THE EFFECT OF DIFFERING LEAF HEIGHTS IN PINUS SP.

ON STOMATAL DENSITY

ALAN MO

James Madison University

ABSTRACT

Stomata are plant structures typically found under leaves that control water loss. Plants

regulate water loss either by opening and closing their stomata or by varying their stomatal

densities. The purpose of the experiment was to see how sunlight exposure affects stomatal

density. Since high light intensity correlates with higher stomatal density (Tichà 1982), it was

predicted that there are more stoma on higher leaves than lower leaves. However, there may be

more stoma on lower leaves if water stress is high; the higher leaves would create shade for the

lower leaves. Therefore, the effect of differing leaf heights in Pinus sp. on stomatal density was

tested. To carry out this experiment, samples of 12 leaves were obtained for each variable, “Low

Sunlight” and “High Sunlight”. Clear nail polish was applied to the samples making an imprint

of the stomata. Clear tape removed the nail polish; the tape with the imprint was put on a slide.

Under 400X power, stomata were counted using the imprinted slide and the density was

determined using a micrometer slide. Then a two sample t-test showed significant results at the

alpha=0.05 level. This supported the alternative hypothesis that different heights of leaves have

an impact on stomatal density in Pinus sp. Yet the prediction that a greater number of stomata

exists at higher levels than at lower ones was not supported; interestingly the opposite was

supported. A possible explanation is that higher leaves create shade for lower leaves so

photosynthesis occurs at lower leaves where water loss isn’t as high. Future studies include the

search for a correlation between plant shape and stomatal density and between shade and

stomatal density.
 INTRODUCTION

Stomata play an essential role in all plants. These openings allow for gas exchange and

water flow throughout plants. Gas exchange occurs when carbon dioxide molecules enter the

leaves for photosynthesis and when excess oxygen molecules leave the leaves. The pores allow

for water flow throughout plants by creating a lower water pressure at the leaves than at the

roots. Since water will move from an area of greater water pressure to one of lower water

pressure, the sun’s induced water evaporation from the plants’ stomata will allow water to flow

from the roots, to the shoots, and finally to the leaves. Also, this process of water evaporation is

analogous to humans sweating, cooling down a plant when temperatures are high to protect

enzymes and cells from overheating. But if stomata are left open, too much water can evaporate

leading to desiccation. Thus, stomata can open and close throughout the day to control water

loss. In addition to regulating water loss through the opening and closing of stomata, plants can

vary the stomatal density.

Greater stomatal density results in the more efficient control over water loss.

Consequently, one would expect to find greater stomatal density in regions of high water loss.

Because the amount of sunlight exposure directly correlates with water loss, greater stomatal

density is expected to be in areas of greater sunlight exposure (Tichà 1982). In one situation, it is

predicted that there is more need for water control in upper regions of a plant than in the lower

regions because the upper regions receive more sunlight. Yet this may not be the case in areas

where water stress is high. For instance, a plant living in a water-stressed environment may

rarely open its stomata in the upper regions and instead open them in the lower regions where the

sunlight isn’t as intense. Therefore it is logical that the plant have more stomata where it is
opening them the most, the lower regions in this case. With respect to the inconsistency of

stomata densities, we question how sunlight exposure affects the stomatal density.

Plants can vary in stomatal density and distribution during development which shows

their resilience to environmental changes (Hetherington, Woodward, 2003). Because high light

intensity correlates with higher stomatal density (Tichà 1982), the following were predicted.

First, leaves at the top of the plant have more stomata than those at bottom. Second, leaves on the

outer part of a plant have more stomata than those on the inner part. Third, leaves facing the

south have greater stomatal density than those facing the north. Fourth, leaves in the shade have

fewer stomata than those in the light. Finally, leaves of plants near water have fewer stomata

than those in drier areas.

In this experiment, the effect of differing leaf heights in Pinus sp. was examined to see if

there would be an effect on stomatal density. The null hypothesis would be that the effect of

differing leaf height in Pinus sp. does not have an effect on stomatal density. It was predicted

that leaves collected in the upper tiers (high sunlight) of a tree would have more stomata than

those at the lower tiers (low sunlight). The plant was selected over others primarily because a

noticeable change in height could be tested. The majority of Pinus sp. grows about 15 meters to

45 meters tall. In addition, this species was used over other pine tree species because it had scale

leaves in which stoma could be counted. Other hypotheses could have been made and tested to

answer the question of how sunlight affects stomata density. Among these are the distance of

leaves from the center of the tree, the direction the leaves are facing, and the presence of shade

on the leaves. These were accounted for by taking the samples of leaves from the inner part and

the south side from the same tree with no obstructions creating shade.
 MATERIALS AND METHODS

On January 8, 2008, samples were collected from the pine tree adjacent to Alumnae Hall

and closest to Keezell Hall at James Madison University’s Quad. The samples that constituted as

“Low Sunlight” were taken from about 7 feet, and those that labeled as “High Sunlight” were

taken from about 15 feet. In addition, the sample was taken from the inner part and south side.

Branches from each of these heights were taken back to the lab and 12 scale leaves from each

variable were randomly obtained and organized by either “Low Sunlight” or “High Sunlight”.

Next, clear nail polish was place on the underside of the scale leaves. After the nail polish

dried, clear tape was used to remove the imprint and then placed on a slide. This was repeated

with the other 23 scale leaves. Then, the stomata were identified on 400X power on a microscope

with the criterion that stomata in the slide had to be either greater than or at least half in the field

of view to be counted as one stoma. Two of the four group members confirmed the stomata

counted which was recorded and repeated with the remaining scale leaves. Later, a micrometer

slide was used to determine the area of the field of view for all microscopes used. This was then

averaged and then used to find the densities (stoma/mm2) of all 24 data points.

A histogram of the frequency of stoma plotted against density was created to observe the

spread of the data. A bar graph was created with error bars to analyze the means of the variables.

Finally, a two sample t-test was conducted to evaluate the significant difference in the means of

the variables at the alpha=0.05 level.

RESULTS

After the stomata were counted and densities determined, the average, variance, and

standard deviation values for both the “Low Sunlight” and “High Sunlight” were determined.

Those values of the “Low Sunlight” were 76.91 (stoma/mm2), 145.17, and 12.05 respectively.
Those values of the “High Sunlight” were 60.84 (stoma/mm2), 153.79, and 12.40 respectively. A

bar graph with error bars, Figure 1, was created as a graphical representation using those values.

Mean Values of Stoma Density from Leaves of Different

Heights on Pinus sp.

100.00
90.00
Density of Stoma

80.00
Figure 1. These bar graphs
(stoma/mm )

70.00
2

60.00 Low Sunlight

50.00
High Sunlight
show the mean values of
40.00
30.00 stomatal density with error
20.00
10.00 bars.
0.00

Low Sunlight High Sunlight

Different Heights (feet)

Figure 2 shows the histogram created to see the spread and normality of raw data points. The

data points ranged from 41.32 (stoma/mm2) to 96.42 (stoma/mm2). Also, the median and mode

values of the “Low Sunlight” were both 75.76 (stoma/mm2) and those of the “High Sunlight”

were 61.98 (stoma/mm2) and 75.76 (stoma/mm2) respectively.

Histogram of Stoma Density from Leaves of Different

Heights on Pinus sp.

4.50
Frequency (number of

4.00
3.50
3.00
Low Sunlight Figure 2. This histogram shows the
stoma)

High Sunlight
2.50
2.00
frequency of stoma as density increases
1.50 from 41 to 100 (stoma/mm2).
1.00
0.50
0.00
41-46 47-52 53-58 59-64 65-70 71-76 77-82 83-88 89-94 95-100
2
Density (stoma/mm )

Finally, a two-sample t-test was conducted under the assumption that randomness and

normality were not violated, and that the sample was robust. At the alpha level of 0.05 and

degrees of freedom of 22, the critical t value was calculated at 1.717144. The t-statistic was

calculated as 3.21958546.
 DISCUSSION

The research hypothesis, different leaf heights in Pinus sp. have an effect on stomatal

density, was supported and thus the null hypothesis rejected; the t-statistic was greater than the

calculated t-value, 3.22>1.72. This means that there is 95% or greater a chance that this

difference in means didn’t randomly happen by chance. However, the prediction that leaves on

higher levels have more stoma than those on lower levels was not warranted. In fact, we are 95%

certain that the opposite is true. Figure 1 shows the difference in means: the mean value for

“Low Sunlight” was 76.91±12.05 (stoma/mm2) and the mean value for “High Sunlight” was

60.84±12.40 (stoma/mm2). Next, the t-test may not have been most ideal for the data that was

collected. The histogram in Figure 2 shows that the data of “Low Sunlight” is pretty much

normal though skewed a little to the left, but the data of “High Sunlight” is not normal and

skewed to the right. As a result, more samples should’ve been taken to increase the robustness of

the data.

Overall, the experiment supports that lower leaves on Pinus sp. have more stomata than

higher ones. This could be because of the plant’s adaptation to water stressed environments over

centuries. Because noon is where water loss is most likely to occur, perhaps the trees have

adapted by opening stomata at the bottom of the tree more often than at the top. The leaves at the

top would create shade for those at the bottom, limiting water loss. Since the stomata would be

opened at the bottom of the tree more often than at the top, more stomata at the bottom would

allow for better water control. In addition, this may explain the fact why the tree is shaped like a

cone on its base, though further testing would be required. At noon, the surface area to volume

ratio of a cone is very low with respect to sunlight, reducing the chance of plant desiccation. It
seems logical for the plant to have more stoma in bottom of the tree where it can actually open

them and without worrying too much about water loss because of the upper leaves’ shade.

The samples were taken from only one species; extending the sample to a greater pool

would make the data statistically more significant and applicable. In addition, increasing the

variety of heights would be very helpful in determining a correlation. Possible future studies

could test if a tree’s shape correlates with stomatal density. Specifically, the pine tree used in the

experiment could be tested to observe whether stomatal density correlates with its cone shape.

Finally the effect of shade on stomatal density would be helpful in determining why there are a

greater number of stomata at lower heights of the Pinus sp. than there are at higher heights.

LITERATURE CITED

Hetherington AM, Woodward FI (2003). The role of stomata in sensing and driving
environmental change. Nature 424, 901–908.

Tichà I (1982). Photosynthetic characteristics during ontogenesis of leaves. 7. Stomata density

and sizes. Photosynthetica 16, 375–471.