Stimulation and Observation of Leaf Stomata
of 800 ppm and RH=64%. Te stoma opened between Events #1 and #2 and closed again aſter Event #2. Te time for the stoma to fully open aſter Event #1 was
and the chamber was again supplied with air having a CO2 level
approximately 80 minutes. Te time for the stoma to fully close aſter Event #2 was approximately 110 minutes. Tus, with all other parameters being equal, the stomatal response to the change in CO2 close when CO2
and humidity took nearly 40% longer to was restored as it did to open when CO2
was
removed. Videos showing the progress through these experiments
can be found at
https://paedia.com/
Stomata.html. Experiment No. 2. In this experiment, temperature, concentration were held constant while the
humidity, and CO2
illumination of the leaf was varied between two extremes. Te following steps were undertaken:
1. Koehler illumination was used. 2. Te R LED light source was activated and set to deliver 300 μmol/m2
light was OFF.
3. A recently irrigated T. spathacea plant was placed next to the microscope’s stage, and an attached leaf was taped across the microscope stage with the underside of the leaf facing upward, toward the objective lens.
4. A heater attached to the stage was activated and set to a tem- perature of 25 °C.
5. Te chamber was placed over the 50× objective and slid downward so that its lower gasket formed a seal against the leaf (Figure 4b) and the upper gasket sealed against the objective lens.
6. A hose was connected from a room air source to the inlet hose connector on the chamber, and room air with RH=65% and CO2
level of 800 ppm was pumped continu- ously through the chamber.
7. A stoma of interest was selected for observation. Te R light was temporarily switched off, and the W light was ener- gized long enough to record an image of the stoma. Aſter picture-taking was done, the W light was switched off and the R light was turned back on. Image stacks of the stoma were acquired and processed at periodic intervals. Stomal opening was determined by measuring the number of pixels between the stomal edges as shown in Figure 5c. Te ver- tical extent of each image was 2048 pixels over a span of 209 μm so each pixel represented 0.1 μm.
Results from Experiment No. 2. During Experiment
Figure 5: A stoma of T. spathacea (a) open and (b) closed (see Experiment No. 1). Image width=250 μm. (c) Opening of stoma showing where measure- ments were made (see Experiment No. 2). Stomal opening extent was deter- mined by measuring the number of pixels between the stomal edges. Vertical height of image=209 µm.
first event, #1 in Figure 6, the leaf was exposed to room air for 40 minutes. Te stomatal opening was closed for the conditions of light, temperature, humidity, and CO2 on the leaf. At Event #1, the CO2
22
#2, a stoma was stimulated to open and close by changing the intensity and wavelength of illumination applied to the leaf. Te height of the stomatal opening was recorded over time as the leaf was stimulated by two events. Te results are shown in Figure 7. Te abscissa is time in minutes, and the ordinate is the height of the stomatal opening in μm. Prior to Event #1, the stoma was allowed to equilibrate
concentration imposed
the chamber was supplied with air that had a CO2 level of 0 ppm and RH=85%. At Event #2, the CO2
scrubber was connected and scrubber was disconnected
at an opening of 5 μm at this level of R light. At Event #1, 15 minutes into the experiment, B light of intensity 100 μmol/ m2
·sec was added to the R light. Te stoma began opening
immediately and continued opening until it equilibrated at a maximum of 10 μm at 61 minutes. It was assumed that this was
www.microscopy-today.com • 2019 July ·sec at the surface of the microscope stage. Te B
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