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Stomata*

Stomata are of great interest for a variety of reasons. Some scientists are comparing stomata in recent and fossil leaves in order to better understand the importance of global warming1, 2. Others propose manipulating stomatal function using combinations of physiological and molecular genetic methods in order to improve water use efficiency of plants.3 This is especially important in the case of droughts. Plants are classified by the sizes, shapes, arrangements, and densities of stomata on leaves.4 Understanding the role and behavior of stomata provides guidance that can lead to improvements in foliar (leaf-applied) fertilization.5 The study of stomatal physiology can help in the discovery of new strategies to prevent pathogens from entering leaves.6 The locations of stomata on leaves can reduce water loss that can lead to fire risk. This can lead to planting of certain kinds of trees in forested areas that are subject to periodic wildfires.7 Detailed microtopographic images like those below can provide guidance for these and other purposes.

Stomata are found on the upper and lower sides of leaves, on flower petals, stems, and on roots. Scientists survey plant surfaces to determine the density and size of stomata and relate these findings to properties of the environment, such as the temperature and amounts of sunlight, humidity, oxygen and carbon dioxide in the air when a leaf is formed. Leaves of terrestrial plants generally have more stomata on their undersides. Floating leaves of aquatic plants generally have stomata only on their upper surfaces while some underwater plants have no stomata at all. The seeds of monocots (grasses, bulbs) have a single embryonic leaf or cotyledon. The major veins in monocots are parallel to the leaf axis. Their stomata are generally oriented similarly to one-another. Dicot seeds (oak trees, roses) have two embryonic leaves. Their leaf veins are in net-like arrangements. They have stomata that are randomly oriented. These differences are seen in the images below.

*See two accompanying articles in Microscopy Today:

Images of Leaf Stomata: Little Things that Matter

and

Stimulation and Observation of Leaf Stomata Using a Light Microscope

**Click here for links to selected T-shirts.

 

Specimen preparation

Epidermal Peel Epidermal Shave Epidermal Cast

These preparation methods are well known. There are many examples on the Internet. All specimens were wet-mounted. A homemade microtome was used for cutting wax-embedded leaf cross-sections.8

Equipment

A Nikon Eclipse LV100 microscope and a Point Grey Grasshopper camera were used. Koehler illumination was used throughout. Image processing and focus stacking software were used where appropriate.

Images

Each set of images includes a picture of a plant, magnified views of stomata, a stereo close-up, a dynamic perspective view, and a cross-sectional view of a stoma. The plants shown here all grow in San Francisco, California.

Click on an image to enlarge it.

Agave hybrid 'Blue Glow'.

Agave 'Blue Glow' is a succulent, monocarpic monocot. The leaf axis is vertical in these images.

 

Beschorneria yuccoides 'Flamingo Glow'.

B. yuccoides is a succulent monocot in the family Asparagaceae, subfamily Agavoideae. The leaf axis is vertical in these images.

 

Podophyllum pleianthum.

P. pleianthum is an herbaceous perennial dicot.

 

Masdevallia Highland Monarch 'Golden Sunshine'.

Masdevallia Highland Monarch 'Golden Sunshine' is a monocot.

 

Clivia miniata, yellow form.

C. miniata is a monocot. The leaf axis is vertical in these images.


In Vitro Time-Lapse Videos

In the images below, a leaf specimen was studied as soon as possible after a leaf was removed from a plant.
Stomata begin closing very quickly after a leaf is cut.

 

Tradescantia zebrina

This is an 8 minute time-lapse video of a closing stoma on a Tradescantia zebrina leaf.
Image width is 250 microns. An image was taken every 2 minutes.
The sequence reverses half way through the video.

   

Salvia microphylla var. 'Hot Lips'

This is a 14 minute time-lapse video of closing stomata on a Salvia microphylla leaf.
Image width is 622 microns. An image was taken every 2 minutes.
The sequence reverses half way through the video.

 

Cyclamen hybrid

This is a 2.5 hour time-lapse video of closing stomata on a Cyclamen leaf.
Image width is approximately 220 microns. An image was taken every 6 minutes.
The sequence reverses half way through the video.

 

Masdevallia hybrid

This is a 1.75 hour time-lapse video of a closing stoma on a Masdevallia leaf.
Image width is approximately 220 microns.
The sequence reverses half way through the video.

 

Veltheimia bracteata

This is a 1.3 hour time-lapse video of closing stomata on a Veltheimia leaf.
Image width is approximately 220 microns.
The sequence reverses half way through the video.

 

Coelogyne mooreana 'Brockhurst'

The center image is a 1.3 hour time-lapse video of opening stomata on a Coelogyne leaf in a CO2-free environment
with red and blue illuminations of 250 and 100 µmol/m2·sec. The sequence reverses half way through the video.
The right-hand image is an orthogonal view indicating heights of various features above the leaf surface.
Image width is approximately 220 microns.

 

Vetch sativa

The center image is a 10 minute time-lapse video of closing stomata on a Vetch sativa leaf. The sequence reverses half way through the video.
The right-hand image is an episcopally illuminated dark field view of the leaf underside showing vascularity connecting with stomata.
Width of the center image is approximately 220 microns. Width of the right-hand image is 620 microns. Click on the right-hand image to enlarge it.

 

In Vivo Time-Lapse Videos

In the images below, leaf specimens were studied while attached to the plant.

Experiment #1: CO2 and humidity transients.

A leaf was secured to a microscope stage that was heated to a constant temperature of 25 deg C. The leaf was illuminated with 100 µmol/m2·sec each of red and blue light and a stoma was selected for observation. The ambient CO2 and humidity levels were then varied. Prior to "1", the leaf was exposed to room air with a CO2 level of 800 ppm and 65% RH for 40 minutes. At "1" the ambient levels were changed to 0% CO2 and 82% RH, causing the stoma to open. The area of the opening stoma was measured every 10 minutes and plotted on a graph. After 80 minutes, the area of the open stoma reached a maximum. At "2", the leaf was again exposed to room air with 800 ppm CO2 and 65% RH, causing the stoma to close. The experiment lasted 250 minutes.




Tradescantia spathacea

Experiment #2: Blue light transients.

A leaf was secured to a microscope stage that was heated to a constant temperature of 25 deg. C. The leaf was illuminated with red light only at an intensity of 300 µmol/m2·sec and a stoma was selected for observation. Ambient conditions were CO2 = 800 ppm and RH = 65%. Prior to "1", with red light only illumination, the height of the stomal opening at its center had equilibrated at 5 microns. At "1" blue light at an intensity of 100 µmol/m2·sec was added to the red light. The height of the opening stoma was measured every 10 minutes and plotted on a graph. At about 60 minutes, the height of the stomal opening reached a maximum of 10 microns. At "2", the blue light was switched off. The opening rapidly closed with an undershoot and an overshoot before settling at 5 microns. The experiment lasted 190 minutes.




Experiment #3: CO2 and humidity transients.

This experiment is similar to Experiment #1 above except that a different stoma was selected. A leaf was secured to a microscope stage that was heated to a constant temperature of 24.5 deg C. The leaf was illuminated with 100 µmol/m2·sec each of red and blue light and a stoma was selected for observation. The area of the stomal opening was measured as ambient CO2 and humidity levels were varied. Prior to "1", the leaf was exposed to CO2-free air and 85% RH for 105 minutes. At "1", the leaf was exposed to room air with 650 ppm CO2 and RH = 65%. At "2", the leaf was again exposed to CO2-free air and 85% RH. At "3", the leaf was again exposed to room air with 650 ppm CO2 and RH = 65%. The experiment lasted 220 minutes.

 

Sequences

Tradescantia zebrina

This sequence shows the closing of a stoma on a Tradescantia leaf. The leaf was removed from a plant and images of a stoma were taken at 0, 10, and 60 minutes.
The top row shows plan views, the second and third rows show oblique views. Image width in the top row is about 220 microns.

 

Tradescantia zebrina

Click on an image to enlarge it.

This sequence shows plan and oblique views of the closing of a stoma on a Tradescantia leaf.
The leaf was removed from a plant and images of a stoma were taken at 4 minute intervals.
Image width in the top row is about 120 microns.