various experiments were designed to test this. Seeds of etr1-1 mutant plants were obtained from the stock center. Simultaneously, the corresponding wild type background plant seeds were also obtained.

3.6.1. Growth of Arabidopsis Plants

3.6.2. Phenotypic Analysis of Plants Stomatal bioassays

1. When the seeds have been obtained, store them at 4°C for at least 48 h, to break the dormancy of the seeds.

2. Sow both wild type and mutant 4 tr1-1 (mutant) seeds in separate trays of Levington's F2 compost with sand, after wetting the compost until it forms clumps in the hand (see Note 9). Sow the seeds evenly spaced out, and singly, using a toothpick with a wet end. On average approximately

15-18 plants can be sown on a 30 x 20-cm tray.

3. Place the trays in a controlled environment growth cabinet with the following settings: 22°C, 80% relative humidity, and

16-h photoperiod with lights of intensity 60-100 pE/m2/s (see Note 10).

4. Water the trays from below until the bottom of the tray is just above covered level (4ee Note 11). Water the plants three times a week, Monday, Wednesday, and Friday, at similar times, to maintain uniformity in plant growth.

5. Let the plants grow for at least 4 weeks until fully expanded rosette leaves have been obtained. Use leaves of similar size from both wild type and mutant plants.

H2O2 causes stomatal closure in wild-type Arabidopsis leaves (4) and this can easily be used as a system to study the function of various signaling proteins. To perform stomatal bioassays, it is of absolute importance that both wild type and mutant plants are grown under exactly the same conditions, preferably in the same growth cabinet. This avoids any differences and variations due to growth conditions.

1. Detach single leaves from different plants that have been grown for 4 weeks (see Note 12).

2. Place each individual detached leaf in a 5-mL Petri dish with 2 mL of MES/KCl stomata bioassay buffer.

3. Cover the dishes and place them in the growth cabinet for 2.5 h.

4. Add various doses of H4 O2 to different Petri dishes (wild type and mutant leaves).

5. Incubate for a further 2.5 h under the same conditions.

6. Blend each leaf individually in a Waring blender with 100 mL of water, for 30 s.

7. Collect the blended epidermal fragments on a 100-pm nylon mesh (SpectraMesh, BDH-Merck, UK).

8. Transfer the epidermal fragments to a glass microscope slide and place a cover slip on top.

9. Measure the stomatal apertures with the aid of an eyepiece graticule on a calibrated microscope. During measurements, make sure to move around and note apertures of at least 25 different stomata, from different fields of view. Also, ensure that the correct focal plane for the aperture is used. As the cell walls of guard cells are fairly thick, it can be difficult to focus on the inner walls of the aperture (see Fig. 7.2). Also, it is very important to repeat these experiments at least three to five times, preferably with different batches of plants. Using this method the results in Table 7.1 were obtained, which clearly indicate that with increasing concentrations of exogenous Hs O2, stomata of wild-type guard cells respond by closing, whilst stomata of the mutant etr1-1 plants do not close in response to H2O2. As the etr1-1 mutant is mutated in the Cys65 residue this confirms that this residue is somehow involved in mediating H2O2-induced closure in Arabidopsis guard cells.

Both exogenous and endogenous H2O2 are known to affect root growth responses in plants (5) and, therefore, one of the pheno-types that can be analysed in mutant versus wild-type plants is root growth in the presence of various doses of Hs O2. To do this, experiments are performed on seedlings, so as to be able to monitor root growth easily. For this, seedlings are grown in tissue culture on nutrient agar plates. Analysis of root growth

Fig. 7.2 Light microscope image of an open Arabidopsis stomate. Each cell surrounding the stomate is a guard cell. The arrow indicates the stomatal aperture (width) which is measured.

Table 7.1

Stomatal Aperture Measurements of Wild-Type (wt) and etr1-1 Arabidopsis I Response to H2O2 (see ref. 3).

Wt SEM etr1-1 SEM

Table 7.1

Stomatal Aperture Measurements of Wild-Type (wt) and etr1-1 Arabidopsis I Response to H2O2 (see ref. 3).

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