Stomatal responses

Stomatal reactions are crucial adaptation processes for plants to survive in a variety of environmental situations. Climate and environmental conditions have an impact on the physiological functioning of plants, which in turn affects their morphology. Stomata density and pore size responses to variables including water availability, temperature, and light are examples of such reactions. Thus, depending on the local climatic conditions, different plants of the same species may have different stomatal densities. Some reasons of the environmental circumstances include fragmentation and the resulting edge effects, which may have a significant impact on stomatal density. The objective of this experiment is aims to compare the stomatal density from leaves of plants from the edges and those from the core or non-edge, and thus to establish the impact of human activities (mainly fragmentation) on the factors that affect the number of the stomata on leaves. The findings indicate that there are significant differences in the averages of the stomatal densities that are noted as 32.3, 33.9, 31.9 and 29.4 for Driftway, Western, North, and Core sites respectively. It can thus be concluded that fragmentation and edge creation through human activities have significant impact on the structures of the plants existing in a landscape as demonstrated by variation in the stomatal densities.

Keywords: stomatal density, edge effects, Turkish HSD

Stomatal Density, Edge Effects and Fragmentation

Background

The leaf is one of the most valuable parts of a plant primarily due to its photosynthetic role as well as for aiding gaseous exchange through the stomata (Nonami, Schulze, and Ziegler 1991, 57). The leaf stomata are the major means of gaseous exchange in plants. Stomata are small pores that exist on the underside of the leaves and can close or open with the control achieved through the utilization of the guard cells that are banana-shaped. The mechanism by which the guard cells assist in the opening and closing of the stomata is based on the osmotic potential. The cells draw in water and become turgid, thus opening the stomata. In case there is no or inadequate water the cells become flaccid and close the stomata in the process.

During the day, the stomata open to allow entry of carbon IV oxide gas that is used for the manufacture of glucose through photosynthesis in the presence of sunlight. At the same time, the stomata assist in transpiration (Zeiger, Farquhar, and Cowan 1987, p. 14). Transpiration is essential as it enables the plants to release excess water into the environment. However the excessive loss of water is dangerous as the plant may end up wilting or desiccating. A study by Gao et al. (2015) indicates that the stomatal behaviour in response to droughts and dry conditions has been a subject of many research works, especially in the light of the increasing climate change and global warming. The responses of the stomata are known to arise due to the fact that the environmental and climatic conditions influence the plant morphology and subsequently their physiological performance. For instance, the stomata density and pore sizes respond to the factors like water availability, temperature, and light (Wang, Chen, and Xiang 2007, p. 1435).

The stomatal density is a subject of environmental conditions like temperature, wind, humidity, and availability of water or CO2 as well as the characteristics of the leaf-like shape, colour, and orientation. It is evident that there are more stomata on the leaf surface in conditions like with low CO2 concentrations, a high degree of moistness, and in areas with excess light. It can thus be observed that the number of the stomata may primarily affect how a plant adapts to the listed environmental conditions (Peñuelas and Matamala 1990, p. 1120). The orientation of the leaf changes the number of the stomata as some leaves like those in eucalypti species are hanging to face the direction either direction of the sun.

Plants utilize the aspects of the stomata like the stomatal density to achieve the control of factors like the uptake of CO2 and loss of water. The stomatal density is the actual number of stomata per unit area of a leaf. The number of the stomata on a leaf has various impacts that have been demonstrated in several studies. Wang et al. (2007), for instance, indicate that changes in the density and size of the stomata have the potential to influence aspects like CO2 uptake and water loss into the atmosphere (p. 1435).

According to Willmer and Fricker (1996), the edge effect is the phenomenon in which the water molecules are lost more rapidly near the edges of a leaf as compared to its centre or core. The authors, therefore, contends that more water will be lost on the sides of the leaf since more water molecules are diffused on such areas as compared to the pores that are towards the core of the leaf. However, the focus of this experiment is to investigate the edge effect as it relates to the fragmentation of landscape. The side effect that results from habitat fragmentation is one of the aspects that influence the stomatal density as the plants respond to the changes in the environment as demonstrated in the works of Kattan and Murcia (2003 p.181). Fragmentation is the phenomenon in which the landscapes are reduced into patches due to the natural or human-made actions. The patches of forests or biodiversity hence remain as a result of activities like construction of power lines, roads, railways, and pathways among others (Fahrig 2003, p. 487). Even though the massive loss of biodiversity observed globally is primarily linked to the increase in the levels of the CO2, it is evident that human beings are responsible for disrupting the natural ecosystem through fragmentation. Fragmentation leads to the edge effects that are observed if two types of boundaries come into contact. Edge effects cause the plants to adapt appropriately especially since the plants are exposed to new environments. It can further be observed that the edges mat consists of diverse organisms that may differ from those that are present in the ecosystem. The end effects are further recognized to be essential in the regulation of the structure of a plant.

Not only does the edge influence affect the plants but leading to ecological change as well due to the alterations of the local environment. Soil moisture is an example of the ecology that may be influenced significantly by the fragmentation (Solé, Alonso and Saldaña 2004, p. 56). It can be noted that there is a steep soil moisture gradient between the core and the edges. Roadways are examples of human-induced fragmentation and creation of edge that has the potential of causing local environmental changes by altering aspects like soil temperature, local temperature, light permeability and canopy height.

Research Question

The experiment was carried out to answer the following question: Is there a difference in stomatal density between the four sites?

Research Objective

This research aims to compare the stomatal density from leaves of plants from the edges and those from the core or non-edge and hence to establish the impact of human activities (mainly fragmentation) on the factors that affect the number of the stomata on leaves. The experiment also evaluates whether there is a change in the structure of a part of a tree due to the boundary effect.

Research Hypothesis

There is a significant difference between the stomatal densities of core and edges sites.

Methods

Description of the Leaf Sampled

The experiment involved the investigation of the leaves of Eucalyptus moluccana Roxb Grey Box. Grey Box that is also known as Eucalyptus hemiphloia F.Muell is a tree that grows up to 25 metres (Plantnet 2017, n.p). The leaves features vary based on the stage of development. The young leaves are dull grey-green and are ovate to orbiculate in shape. The shapes of the adult leaves range from lanceolate to ovate and are 8 and 14 cm long with a width of between 2 and 3.3 cm wide. Further features of mature leaves are observed in the green and glossy colour (Atlas Australia 2017, n.p.).

Leaf Collection

Six leaves were randomly collected from selected Eucalyptus moluccana trees. The leaves from each tree were bagged separately. The bags were then clearly labeled as either edge or core and students’ pair names. The collection was limited to what could be reached by hand. The data was collected and notes regarding the description of the site. A total of four sites were used for the leaf gathering to compare the different stomatal density and fragmentation. Three edge sites and one core site were deployed. The edges were identified as North, Western and Driftway.

Obtaining the Stomatal Impressions

Six leaves were collected from a bag, and the identification of the packet noted. A thick swath of clear nail polish was painted on the side to be examined with the area obtained being slightly more than 1 cm2. Three leaves were utilized to obtain a sample from the upper aspect of the leaves while the remaining were used to determine the stomatal density on the lower side of the sheet. The painted samples were then left to dry. A piece of clear cellophane was then laid on the dry nail polish patch with a finger pressure used to ensure a good contact is obtained between the tape and the dried nail polish. The nail polish patch was then gently peeled off by removing the cellophane to get the leaf impression for examination. The tape and the nail print were laid on top of a mini-grid after which the tape was prevented from sticking on the label by sliding it. The impression was then observed using 10X first and then 40X. The random number table was after that used to select the quadrat from which the sampling was to be done with care taken to avoid damage or unclear regions on the leaf. The 40X objective was then moved to the central part of the chosen quadrat so that the edges could not be observed in the field of view. All the stomata visible on the field of view were then counted and the number recorded. The stomata recorded were only those that fell completely with the field of view (each quadrate). The described procedures were then repeated for all the six leaves samples present in the bags.

Results

On the upper surface of the leaf the stomatal densities were noted as 25.0, 25.6, 28.5 and 24.0 for Driftway, Western, North and Core sites respectively. The North edge has the highest stomatal density followed by the North clearing. The lowest number is noted in the core. Figure 1 below represents the stomatal density as obtained from each of the sites.

Figure 1: Average density values upper leaf surface (per mm2)

On the same note, the stomatal density varied to the same extent in the lower side as observed in the upper leaf. 39.7, 42.3, 35.4, and 35.9 were found for Driftway, Western, North, and Core sites respectively. Figure 2 presents the results obtained.

Figure 2: Average density values per mm2 of Eucalyptus molluccana, lower leaf surface in 3 edge sites: (Driftway, Western & North) and core (3 sig fig)

Analysis of the data obtained using a two-way ANOVA presents the descriptive statistics for the sites as indicated in table below.

Table 1. Two-way ANOVA table of 3 edge sites: (Driftway, Western & North) and core (upper and lower leaf surface) from raw data

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Sample

147831.7539

1

147831.7539

1894.679458

0

3.843949078

Columns

7655.744904

3

2551.914968

32.7065109

7.33215E-21

2.607286388

Interaction

12635.56243

3

4211.854142

53.98105153

3.76238E-34

2.607286388

Within

291500.1955

3736

78.02467759

Total

459623.2567

3743

The Tukey HSD (Honestly Significant Difference) test was used to measure differences in the stomatal densities in the four sites for upper, lower and total number of stomata in both the surfaces. For the upper surfaces the only averages that are equal are between Driftway and Western with absolute difference of 0.528 as well as between Driftway and Core (absolute difference of 1.04). Comparing Driftway to North, Western to North, Western to Core and North to Core shows significant differences with absolute differences of 3.43, 1.57, and 4.48 respectively.

For the lower surfaces equality is observed only in the comparison of North and Core (absolute difference 0.444). Driftway to Core, Driftway to North, Western to North, and Western to Core all have significant differences in the average number of stomata on their surfaces with absolute differences of 2.54, 4.32, 3.88, 6.87, and 6.43 respectively.

Only Driftway and North showed equality in the total number of the stomata with an absolute difference of 0.44. Comparison of Driftway to Core, Western to North, and Western to Core shows absolute differences of 1.53, 2.46, 1.98, 4.00, and 2.01 respectively and hence significant differences.

Discussion

The stomatal densities varied from one site to the other. Each of the three edges was at least 150 metres away from the core site that was in the middle of the forest. All the sites were classified as grassy woodlands with trees measuring approximately 10-30 metres high and foliage cover of approximately 10-30% at the tallest level. Driftway borders the main road with the existence of houses and other urban developments. The north and western-facing edge both border agricultural and farmlands. Driftway has a more human impact as compared to the other sites while the core has the least influence from artificial activities. The north and western-facing edges have some human activities but not as much as in the Driftway.

Cause of variations is thus mainly noted to rise from the plants’ response towards the control of the rate of water loss through transpiration in the regions of interest. Transpiration is the process through which water from a plant is evaporated through different parts (Zeiger et al. 1987, p. 14). 95-99% of all the water that is absorbed by the plants is lost (transpired) into the air via the aerial parts of a plant like cuticles, lenticels, and stomata.

However, the stomata are the main point through transpiration takes place as approximately 90% of the water is lost through it (Zeiger et al. 1987, p. 14). The plants must regulate the water loss. In soils with lots of water, the rate of transpiration is enhanced by increasing the stomatal density. Likewise in the stomatal density is expected to be lower in dry, hot, windy, and in sites with low humidity. The agricultural edges Western and North have the highest number of stomata, averagely 28.5 and 25.6 respectively on the upper side of the leaves. It can, however, be observed that more stomata are available on the lower surface than on the upper surface. There are more stomata on the lower surface of the leaves to avoid direct exposure to the environmental aspects that increase the rate of transpiration and water loss. The maximum density is further demonstrated in 42.3 and 39.7 for the cases of Western and Driftway respectively in the lower surface of the leaves. Agricultural fields are normally associated with activities like irrigation, especially during the dry season. The soil water contents from the two edges are therefore different from the Core and Driftway patches.

Significant differences are observed in comparing the stomatal densities in all the sites for both the upper and lower surfaces of the leaves as well as the total number of stomata in the leaves. Using the Tukey HSD test, equality or unequal averages were evaluated by comparing the absolute differences between two sites and the computed HSD value. A significant dissimilarity is concluded when the absolute difference is more than the HSD value. The distinct natures between any two selected sites contribute to the major differences. On the same note, it can be observed that the significant differences may result from the differing factors that affect the stomatal density in the regions. For instance, the substantial difference between the Driftway and Western edges occurs due to the variation in soil water in the two regions. Furthermore, a factor like light exposure and permeability may significantly affect the stomatal densities between the sites. Lower sunlight permeability as expected in the core is supposed to have fewer of stomata as compared to a site like Driftway that borders the main road and urban development and is thus significantly exposed to the sunlight. The Core may have a relatively low sunlight exposure as the leaves may be concealed by other leaves and hence leading to small stomatal densities to reduce the rates of photosynthesis. Further differences are observed between the stomatal densities in the Core and Driftway due to the extent of human activities in the respective sites. In Driftway human activities in the urban development and the main road may expose the site to water run-offs and hence the necessity by the trees to adapt by having more stomata to loss excess water being absorbed. On the other hand, the Core has minimal human activities like exposure to intense water runoff and hence the lower number of stomata to assist in minimizing the rates of transpiration and unnecessary water runoffs. Even though both Western and North edges are both agricultural areas, it is evident that there is a significant difference in the number of stomata in the two regions. The variations may be explained regarding the differences in exposure to light and the nature and extent of farming activities that are taking place in respective areas.

Similarities and differences can be observed in comparing the 2017 experimental data to the outcomes obtained in 2015/16 experiment. First, the Northern edge was not present in the 2015/16, and hence no comparison can be done on the treatments and findings from the two periods. Secondly, the Road edges in both the 2015/16 and 2017 experiments cannot be compared even though they have similar features. The Blacktown Road sample (2015/16) was more shaded because of the lesser degree of tree removal on both sides of the road as compared to Driftway population (2017) that were more exposed and hence less shaded due to the existence of urban development in the area.

Thirdly, the ‘western edge’ site in the 2017 sample is compared to the ‘power line edge’ of 2015/16. The ‘western edge’ was primarily populated by shoots that had resprouted from root stocks. The growth was very vigorous (to 4 metres high) as compared to the power line edge sample of 2015/16. Lastly, the core in 2017 sample was obtained mainly from plants that were heavily shaded and many of which were smaller in size and hence forming a significant composition of the understory. On the other hand, the core from 2015/16 had samples collected mostly from taller trees with fewer shades.

In comparing the two data sets, it is observable that in the 2015/16 the Core had the highest average total number of stomata at 32.75. However, 2017 experiment shows that the Core site had the lowest stomatal density at 29.9. The variations in the datasets from the two periods have arisen due to two main reasons that include the nature of the trees from which the samples were collected and the varying nature of the edges as already mentioned. The major causes of discrepancies between the two datasets are observed in the effects like shading as relating to the various edges under consideration.

Conclusion

Retrospectively, it can be found that fragmentation and edge creation in ecosystems that are dominated by people lead to the significant changes in the structures of the plants existing in a landscape. The results shown in the experiment indicates that human activities have the potential to influence ecological changes on a local scale. Since there are significant differences in the stomatal densities between the core and edges the hypothesis is thus proven. The findings of the experiment imply that the differences caused by the edge effect from the fragmentation of landscape have the ability to loss of biodiversity as species are forced to change with the alteration of the ecology. A recommended future study is to determine the impact of the edge effect on the structural changes in plants.

References

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Wang, Y., Chen, X. and Xiang, C.B., 2007. Stomatal Density and Bio‐water Saving, Journal of Integrative Plant Biology, vol. 49, no. 10, pp.1435-1444.

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