How do I measure stomatal density?

There are a number of ways of doing this. Because of the size of stomata, you will need a reasonably good microscope for this. Your choice of magnification will depend on the leaf material that you are using, and the size of the stomata

• Prepare an epidermal impression by coating the leaf surface with nail varnish. Peel off the dried layer of nail varnish by using sellotape and stick this onto a slide.
• Alternatively, with some plants you can peel off an epidermal strip directly, which you can mount in water on a slide and place under the microscope.
• If you have an eyepiece graticule which you can use, you can work at a relatively low power, and you can count the number of stomata within different squares to act as replicates.
• If you do not have an eyepiece graticule, you can work at a higher magnification and count a number of different fields - the area visible under the microscope - at any one time.

Make sure that:

1. You get enough counts to be able to analyse your results statistically,
2. You calculate the area of leaf which you are counting in order to give a quantifiable result e.g. stomata per square mm. You will need to calibrate the size of the field of view, or the size of individual squares within a field, using a stage micrometer to do this.

M. MacDonald, SAPS Cambridge.

One of the best plants for doing epidermal peels is the red hot poker plant Kniphofia. Being a monocot its stomata are highly ordered in rows, but they are big and great for stomatal opening and closing using solutions of different concentrations.

Almost as good is the Elephants Ear Saxifrage Bergenia. This also peels very easily, but the stomata are smaller although clearly visible at x100 magnification. This is a dicot so the distribution is more random.

B. Meatyard, SAPS Warwick.

What factors influence the opening and closing of stomata?

There are many factors which lead to stomata opening and closing.

i) There is an endogenous rhythm (a biological clock). Stomata open during the day and close during the night. (Though certain succulents which are native to hot, dry conditions have a reversed rhythm to enable them to economise on water loss.) However, stomata continue to open and close on an approximately 24 hour clock (circadian = about a day) even when switched to continuous light. The phase of this opening and closure can be shifted (made to occur at other times of the day) by contol of the end of the dark period.

ii) The water balance of a plant affects stomatal apperture. Wilting plants close their stomata. The plant growth regulator abscisic acid (ABA) seems to act as a mediator under these conditions. Water stress in the roots can transmit (in xylem?) its influence to stomata in leaves by the signal of ABA.

iii) Low concentrations of CO2 cause stomata to open. If CO2-free air is blown across stomata in darkness, their stomates open. High CO2 causes stomates to close.

iv) Light causes stomates to open. The minimum light level for opening of stomates in most plants is 1/1000 to 1/30 of full sunlight, just enough to cause some net photosynthesis. Blue light (430-460nm) is nearly 10 times as effective as red light (630-680nm). The wavelengths that are effective in the red part of the spectrum are the same as those that are effective in photosynthesis ie is absorbed by chlorophyll. However, the blue light effect is quite independent of photosynthesis. Photosynthesis will change intercellular CO2 concentrations and may have its effect through number iii) above.

So, how are these movements brought about?

Blue-light wavelengths of daylight, detected by zeaxanthin (a carotenoid) activate proton pumps in the guard cell membranes, which proceed to extrude protons from the cytoplasm of the cell; this creates a "proton motive force" (an electrochemical gradient across the membrane) which opens voltage operated channels in the membrane, allowing positive K ions to flow passively into the cell, from the surrounding tissues. Chloride ions also enter the cell, with their movement coupled to the re-entry of some of the extruded protons (Cl/H symport) to act as a counter-ions to the potassium. Water passively follows these ions into the guard cells, and as their tugidity increases so the stomatal pore opens, in the morning. As the day progresses the osmotic role of potassium is supplanted by that of sucrose, which can be generated by several means, including starch hydrolysis and photosynthesis. At the end of the day (by which time the potassium accumulation has dissipated) it seems it is the fall in he concentration of sucrose that initiates the loss of water and reduced turgor pressure, which causes closure of the stomatal pore.

ABA also seems to trigger a loss of K ions from guard cells. Some workers suggest that in some species, ABA alters turgor pressure without changing solute potential or water potential.

There is evidence of a role for increased cytoplasmic calcium (Ca2+) in closure, possibly by effects on opening/closing of ion channels at the plasma membrane.

Starch break down to phosphoenol pyruvate (PEP) is stimulated by blue light. This PEP then combines with CO2 to fom oxaloacetic acid, which is converted to malic acid. It is H ions from the malic acid which leave the cell in the mechanism outlined above. Thus the intake of K ions is matched by formation of anions from malic acid in the guard cells. This causes an increase in osmotically active substances in exchange for the breakdown of starch in guard cells.

References:-

Hart, J.W. in Light & Plant Growth (1988), 2nd Impression 1990. (pp 135-6).

Taiz and Zeiger in Plant physiology - 2nd ed.(1998) published by Sinauer, ISBN 0-87893-831-1 (pp 522-530)

Salisbury and Ross in Plant Physiology - 4th ed (1992) published by Wadsworth, California, ISBN 0-534-15162-0

John Hewitson, Roger Delpech and Richard Price

Do all stomata on a leaf or plant open at the same time?

Our previous answer (above) explains that the opening/closing of stomata is a complex process controlled by more than one variable. In addition to what is mentioned there, Abscisic acid (ABA) acts as a hormone and causes stomata to close.

So .... we don't know the true answer to your question!

If ABA is the mechanism (as, I believe, it is under drought conditions) then I would predict that all stomata would move in unison as the hormone spreads throughout the leaf/plant.

On the other hand, Barry Meatyard comments:-
Since stomatal opening is controlled by the turgor of guard cells and turgor of guard cells is presumably controlled by the local water relations in the environment of the stoma itself (rather than by remote control from the rest of the plant), then I could see a situation where if one leaf was in humid conditions and another in a locally drier environment the stomata on these leaves would vary in the degree of opening. Such a differentiated environment would presumably occur as a gradient from the 'surface' of a bush into its centre. As a hypothesis it would be worth testing with some epidermal peels or nail varnish 'casts'. How about a sample of leaves varnished in vivo in a privet hedge?

This is a good opportunity for some real open ended discovery based on a real experiment.

If you do the experiment, please let us know what you find out!

Will stomata density be greater in dicots or monocots and why?

We wonder why you ask this question? Do you have reason to believe that stomatal density is related to whether a plant is a dicot or monocot?

The most significant difference between the stomata of monocots and dicots is the design of the guard cells - the monocots having the dumbell type, and dicots the pair-of -sausage type. Also the monocots have them arranged in regular arrays, whereas the dicots have more of a crazy-paving of them!

The role of stomata is to enable gaseous exchange whilst trying to minimise the consequent water loss.

We guess that stomatal density stated in terms of "number of stomata per square mm" would also depend on the size of the stomata. Some plants may have many small stomata whilst others have few large ones. However, each would aim to achieve the same effect in terms of gaseous exchange and water evaporation.

Monocots have stomata on both the "upper" and "lower" surfaces of their leaves, whilst SOME (but not ALL) dicots have stomata on only one surface (usually the lower one), so on this basis, to achieve the same effect, a monocot may need half the stomatal density of a dicot of a type with stomata on only one surface. However, many dicots have stomata on BOTH surfaces and some aquatic plants with floating leaves have stomata on the upper surface, so it is not possible to generalise about ALL monocots and ALL dicots.

However, as a "stoma" is strictly speaking, the hole in the structure, we might guess that the total stomatal area per square mm of leaf surface would be the same for the same amount of gaseous exchange/transpiration.

We would normally expect stomatal density to be related to the climate in which the plant is adapted to grow. Some cacti have no leaves, but green stems instead, perhaps because there is a lower stomatal density on stems than on leaves - the leaves have been reduced to the spines on the cactus. The stomata on such xerophytes may be in deep pits or in the folds of leaves. We would guess that xerophytes have a lower stomatal density than mesophytes.

A monocot which is good for studying is Red Hot Poker (Kniphofia) becasue the epidermis peels off so easily.

John Hewitson, Barry Meatyard, Roger Delpech and Kath Crawford

What is the relationship between increase in stomata density and rate of transpiration?

Salisbury and Ross. Plant Physiology. Wadsworth Publishing Co. 4th Ed Chapter 4

"Nature often proves to be more complex than we expect. Suppose we compare the evaporation rate from a beaker of water and from an identical beaker that is half covered, say with metal strips. We would expect evaporation from the second beaker to be about half that from the first. Now let's cover all but about 1% of the second beaker. We will use a thin piece of foil with small holes making up about 1% of the total area. Will we measure about 1% as much evaporation? Not if the holes have about same size and spacing as the stomates found in the epidermis of a leaf. We will in fact measure about half as much evaporation (50%) as from the open surface.

"How can this be? Why isn't evaporation directly proportional to surface area? It certainly seems paradoxical that stomatal openings on the leaf make up only about 1% of the surface area, whereas the leaf sometimes transpires half as much water as would evaporate from an equivalent area of wet filter paper. We resolve this apparent paradox be realising that evaporation is a diffusion process from water surface to atmosphere. Simply stated, diffusion is proportional to the driving force and the conductivity. In our example, the driving force is the same for both beakers: the difference in vapour pressure (or density) between the water surface (where the atmosphere is saturated with vapour) and the atmosphere some distance away (where it must be below saturation if evaporation is to occur).

"The different evaporation rates depend on different conductivities to diffusion. Part of the conductivity is a function of the area, and this value is much lower above a beaker covered with porous foil, which is what we expected. But the other part of the conductivity depends on the distance in the atmosphere through which the water molecules must diffuse before their concentration reaches the atmosphere as a whole. The shorter the distance, the higher the conductivity. This distance can be called the boundary layer, and it is much shorter above the pores in the foil than above the free water surface. Molecules evaporating from the free water will be part of the relatively dense column of molecules extending some distance above the surface, whereas molecules diffusing through a pore can go in any direction within an imaginary hemisphere centred above the pore. In the hemisphere, the concentration drops rapidly with distance from the pore, which is to say that the concentration gradient is very steep because the boundary layer is very thin. Of course, if pores are closer together than the thickness of their boundary layers, these hemispheres overlap and merge into a boundary layer.

"Many empirical studies were made several decades ago to determine the effects of pore size, shape and distribution on diffusion rates (eg Brown and Escombe 1900, Sayre 1926, reviewed by Meyer and Anderson in 1939). Stomates of typical plants proved to be nearly optimal for maximum gas or vapour diffusion. Thus, plants are ideally adapted for CO2 absorption from the atmosphere - but also for loss of water by transpiration. The stomates can close, however, and in most plants they are adapted to close when photosynthesis and CO2 absorption stop (for example in darkness)."

How can I investigate whether size of stomata affects transpiration rate?
(for an AS level investigation)

You need to think at a basic level:-
1) How can you measure transpiration rate? Remember that a bubble potometer (for instance) measures water uptake whereas a weighing potometer (weighing a plant in a sealed water reservoir) should give transpiration. (Changes in mass due to photosynthesis and respiration can probably be ignored.) It may be sufficient to simply hang up leaves of different types and measure their loss in weight over a period of time. You can cover surfaces with vaseline if you wish to compare how much is lost from the lower surface with stomata with how much is lost from the upper surface.

2) How can you measure stomatal size? With most leaves, you can paint the surface with nail varnish and leave it to dry, then press selotape to the leaf surface and peel off the nail varnish impression and stick it to a microscope slide to measure the size of the stomata under a calibrated microscope. Now, this is almost impossible to do as the stoma is actually the hole between the guard cells and you rarely get the chance to measure the diameter of the hole - perhaps you will just have to measure the length of the closed hole and make some assumptions from there - perhaps you will just have to measure the diameter of the guard cells and make some even bigger assumptions from there.

If you haven't got the names of some varieties of leaves which you can get hold of, I would simply choose some mesophytes and xerophytes and get measuring. I happen to know that hairy leaves are no good for the nail varnish method, so several xeromorphs may be ruled out. The value of an AS project is to a) plan an experiment which will test your chosen hypothesis, b) choose appropriate apparatus and show you can use it skillfully c) take measurements and keep sensible records d) analyse the experiment and write your conclusions and e) comment on the reliability and accuracy of your experiment with comments on how the experiment could be improved. What is NOT marked is how well your data fit your prediction! ie, there are not many (if any) marks which depend on choosing the right leaves.

John Hewitson

Does wind speed affect the opening of stomata?

I am exploring the effect of wind speeds in light and dark environments on transpiration. If the wind speed is high enough during the night time will this cause the opening of stomata and transpiration, even though it is against the plants bioloical clock to do so?
I've got 3 different wind speeds on the fan and I'm keeping the temperature constant in both light/dark environments.

Wind speed does not normally cause stomata to open. Indeed, high wind speed may cause stomata to close a) because of the high rate of transpiration leading to water stress but also b) (when photosynthesis rate is high) gentle breeze can bring more CO₂ close to the stomata, increasing the diffusion of CO₂ into the leaf, causing guard cells to become less turgid.

Stomates will normally be closed in the dark. Granted, there may be a tendency for the stomata to be open if you are doing the experiment during daylight hours and the plants have not had a chance to reset their biological clock. On the other hand, I would expect the biological clock mechanism to be over-ruled by the other environmental conditions you are imposing on the plants which (I guess) will have a stronger effect on the stomatal aperture than the diurnal rhythm. Opening of stomata at sunrise generally requires about an hour and closing is often gradual throughout the afternoon. Stomates close faster if plants are suddenly exposed to darkness. The minimum light level for opening of stomates in most plants is about 1/1000 to 1/30 of full sunlight - just enough to cause some net photosynthesis. Higher irradiance levels cause wider stomatal apertures.

This should be an interesting series of experiments. Will you measure the leaf area and quote your results in terms of g water/mm² of leaf? How long will you need to wait after you have changed the conditions before making your critical measurements (an hour?) Will your kale be planted in soil, or will you be removing a kale leaf from the plant for experimentation?

John Hewitson

Why does polyploidy result in increased stomatal size? I have been doing an investigation into stomatal size in Tagetes species and found that 4n Tagetes have fewer stomata per unit area than 2n Tagetes species.

This is a fascinating topic and I have not been able to find a clear answer in the literature.

Indeed, it is well known that stomatal size is bigger in polyploid plants.  I was involved with a project where we scanned hundreds of plants treated with colchicine by looking at the size of their stomata, in the hope of identifying any polyploids the colchicine had produced.

This article confirms your findings (but not in Tagetes) :-

This article suggests that diploid and polyploid plants are quite different in their water content:-

Another article suggested that the osmotic potential of diploids and polyploids is different.

All these features might be expected to influence the transpiration properties of the leaves and the changes in stomatal size and stomatal density might be (in part) a response to this.

Ottoline Leyser tells me that "There is a well-established correlation between ploidy level and cell size - polyploid cells are bigger.  It seems to me that if you have bigger guard cells you would have to have bigger stomatal pores. If the pores are bigger then the plant would make fewer to compensate, because they make the minimum for sufficient CO₂ uptake."

John Hewitson