairie at the Conard Environmental Research Area (CERA).  These may or may not be native species.   You should make measurements of photosynthesis and respiration as you did in Part I last week.  Make these measurements on three different samples of leaf tissue from your plants to see how variable these replicates may be.  Save the tissue from all three replicates.  While making your measurements make note of the relative humidity levels in your reference air (Reference relative humidity) and during your photosynthetic measurements (Analysis relative humidity) and record these in the Results section.  With the relative humidity values you will be able to calculate a transpiration rate for your leaf tissue and use that value to determine the water use efficiency for your plant, that is, how many molecules of H₂O it takes to fix one molecule of CO₂.

After completing your measurement of the third sample (while the tissue is still in the leaf chamber), proceed to do a light response curve on it as you did in Part II last week.  While members of your group are determining these rates, others may begin to extract chlorophyll from the two previous samples already measured.  Extract chlorophyll from the third sample when measurements on it are complete.  Be sure that each person in your group determines the chlorophyll content of one of the leaf samples.

Chlorophyll Determinations

Before extracting chlorophyll, cut out the 9 cm² of tissue that you used for your photosynthetic measurements from the rest of the leaf tissue with a razor blade.  Determine the fresh weight of this tissue and record it.  Use this tissue sample for your chlorophyll determination.

Grind the 9 cm² of leaf tissue in 9 ml of 80% acetone until fully extracted.  Centrifuge the sample at full speed for 5 min in a clinical centrifuge to remove debris.  Dilute the supernatant to 10 ml with 80% acetone.  First do a scan of the visible spectrum (400-700 nm) using the Cary 50 spectrophotometer and then read the absorbance of each sample at 652 nm (or 645 and 663 nm if separate chlorophyll A and B concentrations are desired).  Use 80% acetone as your blank for all of these measurements.  Use the following formulas to calculate mg chlorophyll per gram fresh weight:

Total chlorophyll

mg/ml =  A₆₅₂ / 34.5

OR

mg/ml = 0.0202 A₆₄₅ + 0.00802 A₆₆₃

For separate chlorophyll A and B concentrations:

Chlorophyll A (mg/ml)  = 0.00802 A₆₆₃

Chlorophyll B (mg/ml)  = 0.0202 A₆₄₅

To calculate mg chlorophyll per gram fresh weight:

[mg. chlor./ml   X  volume of extract (ml)] ÷ fresh weight (gm)

After you have determined the chlorophyll content of your leaf tissue, use the photosynthetic rate that you measured earlier to calculate the photosynthetic rate per mg chlorophyll as follows:

µmol CO₂/sec  ÷  fresh weight (gm)  =  µmol CO₂/gm fresh wt/sec

µmol CO₂/gm fresh wt/sec   ÷  mg chlor/gm fresh wt  =  µmol CO₂/mg chlor./sec

To determine the water use efficiency of your plants, first calculate the transpiration rate of the leaf tissue in your sample using the reference and analysis relative humidity values that you recorded and the calculation template demonstrated by your instructor.  The water use efficiency is the ratio of transpiration rate to photosynthetic rate:

This ratio can provide information about how efficient a particular plant is in its fixation of CO₂.  Some plants may be more efficient than others, requiring fewer water molecules per CO₂ molecule.  Greater efficiency may, in turn, allow some species of plants to grow in an environment where water is limiting the growth of less efficient species.  This would have ramifications for the productivity of a plant or crop species in terms of its adaptation to a particular environment.  A comparison of C₃ and C₄ plants in this regard may provide insights into their regional distribution.

Results

Plant Species _____________________          Air Temperature ________ ° C

Sample 1

Leaf Area =    ________ cm²                  Fresh Weight = __________ gm

Reference CO₂ concentration =  ________________ ppm

Analysis CO₂ - light  =  ________________________ ppm

Analysis CO₂ - dark  =  ________________________ ppm

Reference relative humidity = __________________ %

Analysis relative humidity = ___________________ %

Sample 2

Leaf Area =    ________ cm²                  Fresh Weight = __________ gm

Reference CO₂ concentration =  ________________ ppm

Analysis CO₂ - light  =  ________________________ ppm

Analysis CO₂ - dark  =  ________________________ ppm

Reference relative humidity = __________________ %

Analysis relative humidity = ___________________ %

Sample 3

Leaf Area =    ________ cm²                  Fresh Weight = __________ gm

Reference CO₂ concentration =  ________________ ppm

Analysis CO₂ - light  =  ________________________ ppm

Analysis CO₂ - dark  =  ________________________ ppm

Reference relative humidity = __________________ %

Analysis relative humidity = ___________________ %

Calculated Values