In most plants about 98% of the water taken in by the roots is transpired from the leaves' surfaces. To give some idea of the magnitude of water movement, it has been calculated that during the day a 15 meter high Silver Maple (Acer saccharinum) can lose up to 220 liters of water per hour through transpiration.
Carbon Dioxide & Light:
Plants contain specialized structures within the epidermis of their leaves which allow for the uptake of carbon dioxide (used as the carbon source for photosynthesis) and the release of water. These structures are composed of openings, known as stomata’s, and guard cells which regulate the size of the stomata’s. Guard cells respond to various environmental stimuli by shrinking and swelling which, in turn, regulates the size of the opening.
Within plants most stomata are closed in darkness yet are stimulated to open in light. This is similar to the method for plants when carrying out photosynthesis. This method is most common when looking at stomata openings but is not the only method, some plants are able to open at night. Although this is because the particular plant is not focusing on the amount of light available but the amount of carbon dioxide available. This carbon dioxide gained during the night is stored by the plant and fed into the clavin cycle during day time. A low concentration on carbon dioxide causes the stomata to open; a high concentration leads to their closing. The activity of photosynthesis taking place in the guard cells where chloroplasts are found is related to osmotic value and thus also related to the opening of the stomata.
Light is able to exert its effects mainly by decreasing intercellular and intracellular concentration of carbon dioxide. Carbon dioxide is faster consumed than supplied due to mesophyll’s photosynthesis. This causes an intercellular deficit close to the stomata’s opening. The photosynthetic activity within the guard cells themselves leads to a decrease of the intracellular level of carbon dioxide, and causes simultaneously that water is drawn from the subsidiary cells. The more light available the more stomata you would find located on the epidermis of the plant.
The number of stomata within a given area of a leaf varies depending on the species of the plant. The average number of stomata/in a leaf is about 30,000.There is also often a large difference between the number of stomata on the upper and lower surfaces of a leaf. For example, the tomato leaf has approximately 13,000/on it’s lower surface but only 1,200/on its upper surface.
Stomata are commonly located on the lower surface of the leaf, and in many trees and shrubs they are absent altogether from the upper epidermis. In other plants where the leaves are found held vertically, stomata is located on both sides of the stomata. One reason that the stomata is often only located on the lower epidermis is that the guard cells and substomatal air spaces would tend to scatter incoming light if they were on the upper epidermis. As well as this the slightly lower temperature on the surface may reduce transpiration.
When heated plants may denature to avoid this an individual plants may open its stomata and evaporate water which will lower the leaf temperature. One may hypothesize that leaves in the sun should have higher stomata density than do leave in the shade all else being equal.
If water is not available, for example in drought conditions excessive conditions may lead to desiccation and an equal amount of severe denaturisation taking place in the plant.
Thus one might expect plant leaves exposed to drought conditions have fewer stomata in sunlit environments. This knowledge I have vacated on the effects of light and temperature would support and help to prove my prediction the idea that the environment in which my leaf types were found would have an effect n the amount of stomata found.
Within this investigation I shall look at the stomata numbers within the following leaves where I have carried out particular leaf types:
Ivy
There are certain types of plants which have the ability to climb up various plants and structures. Such plant types are known as climbers they come from a range of plant families they are nearly all dictyledons. In most habitats where climber plants are located in particular dense jungles, the climbing ability is necessary as a means of reaching sunlight and reaching their full potential in making photosynthesis to survive. This act as a continuous circle of life for the climber plants where by the energy saved by the plant by climbing can be used to enable the plant to grow faster, farther and higher than neighbouring plants which gives me the plant a positive advantage over others to be in access of the sunlight and gain even more energy. The particular leaf type I chose for my experiment from this climber family was the ivy plant type. I recovered this leaf from my local school court where it was located carrying out this climber trait upon a wall structure where it was entwined among other of its leaf type all competing for available light.
From this above evidence I have vacated a prediction that the Ivy leaf type having the climbing ability would contain an average amount of stomata on their leaves since they don’t live in really dry or very moist conditions. Their great requirement for sunlight’s which supplies its energy, suggest that they would have fewer stomata on the top of the leaf that the bottom which would provide less interference with the photosynthesis.
Holly Leaves
These are adapted to growing in boreal zones and on mountains, where there may be a lack of water when the ground is frozen in winter. In addition their pyramidal shape ensures that snow is more likely to slide off the branches that collect on then, possibly breaking them. The conifer leaves are constructed to resist drying out, with a thick waxy cuticle and, usually small in size.
From such evidence I predict that the holly leaves will have a similar figure of stomata on both top and bottom epidermis layers of the leaves. Reasons for such a prediction are that these particular leaves survive through winter time where sunlight is scarce. Therefore a large surface area is not necessary for photosynthesis, the larger the surface area the larger the number of stomata. In addition the area of the holly leaf is quite moist; they can afford to lose large amounts of water, meaning their number of stomata is large.
Privet from a bush
Privet is a common ornamental shrub. This plant, a mesophyte, is adapted to a moderate habitat that is neither very wet nor very dry.
A diagram of the stomata found within Privet Bush:
Privet Transpiration:
Variegated Holly:
Method
A convenient means of explaining stomatal distribution is to make a replica of the leaf surface using the brush in the bottle. After applying the nail polish, I will allow it to dry, I then was able to peel off the replica slowly using forceps. I will then mount it onto a slide and use a cover slip. By examining the replica slowly under a microscope, the stomata in a given field of view of the microscope will be determined by the measuring the diameter with a calibrated slide or transparent ruler. The amount of stomata will then be calculated by using the formula πr2 (where r is the radius and the π = 3.142). After gaining a mean value of the stomata found, comparison between the distribution of stomata in the upper and lower in different species epidermis will be made.
- Make a replica of leaf surface using the brush in the bottle.
- Allow it to dry, peel off the replica slowly using forceps.
- Mount it onto a slide and use a cover slip.
- Examine the replica slowly under a microscope, the stomata in a given field of view of the microscope which is determined by the measuring the diameter with a transparent ruler.
Equipment / Apparatus:
Clear Nail Vanish- This was required in order to make and exact replica of the plants epidermises clear small and could be easily peeled off and place onto a slide it was the best replication method available to me.
Slides- These were required as a base to place my replica upon n order to see the replica under the microscope.
Cover Slips- These were required in order to keep the replica on the slide in order to easily see the replica on the microscope so that the replica nail polish didn’t curl up or com off the slide.
Fine Forceps- These were required to peel of the nail vanish once dry from the leaf surface and placing it onto the slide without it curling up or having my finger prints over the replica and thus being unable to see the replica.
Microscope- This was required in order for me to visualise a good picture of the replica to give me a big enough magnification of the stomata on each leaf surface.
Materials:
Holly Leaf,
Privet Leaf,
Variegated Holly Leaf,
Ivy Leaf
Fair Test
The variables: In order to make this investigation a fair test, the test will be carried out on different types of leaves to see if this will affect the number of the stomata depending on the location the leaf type came from this will therefore be the only variable to be changed. There fore the key variables involved include the leaf type the epidermis layer which I will also be changing and the different environments which my leaf types came from. By having three different variables I am able to compare and contrast the distribution of stomata in each leaf to give e more reliable and accurate results.
I will use the same size slides and cover slips, I will also use an equal amount of nail vanish on each leaf surface by using one large sweep of the brush onto the surface.
Three different people will count the number of stomata on each replication, to get an unbiased number and then an average will be taken. Only the stomata in the field of view will be counted, to ensure everyone is counting the same surface area and it is equally fair for each replication. The same magnification will be used when viewing under the microscope.
Safety
- Safety goggles were worn when looking down the microscope, to prevent serious accidents in case someone is pushed.
- All hair should be tied back all lose clothing tucked away and always wearing an apron.
- Being careful and aware of surroundings at all times, expensive equipment is being used i.e. the microscope.
- Taking care when cutting the leaf types chosen with pliers no to damage the plants or your hands.
- Using caution when dealing with the slides and cover slips as although they are glass still very fragile and if broken can be very dangerous as sharp.
- Should be aware of the chemicals being used as nail vanish is a solvent, so you should be careful not to inhale too many vapours when carrying out experiment.
- When using the particular leaf types one should be careful as some have sharp edges which are part of their protection in their initial environment i.e. the holly leaf.
Detailed Procedure for Obtaining Stomata Impressions:
I firstly began my investigation by obtaining the particular leaves upon which I wished to census stomata. I then decided upon the side I wished to censer the stomata, typically the leaf underside although my investigation involved comparing the two and thus I would eventually censure both sides of plant. I chose to begin censuring the upper epidermis here I painted a rather thick swath of clear nail polish upon the surface of the plant. Once the nail polish had dried taking several minutes, I GENTLY, peeled my nail polish swath from the leaf using the forceps making sure all nail polish was completely peeled off the leaf surface. Here I was aware I was carrying out my method of replication correctly when I saw a cloudy impression of the leaf surface attached to the nail polish swath. Hereafter my complete swath could be referred to as my "leaf impression".
I next carefully placed my leaf impression onto the centre of a VERY CLEAN slide using the same forceps required for peeling off my impression. I found it particularly effective to use two forceps if having difficulty placing impression on slide flat and well spread out. I then used a pen and wrote an ID code signifying the treatment group name (e.g. the leaf type and which epidermis side impression taken from) onto a tissue and placed it above the slide. Once the replication was placed flatly on the slide I then placed carefully a cover slip over the top to hold the replication in place.
Once cover slip was in place I was required to place my leaf impression in the centre of the stage of the microscope followed by placing the metal holders over the slide. Next the important and for some the more difficult process of this method to focus my leaf impression under the microscope at least 400x power and observe the stomata. Next I search around on your impression to find an area that subjectively appears to have a high density of stomata. That is, move the slide around until the field of view is away from the edge of the impression and so that there are no dirt blobs, no thumbprints, no damaged areas, and no big leaf vein impressions in view.
I subsequently counted all the stomata I see and record the number neatly on a clearly labeled data sheet. The design of my data sheet on which I recorded my stomata counts was clearly designed to indicate which data corresponds to which leaf and epidermis side of plant. Example of tables used:
I repeat the method six times per epidermis, of which I had four different leaf types in all I had 96 slides of which I viewed and counted the stomata counts. From all six stomata counts I recorded I took an average which was calculated by adding up the results of each six readings and dividing this sum by six.
You should use a stage micrometer so that you may convert your data from units of "stomata number per field of view at 400x" to units of "stomata per “mm²." There are subtle differences among microscopes in the exact size of the field. You must convert your data to units of "stomata/ mm²." I carried this out by recalling that the area of a circle = pi * radius^2. To work out your measurement of the area of the "field of view" at 400x should be about 0.12 mm^2.
Epistomatic: Number of Stomata confined to the Upper Epidermis
Hypostomatic: Number of Stomata confined to the Lower Epidermis
To work out the Magnification for the area of view:
The magnification chosen to be used from the microscope was x40 which was equal to 5mm diameter. From this information I could work out the area = πr2 the radius/r is equal to half the diameter = 3.14 x 2.52 = 19.62m2 as we are looking at a x 400 magnification I then divide the found area by 10 (19.62/10) = 1.962mm2
To work out the number of stomata found in area of view you divide the average number of stomata sound by the area found i.e. 23.6/1.962 = 12.02mm2
Conclusions& Discussions:
As you can see from above graph there were actually no stomata located on any of the leaf types which is a big difference when compared to the number of stomata located on the lower epidermises of each leaf type I investigated.
On the above graph you can see that I found a substantial amount of stomata located on the lower epidermis or the hypostomatic side most stomata located on the privet leaf at 62.83 rounded to 63 stomata found, the least number of stomata found was on the variegated holly.
As stated in my conclusion I discovered that out of all four leaf types the Privet leaf had the most number of stomata on its lower epidermis. There are several reasons for this these include the environment in which I took the privet from and the personal characteristics this particular leaf type has. As stated in my background knowledge the privet leaf is well adapted to a moderate habitat that is neither very wet nor very dry. Thus the stomata within the Privet would be well adapted to opening and closing in the particular areas where it’s wet or dry as the environment in which I picked the privet was neither dry nor wet in between.
The privet leaf it adapted for the process of photosynthesis with broad leaved plants, the leaves are large, thin, flat structures. There leaves are large in fact larger than that of the other leave type and thus have more stomata, in order to trap lots of light energy. Their leaves are also thin so that the carbon dioxide can diffuse into the leaf from the surrounding air. With more carbon dioxide available in their surroundings compared to that of other leaf types investigated there is a greater number of stomata simulated to be within the plant in order to intake the maximum amount of carbon dioxide available and allow the least amount of transpiration to take place. The second highest was the holly leaf type at the average stomata count of 33.037 stomata’s per field of view reasons for this include that they do not have as big a surface area as the privet leaf as it is not necessary for photosynthesis as plant is adapted to living in an area of scarce light.
From the graph it can be seen very clearly that most of the stomata will be found on the lower epidermis, as there is no variation depending on the type of leaf as the average number of stomata on the upper epidermis for each leaf type was 0. Although on each of the lower epidermises there is more than double the amount compared to that of the upper epidermis the maximum at an average of 60.867 on the privet leaf. The geranium was the only leaf that did not show a great variation but a clear conclusion can not be drawn from this as the number of stomata was only counted once on both the upper and lower epidermis. The spider plant and begonia had no stomata on the upper epidermis and although the grape ivy showed an average of 2.5 stomata, group 1 only counted 1 stomata but group 2 counted 9, a clear conclusion cannot really be drawn from this because group 2 only carried out the test once.
My prediction that the greatest number of stomata will be found on the lower epidermis was proved correct, as was seen by all the leaves inspected. All the leaves proved without doubt that the greatest number of stomata are found on the lower epidermis. The ivy, holly, variegated holly and the privet were all thin leaves without a visible waxy cuticle so the stomata are located on the lower epidermis to prevent excessive water loss as they have no waxy cuticle to protect them. Also they are relatively thin leaves so the exchange of CO2 and O2 can occur relatively quickly and easily through the stomata of the lower epidermis.
Evaluation
The sources of error in my investigation could have been; the fact that different people counted the number of stomata. The error could have occurred if someone did not know what stomata looked like or they did not look in the same field of view as the last person. To try and overcome this error everyone was given a picture of the stomata before the investigation. Magnification was a variable which was kept constant, although someone may have adjusted it. Another error in my investigation included the when peeling the nail varnish, in some cases it was rather difficult to peel the replication away from the leaf surface completely. If the replication was not peeled completely the result would mean a poor slide being produced making it difficult to see the stomata. There was a chance of mixing up slides, which would need to be over come if a good investigation was to be carried out. Other Problems with stomata impressions: some leaves are prone to damage from the solvent in the nail varnish. The leaves absorb it, turn brown, and fail to produce any impression. Pupils lose interest and get frustrated because their leaves 'aren’t working'.
Another similar experiment, which could be carried out, is using cobalt thiocyanate. In the anhydrous state cobalt thiocyanate is blue, but when hydrated it turns pink. A piece of cobalt thiocyanate paper is placed on each side of a leaf and sandwiched between two glass slides clamped together, and then a stop clock started you would measure the time it takes for the cobalt thiocyanate to go pink as this indicates that water has escaped out of the leaf. The time varies in which the colour change takes place depending on the temperature and humidity. Generally the pink colour develops more rapidly on the lower epidermis of the leaf than upper surface, the reason already being discussed in the investigation.
A successful alternative is to use a clear water based varnish. A half litre tin is cheap, and can be divided up into smaller amounts for ease of use. Paint the opaque varnish thinly on to the leaf to produce a clear film. Leave it to dry as usual. This particular water based varnish takes longer to dry, so if the leaves are coated during one lesson, the impressions can be peeled off and examined the next. The varnish is non toxic, so can be used on living plants without removing the leaves this means that plants do not have to be denuded for this experiment. In addition to revealing the stomata, the cell walls also show up for a clearer image.
Other suggestions include producing impressions on acetate film, by placing a leaf in propanone and then pressing it onto the acetate. Although this does not work for some plant leaves, especially those that have an uneven surface and the leaf still has to be removed from a plant. Another method is to rub a board pen over the surface. The solvent-based ink permeates the leaf, showing up the stomata. However, this seems to work only with certain types of pen probably related to the strong solvent in the pen. Although this raises more severe health and safety issues.
Further Work:
If this investigation was to be carried out again I would use a greater variation of leaves, different shapes, sizes, thickness and leaves from different habitats to see what affect this would have. Also when peeling off the nail varnish the area would be calculated so that everyone was counting in the same area also make sure that everyone repeated the test. Attempts should be made to carry out similar investigations