Previous Next

Plant Detectives Manual: a research-led approach for teaching plant science

Activity 11: Stomata and the effect of the hormone ABA

11.1) Introduction and objectives

In this activity we will look at the effect of the hormone abscisic acid (ABA) in promoting stomata closure. Stomatal response to the environment allows plants to control the loss of water (by closing stomata down) or intake of CO2 (by opening stomata). ABA is produced under abiotic stress conditions, such as high light, water deficit or osmotic shock. One of the many effects of ABA is the promotion of the reduction of guard cell volume, thus inducing the closure of the stomatal pore. It is possible to artificially treat leaf peels containing both epidermal and guard cells with ABA and study the hormone’s effect on stomata closure (Fig. 11). The protocol described below may be conducted in conjunction with the microscopy exercises described in Activity 10, or on its own.

 

The objectives of Activity 11 are to:

  1. visualise and identify epidermal and guard cells
  2. compare the response of the wild type and mutant stomata to ABA.

 

Image

Figure 11. Micrograph of epidermis and stomata in Arabidopsis

A) Surface view of the Arabidopsis epidermis showing irregularly shaped epidermal cells and guard cells and the guard cells (GC) outlining the stomatal pore. B) Close up of the guard cells (GC) surrounding the pore. Incubation with ABA promotes stomata closure. Image courtesy of Nok Pornsiriwong (The Australian National University).

Image

11.2) Materials

  1. microscope slides
  2. microscope (one)
  3. sticky tape (Sellotape)
  4. Petri dishes
  5. thin forceps (one–two)
  6. razor blades (one–two)
  7. opening buffer (50 mM KCl, 0.1 mM CaCl2, 5 mM Mes, pH 6.5) without or with 50 micrometres (µM) ABA

11.3) Procedure

This protocol is based on the ones described in (Roelfsema and Prins 1995; Desikan et al. 2002).

Materials

Method

Sellotape, scissors, plant

  1. Cut one leaf from each of five plants of between five- and six-weeks old and remove the midvein with a razor blade. CAUTION: place the top side of the leaf (adaxial) down onto a strip of sticky tape.

 

  1. Cover the other side of each leaf (the bottom or abaxial side, where most of the stomata are) with another strip of sticky tape, pushing gently with your fingertips.

Razor, scissors

  1. Trim the sides off before gently removing the strip to recover the leaf peel from the abaxial side. Trim the excess sticky tape with scissors if necessary.

Petri dish, ABA, OB

  1. Place the leaf peel attached to the sticky tape strip into a Petri dish containing opening buffer.

 

  1. Incubate for 30 minutes under a lamp at 150–200 micromoles (µmol) s–1 m–2

Petri dish, ABA, OB

  1. Place half of the strips of each genotype into another Petri dish containing the opening buffer plus 50 µM ABA. Leave the other half in opening buffer only.

 

  1. Incubate for 1.5 hours on the bench (no lamp).

Microscope

  1. Begin looking at the control samples (those in the opening buffer) towards the end of the incubation time.

 

  1. Look at the microscope and record the number of stomata and the number of epidermal cells. Note how many stomata are open, semi open, or closed, and any other feature that you think is relevant. If possible, take photos of one or more microscope field views for each sample to count and analyse stomata. If you want to quantify stomata closure, measure the stomata width and length for a set number of stomata from one leaf on each of three plants per genotype using a calibrated micrometre scale. Express the stomata aperture as the width-to-length ratio.

 

  1. Examine the samples incubated in the presence of ABA under the microscope after 1.5 hours.

 

  1. If you take photos of your samples to analyse later, be sure to label them with your group number, date, genotype and plant number. For example: 01–20100705–Col–0–05, is the photo of plant 05, genotype Col–0, taken on 05/07/2010 by group 01.

 

IMPORTANT: you may want to practice this technique on spare plants before applying to your samples.

 

During the long incubation, take more leaf peels and observe the anatomy of the epidermal cells under the microscope. This will familiarise you with the epidermal cells (i.e., examine and differentiate guard cells from pavement epidermal cells) when you start to examine the samples in the buffer. This should facilitate the final analyses of the effect of ABA in the stomata closure between genotypes after the two-hour incubation.

11.4) Expected outcome

  1. Compare the epidermal peels of the two genotypes. Specifically, look at the epidermal cells and guard cells. Is there the same number of stomata? Are they closed or open?
  2. Calculate the following for each photo of the microscope field view:
  1. Stomatal Index; SI=S/(S+E), where S is the total number of stomata, and E the total number of epidermal cells.
  2. Stomatal Density; SD=S/A, where S is the total number of stomata present in area A (how would you determine the area?).
  3. Measure the length and width of a few guard cells in each peel, expressed in µm to assess closure.

Compare the effect of ABA on stomata closure between wild type and mutant. Are the stomata closed in both genotypes after ABA incubation?

Assess whether the differences in stomatal size, number or closure are statistically significant between ABA and control treatments for the wild type and mutant. Remember, this is a factorial design so will require two-way statistical tests (See Appendix B).

 


Previous Next