|Dark Stomatal Response and Carbon Dioxide Levels in Dudleya lanceolata at Various Humidities
Jennifer A. Oberholtzer and Amanda C. Swanson
Department of Biological Sciences
Mission Viejo, California 92692
Crassulacean Acid Metabolism (CAM) is a photosynthetic adaptation carried out by various plants that thrive in arid conditions, such as cacti and succulents. In order to reduce water loss, CAM plants undergo a diurnal cycle of opening their stomata at night and then closing them during the day. This experiment was constructed to determine whether humidity levels, in the absence of light, would affect the carbon dioxide consumption and stomatal response of Dudleya lanceolata. The leaves of eight D. lanceolata plants were acclimated to four different humidities. Carbon dioxide levels and stomatal imprints were taken. The metabolic rates at each humidity were averaged and no statistical significance was found (p=0.655, ANOVA).
There are several methods of carbon fixation a plant can use to convert carbon dioxide into glucose. In C3 plants carbon fixation is initially performed via the enzyme rubisco. Rubisco takes carbon dioxide and adds it to ribulose bisphosphate, initiating the first step of the Calvin Cycle. During the presence of light, these plants open their stomata during the day to allow for gas exchange. Stomata then close at night when light is no longer available (Hartsock & Nobel, 1975). Crassulacean Acid Metabolism (CAM) is a photosynthetic adaptation carried out by various plants that thrive in arid conditions, such as cacti and succulents. In order to reduce water loss, CAM plants undergo a diurnal cycle of opening their stomata at night and then closing them during the day. Since carbon dioxide consumption does not occur during the day, it is stored primarily as malic acid in vacuoles until light is available. Once light is present, carbon dioxide is released and the Calvin cycle begins (Ting, 1985).
In C3 plants, light stimulates potassium ions to enter guard cells causing them to become turgid as this a hyposmotic effect and causes water to move into the cell; resulting in open stomata and the entrance of carbon dioxide into the cell. On the other hand, CAM plants have an endogenous “photoperiodic circadian rhythm” meaning they perform a 24-hour daily cycle in which they open and close their stomata with the influence of external cues, the presence/ absence of light and water (Lee 2010). There are numerous plant species that exhibit the ability to “switch” from CAM to C3 or take on characteristics from both methods of carbon fixation. Plants that possess the ability to switch from CAM to C3 are termed facultative CAM plants and can open and close their stomata either during the day or night. This switch in carbon fixation is dependent on various environmental factors such as availability and salinity of water, temperature, humidity, or photoperiod (Sayed, 2001; Cushman, 2001 and Lange & Medina, 1979). It is known that several species of Dudleya are facultative CAM plants (Nobel & Brutta, 2007). This experiment was constructed to determine whether humidity levels, in the absence of light, would affect the carbon dioxide consumption and stomatal response of the facultative CAM plant Dudleya lanceolata.
Materials and Methods
Eight small D. lanceolata plants were purchased from Tree of Life Nursery (San Juan Capistrano, CA). The plants, all similar in size, were placed outside in direct sunlight during the day and then moved inside at dusk to prevent freezing. They were watered every other day in the evening, at seven pm, with 25 mL of deionized water.
Trials began on March 24, 2010 and continued through April 4, 2010. The testing was conducted at the house of Amanda Swanson (Laguna Hills, CA). Located on site were four Pasco GLX data loggers with carbon dioxide probes and photosynthesis chambers provided by Saddleback College Department of Biology. A photosynthesis chamber is a two chambered tank in which the center, inner portion can be sealed off, via a rubber stopper, to create a sealed, isolated environment.
The four probes were set up and calibrated, prior to each testing, to verify that all equipment was functioning properly.
At the beginning of the experiment, each plant was given a number for identification and a control was established to ensure that the plants were not performing carbon fixation. The control was set up during the daylight at ambient temperature and humidity (35%). For the control, a leaf from each plant was removed and placed into the photosynthesis tank and the carbon dioxide levels were measured.
The appropriate saturated solution for each humidity was determined using tables that indicated which solution (at a temperature between 20 and 25 ْC) would create which humidity in a closed system; tables from different sources were referenced to ensure accuracy (Greenspan, 1976; Sweetman, 1933; Winston & Bates, 1960). The saturated solutions were then prepared. The 0% humidity environment was produced by placing 15 mL of drierite in the bottom center portion of the photosynthesis chamber to absorb all the moisture. A rubber stopper was placed in the middle of the drierite so that the leaf was not directly in contact with the drierite. To produce a 33% humidity environment 15 mL of saturated MgCl was placed at the bottom portion of the chamber. The 75% humidity environment had 15 mL of a saturated NaCl solution. The 100% humidity level was obtained by placing 15mL of deionized water in the center portion of the tank. Each humidity condition had a rubber stopper in the tank so that the leaves could rest in the chamber without contamination or damaged from the solutions.
Each leaf was removed from the plant to eliminate soil-water potential from directly affecting stomatal response during the acclimation period through data collection. One 7cm length of leaf was removed by cutting with scissors. The cut portion of the plant was covered with parafilm to prevent any potential water loss. With no light present, the leaves with the parafilm were weighed and then placed into the center portion of the appropriate photosynthesis chamber. Two leaves, from separate plants, were placed in a photosynthesis chamber to ensure that sufficient CO2 levels could be detected. The leaves were paired consistently. After the leaves were allowed to acclimate for three hours at the respective environment condition, the Pasco data logger CO2 probes were turned on to record data for an additional three hours.
Once the CO2 data were collected, the leaves were removed from the photosynthesis tanks and immediately reweighed. Stomatal imprints were then obtained by firstly adding a drop of Superglue to a blank glass slide. The top of the leaf was then firmly pressed into the wet glue and held there for 10 seconds before being slowly pulled off. The glue was given time to dry and then later analyzed under a compound light microscope magnified at 100x. The trials were repeated with different freshly cut leaves and parafilm for four trials to ensure that each plant was rotated through every humidity level. Stomatal data were analyzed by photographing the stomata and counting the number of open stomata compared to the total number of stomata visible (an area of 1 mm2 was visible).. Statistical analyses were run using Microsoft Excel; all data were analyzed by converting parts per million (ppm) of CO2 to grams of CO2 produced per gram of plant.
The metabolic rate of each humidity was averaged and graphed (Figure 1). There was no significant statistical difference between plant metabolic rate and humidity level (p=0.655, ANOVA). At 0% humidity, the average metabolic rate was 7.25x10-7 ± 4.18x10-7; at 33% humidity, average metabolic rate was 1.20x10-6 ± 8.62x10-7; at 75% humidity, average metabolic rate was 9.50x10-7 ± 1.43x10-6; at 100% humidity, average metabolic rate was 1.58x10-6 ± 2.59x10-6. Stomatal imprints indicated that 44% of the stomata were open for 0% humidity, 41% open for 33% humidity, 50% were open for 75% humidity and 54% of the stomata were open for 100% humidity (Figure 2).
. The mean metabolic rates for each humidity. ANOVA shows no significant difference between humidities (p=0.655). Error bars indicate mean ± SEM.
Mean percentages of stomata opened for each humidity.
The results of this study do not support the hypothesis since there was no significant difference between CO2 levels or stomatal response and humidity. However, other research suggests that humidity does in fact play a role in stomatal response; however, there are mixed results regarding the effect of humidity on CO2 consumption (Lange and Medina, 1979; Griffiths et al., 1986; Luttge, U. et al., 1986). A study done by Herppich (1997) proposed that stomata do in fact respond to humidity, but that this stomatal reaction was not absolutely linked to CO2 consumption at night in Plectranthus marrubioides. The research showed that drought stress played a large role in the plant’s ability to fixate carbon. When well watered, there was no link between CO2 uptake with stomatal response and humidity levels. However, in extreme drought situations, humidity levels did affect CO2 consumption (Herppich, 1997).
Guard Cell Turgidity
Stomatal opening is greatly influenced by turgidity within the guard cells; the more turgid the guard cells, the more open the stomata will be. Turgidity is determined by the amount of ions either entering or leaving the cell (MacRobbie, 2006). The movement of these ions follows an osmotic gradient and in order for the guard cells to become turgid, the cell must also take up water from its surroundings. The influx of water and ions into the guard cell vacuole creates pressure and the stomata then opens (Sheriff & Meidner, 1975). The leaves were removed from the plants. Therefore it is likely that there may have been a decrease in the overall water content within each leaf over the six hour period. Upon weighing the leaves after the six hours, there did appear to be a decrease in the weight of each leaf. If this weight loss was in fact water loss, then the guard cell vacuoles may have been prevented from gaining enough osmotic pressure to become turgid and to fully open the stomata.
Although the leaves were allowed to acclimate for several hours, it is possible that this acclimation time was not sufficient. CAM plants are extremely adaptive to their environments and it has been shown that upon the introduction of an environmental change, it takes a longer period of time for them to show any significant change in their physiological behavior (Szarek, et al. 1987; Hartsock & Nobel, 1976)). In several studies, the acclimation time in which the plants were exposed to experimental changes minimum of two weeks. The independent variables in these studies were CO2 levels (Szarek et al., 1987) and water availability (Hartsock & Nobel, 1976). These leads to the possibility that the amount of time allowed for the leaves to acclimate may have been insufficient.
Another interesting factor that may have contributed to this study was the age of the leaves. A study done by Jones (1974) showed that leaf age contributed to CO2 exchange in Bryophyllum fedtschenkoi, another CAM plant. Young leaves did not display CAM and displayed CO2 output during the night. Mature leaves on the other hand, did perform CAM. They suspected that the older leaves may have had more vacuole space and were able to store higher quantities of CO2 as a result. Although the leaves used from D. lancolata were all the same length, it is possible that there was variation in leaf age and maturity level.
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