EDUCATION

Educational activity for K-12 and college students that demonstrates the principles of Crassulacean Acid Metabolism using an inexpensive and easy to perform assay:  

Protocol for demonstration of Crassulacean Acid Metabolism - Induction in the facultative halophyte Mesembryanthemum crystallinum

1. Introduction

Crassulacean Acid Metabolism (CAM) is an ecologically important adaptation of the C₃ photosynthetic pathway that greatly improves water use efficiency and drought tolerance. In CAM plants, CO₂ is first fixed during the night, when the stomates are open, into C₄ acids, that are then decarboxylated during the day, with the CO₂ released being fixed to carbohydrates in the C₃ photosynthetic mode. While some plants are obligate CAM (i.e. always perform CAM photosynthesis), others only shift into CAM when growth conditions become stressful (e.g., drought, salinity, etc.). These plants are said to be facultative CAM, as in the case of the common ice plant (Mesembryanthemum crystallinum) that has a developmentally programmed switch from C₃ photosynthesis into CAM that is promoted by a series of environmental stresses including drought and salinity. This experiment demonstrates the induction of CAM in the common ice plant by salinity stress. If the plant is performing CAM, during the night, when CO₂ is fixed into mainly malic acid, the overall cell pH drops to 4-5, while during the day the pH increases to 6-7 due to the decarboxylation of malic acid. A simple measurement of the pH of leaf-discs collected at pre-dawn and pre-dusk is sufficient to evaluate the plant's ability or inability to perform CAM as indicated by nocturnal acidification resulting from malic and citric acid accumulation in the vacuole.

Figure 1: The Biochemical Pathway for CAM (from Taiz and Zeigler, 2003):

Night	Day

2. Materials per student (please note: seeds, plates, and chlorophenol red can be obtained by contacting Dr. Cushman by email JCUSHMAN@UNR.EDU,  other materials are readily available).

• Twenty wild type seeds of Mesembryanthemum crystallinum
• Four 4-inch pots filled with Metromix 200 (Scotts Sierra Horticultural Products, Marysville,   OH)or any well draining commercial greenhouse soil mix; cactus soil mix works well.
• One single-paper-hole-puncher.
• One 24-well cell plate (small test tubes can be substituted if necessary).
• Chlorophenol red solution (125 mg/500 ml), Aldrich Chemical Co. (Tel: 414-273-3850) 19-952-4, Indicator grade (not the water soluble form) or equivalent.
• To prepare the Chlorophenol red solution, start by dissolving the 125 mg of the powder in a small volume of EtOH and than bring the volume to 500 ml with distilled H₂O. Keep the solution in a brown glass bottle.
• Nutrient solution (Peter’s Fertilizer works best and is widely available in home and garden centers or hardware stores).
• 0.5 M NaCl prepared in nutrient solution (table or Kosher salt can be used).

3.  Procedure

A. Seed sowing and plant care:

1. Fill pots with soil mix; wet soil until fully saturated.
2. Sow 5 seeds per pot and water again to situate seeds into the soil, but carefully not to displace the seeds out of the pots! Cover pots with Saran wrap.
3. Set the pots under fluorescent (and incandescent) or plant growth lights (very important: light intensity must be greater than 350 µE•sec-1•m-2) set to a 12 h day/12 h night photoperiod.
4. The seedlings should emerge in 2-3 days. Remove plastic wrap. Water daily with 1/4 strength nutrient solution, being careful not to displace the seedlings.
5. When seedlings are 7-10 days old, transplant or thin seedlings to a single seedling per pot. Continue to water plants daily.
6. After 4-5 weeks of growth the plants are ready for CAM induction. Note that it is very important that the plants do not experience any drought stress during this period, as this will induce CAM.

B.     Induction of CAM by salt-stress2

To induce CAM in the facultative M. crystallinum, water two of the plants (stressed) daily with a 0.5 M NaCl solution for 7 days. Water the other two plants as usual with regular nutrient solution (control).

C. Assay of leaf pH

1. Fill 8 cells of the 24-well plate or test tubes with 400 ml of the Chlorophenol red solution. Mark two of the cells (or tubes) for the pre-dawn sampling and two others for the pre-dusk.
2. One hour before the lights go ON (Pre-dusk), punch a leaf disk from a well-developed mature leaf from all of your plants, both stressed and unstressed. Immerse the leaf disk in the Chlorophenol red and vacuum infiltrate for 5 min (alternatively, one can skip the vacuum infiltration and just let the color develop on the bench).
3. After 2 hours (or the next day, whatever is convenient), compare the color of your wells with the color standard supplied. (Yellow is pH 4.0; Orange is pH 5.0; Red is pH >6.5). What is the pH of the leaf disk as indicated by the assay? Record your observations.
4. One hour before the lights go off, punch a leaf disk from the same well-developed mature leaf from all of your plants, both stressed and unstressed, used for the Pre-dusk punch. Immerse the leaf disk in the Chlorophenol red and vacuum infiltrate for 5 min (alternatively, one can skip the vacuum infiltration and just let the color develop in the bench). Alternatively, students can take a time course of samples every four hours throughout the 24 hour period of time.
5. After 2 hours (or the next day, whatever is more convenient), compare the color of your wells with a standard pH curve you make yourself or as shown below. (Yellow is pH 4.0; Orange is pH 5.0; Red is pH >6.5). What is the pH of the leaf disk as indicated by the assay? Record your observations and compare with your pre-stress observations. How did the pH of the leaf disks changed? Explain the biochemical basis of how this assay demonstrates CAM induction?
6. Optional: the assay can be made quantitative by reading the color absorbance (O.D. at 574 nm) of the Chlorophenol red solutions using a visible light spectrophotometer. Students can make up a standard pH curve and compare their results with the curve (see Figure 3).

   Figure 2:
   

   

   Figure 3:
   

D. Lab report:

Have students write up a full lab report. Discussion should include an overview of the biochemical pathway responsible for CAM, under what conditions is CAM a benefit to a plant, the ecological distribution and consequences of such an adaptation (are CAM plants found in the desert?), and potential for exploiting this type of adaptation for genetic engineering of crop plants with improve water use efficiency.

E. References:

Taiz L., Zeiger E. (2003) Plant Physiology. Third Ed. Sinauer and Associates.