A Comparative Analysis of CAM, C-4, and C-3 Photosynthesis on Carbohydrate and Protein Production.

We sought to determine the effects of photosynthetic efficiency on carbohydrate and protein production, and light absorption in crassulacean acid metabolism, or CAM (Pachypodium lamerei), C-4 (Dracaena marginata), and C-3 (Petroselinum neapolitanum) plants.

This experiment utilized three units of testing; Carbohydrate, Photosynthesis, and Enzyme. The isolated variable of testing was photosynthesis and control groups of sugars, water, and alcohol were used as appropriate. Photosynthetic efficiency was determined via comparison of light absorbance and type of pigments present, using the Absorbance Spectrum versus a qualitative analysis of carbohydrate production and a quantitative analysis of protein concentration.

Multiple tests were utilized for each unit. For the carbohydrates we used Benedictās, Barfoedās, Selivanoffās, Bialās, and the Iodine test as qualitative measures of the carbohydrates contained in our test groups. For photosynthesis we used Paper Chromatography, and the Absorption Spectrum to determine the comparative photosynthetic efficiencies of the three different plants. Finally, for the enzymes we used a 0.1% catechol solution, and the Protein (Bradford) Assay to test for the presence of polyphenoloxidase, and protein concentration, respectively.

The results of the carbohydrate tests indicated similar carbohydrate presence among the three plant types. The results of the photosynthesis tests, absorption spectrum and paper chromatography, indicated an increasing photosynthetic efficiency from the C-3, to the C-4, to the CAM plants, respectively. Finally, the Enzyme tests indicated the presence of PPO in the CAM plant, and the Protein Bradford Assay indicated average protein concentrations of 164.605µg/µL for the CAM, 142.353 µg/µL for the C-4, and 102.477 µg/µL for the C-3 plants.

The understanding of the relationship between photosynthetic efficiency and protein and carbohydrate production is potentially applicable in the growing of plants to produce maximum carbohydrate and protein yields.

Research has suggested that CAM and C-4 carbon-concentrating mechanisms evolved as adaptations from the basic C-3 form. This adaptation developed from the necessity to meet strenuous conditions of little water and high CO2 coupled with the need for the plants themselves to create large amounts of energy in a process called photorespiration (Keeley and Rundel, 2003) (Poorter and Navas, 2003). It has been further suggested that the photosynthetic yield of plants has become increasingly more efficient with further adaptations to environment over time. C-3 in strenuous conditions evolved to C-4 and then to CAM photosynthesis, which increase in efficiency respectively. This research relating efficiency to photosynthetic adaptations was derived from various models of photosynthetic quantum yield at different temperatures and CO2 concentrations. It is also suggested by the fact that C-4 photosynthesis only occurs in about half the number of families of CAM (Keeley and Rundel, 2003). Further, Freemanās text shows that the photosynthetic cycle of the C-3, C-4, and CAM families results in the production of carbohydrates (Freeman, 2002). Given this background information, Team Absolute Zero hypothesized that the photosynthetic efficiency of a plant family and its output of carbohydrates and enzymes, as well as its absorbency of light are directly related.

We made several predictions regarding our hypothesis. First, we predicted that carbohydrates and enzymes will be produced as a part of the photosynthetic process. Second, we predicted that as photosynthetic efficiency increased, the carbohydrate range and enzyme production will increase. To show that photosynthetic efficiency increased, we compared the photosynthetic types with their absorption spectrum and the variation in photosynthetic pigments as well as the amount of proteins that they produced (Bradford Assay, Figure 21). It was expected that the most efficient photosynthetic process will yield the most products overall. Since one of each type of plant (CAM, C-4 and C-3) was placed under continuous light, no light, and natural light conditions, we predicted that each would respond with varying protein, carbohydrate and enzyme production.

Our research was performed in three stages: carbohydrates, photosynthesis and enzymes, as was pre-structured in the LBS 145 Laboratory Manual (Krha et al., 2004). In the first section we performed five carbohydrates tests on the nine plants. One plant of each of the three photosynthetic types was grown under continuous light, no light and natural sunlight for a span of two weeks prior to our tests. The variation in light produced secondary effects of differing soil moisture, and a temperature change. The preceding environmental conditions would greatly affect normal C-3 plants (Keeley and Rundel, 2003). Consequently, by varying the light the plants were exposed to, we altered the intake of the key constituents of their photosynthetic processes.

We first attempted to dilute our stock solution of 2 grams of plant to 150mL of water. At this dilution, however, all the results for the natural light tests came out negative for any sugars. Because we learned in past research that sugars are a result of photosynthesis (Freeman, 2002), and therefore present in all photosynthetic plants, we deemed our results to be inconclusive due to inaccuracy. Next, we attempted to dilute our stock solutions with 50 mL of water, but again we were left with inaccurate results. The same was also true for our dilution to 10 mL. Consequently, our results for our natural light plants were inconclusive, pending further testing.

Past research informed us that CAM plants contain substantial amounts of starch (Keeley and Rundel, 2003). At this point, we decided to utilize this information to deduce the proper concentrations for our stock solutions. We started by not diluting the plant at all and adding iodine directly onto the plant tissue. This test supported our predictions when it changed to a brown color. We then worked backward from an overly diluted solution to deduce a concentration that would produce an indication of reaction. This was accomplished by starting with a 1g/10mL concentration and increasing the amount of tissue until it showed a result with the iodine test. This occurred at a concentration of 6g/10.3mL. We then diluted the C-4 and C-3 solutions to equivalent concentrations and performed the remaining tests.

CAM plants kept in the dark, natural light and continuous light resulted in a red precipitate in Benedictās and copper precipitate in Barfoedās tests indicating the presence of reducing monosaccharides (Figuress 4-5). Monosaccarides such as glucose are essential constituents of larger sugars and play significant roles in the biological functions of plants. Therefore, our past research offered a premise for support of the preceding results. C4 plants exposed to continuous light produced red precipitate indicators, while the C4 plants in the dark did not. We know that the glyceraldehydes that are produced in the Calvin Cycle become glucose after they leave the chloroplast (Freeman, 2003). We believe that the lack of indication in the C4 dark plants was due to such low levels of the reducing sugars in the plant that they failed to invoke a profound reaction in Benedictās reagent. That is to say, the low levels of reducing sugars would be an indication of the limited photosynthetic activity in the plant which was deprived of light for two weeks. The C-3 plants produced a red precipitate in Benedictās and a copper precipitate in Barfoedās test after being exposed to all three conditions. Lesser resilience to the light elements was to be expected since C-3 plants are less equipped to handle the more severe environmental conditions that the C-4 and CAM plants are adapted to (Keeley and Rundel, 2003)( Poorter and Navas, 2003). The C-3 plants that were in the dark indicated the presence of starch, however, the results were not as prominent as observed in the C-3 plants kept in the continuous light and natural light. It is then implied that C-3 plants contain reducing monosaccharides under all three conditions, but in decreased quantities within the C-3 plants kept in the dark.

In Bialās test we used xylose and fructose as controls for a pentose furanose ring and a hexose furanose ring respectively. The C-4 in all three conditions turned a deep green (Figures 8-9), although, it was a slightly different hue than the xylose, which we presume was due to the potency of the straight sugar in comparison with the naturally more diluted sugar in the plant. The CAM plants in the dark, natural and continuous light conditions indicated the presence of hexose furanose ringed sugars (Tables 1-3). The C-3 plants also produced an olive colored solution as a result, which indicates the presence of hexanose furanose rings. Due to limited time and methods available, we were unable to determine the precise hexose and pentose rings present.

Selivanoffās test indicated the presence of ketoses or aldoses. We used fructose for our control containing a ketone and glucose as a control containing an aldehyde. The ketoses changed color the fastest, resulting in a very dark red color by the end of 2.5 minutes. In contrast the aldoses took considerably longer, with only a color change to a light pink. The C-3, C-4 and CAM plants in continuous light, natural light and dark conditions contained ketoses (Figures 6-7). Because the ketoses changed faster, this indication does not mean that there are not aldoses also present in the plants. The C-3 plants produced a slight change in hue in the plants from the dark and light conditions. There was a barely noticeable orange hue in both plants after about 1.5 minutes. We believe the very faint hue was an indication of aldoses, but no indication of any ketoses. Because sucrose is a non-reducing sugar composed of glucose and fructose, that is used as a transport sugar from the sources to the sinks of the plants (Freeman, 2003), it follows that sucrose is a necessary sugar in most plants. Fructose has a ketose group; therefore we speculated that ketoses should be present in plants. Our data supports this theory because all three types of plant contained ketoses.

The Iodine test was used to test for the presence of starch. Starch was present in the CAM plant kept in all conditions (Figures 10-11). CAM plants have the capacity to store large amounts of fuel because it is exposed to the most severe conditions of the three types of photosynthesis. The C-4 plants, which were exposed to large quantities of light, revealed the presence of starch, however, the dark plants did not (Figures 10-11). We can predict that the C-4 plants kept in the dark used much of their stored energy to stay alive and were not taking in new energy for storage because there was no light. Since light is necessary for plants to synthesize chemical energy, the C-4 plants kept in the dark were unlikely to have much energy stored in the form of starch.

In the photosynthesis stage of our research we used the spectrophotometer to plot the absorption spectrum of the C-3, C-4 and CAM plants. Our results indicated absorbency peaks at 0.8 for the 445 nm wavelength and 0.42 for the 685 nm wavelength of the dark C-3, 0.68 for the 445 nm wavelength and 0.31 for the 685 nm wavelength of the normal light C-3, and 0.61 for the 445 nm wavelength, 0.3 for the 575 nm wavelength, and 0.37 for the 685 nm wavelength of our C-3 kept in constant light. We also found absorbency peaks at 0.87 for the 445 nm wavelength and 0.47 for the 685 nm wavelength of the dark C-4, 0.83 for the 445 nm wavelength and 0.65 for the 685 nm wavelength of the normal light C-4, and 0.84 for the 445 nm wavelength, 0.27 for the 575 nm wavelength, and 0.49 for the 685 nm wavelength for our C-4 kept in constant light. Finally, we found absorbency peaks at 0.94 for the 445 nm wavelength and 0.38 for the 685 nm wavelength of the dark CAM, 0.65 for the 445 nm wavelength and 0.33 for the 685 nm wavelength of the normal light CAM, and 0.91 for the 445 nm wavelength, 0.7 for the 575 nm wavelength, and 0.49 for the 685 nm wavelength for our CAM kept in constant light. We observed that the CAM plant seemed to have the broadest range of absorbance in the absorption spectrum. We believe the large range results from CAMās need to store large amounts of light energy during the day so that it may use the energy in photosynthetic processes during the night. The C-4 and C-3 also follow in accordance to correlation with their environmental light conditions.

In the enzyme stage of our research we tested for the presence of polyphenoloxidase (PPO), using a 0.1% catechol solution. Our results were intended to indicate the presence of PPO in our C-3, C-4, and CAM plants kept in continuous light, dark, and natural light. Our C-3 and C-4 plants didnāt show any PPO, but this may not be the case. It was realized that given the process of finding the PPO, it is necessary to find subsurface tissue. Both C-3 and C-4 were too thin to open up properly, and therefore may have neglected to show PPO even if they had it. For the CAM though, the constant light condition showed the most PPO, followed by the dark plants, and finally the natural light plants.

The Protein (Bradford) Assay, was used to determine the total protein concentration in the plants. Our results indicated concentrations of 94.20 µg/µL, 89.35 µg/µL, and 185.95 µg/µL in our C-3 no light, natural light, and continuous light respectively. The C-4 plants in no light, natural light, and continuous light showed concentrations of 151.96 µg/µL, 155.91 µg/µL, and 185.95 µg/µL. Finally, the CAM concentrations under no light, natural light, continuous light produced concentrations for this assay of 148.60 µg/µL, 152.65 µg/µL, and 125.81 µg/µL. These results are consistent with our predictions in that it shows an increase in protein production from the CAM plants followed by the C-4 plants and finally the C-3 plants. This could be due to the structure of the plants. The CAM, being a cactus was thicker and more needing of protein structure than either the C-3 Italian parsley or the C-4 Red-edged Dracaena.

Our results show the carbohydrate structures, absorbency values, protein concentration and presence/lack of PPO in each of the tested C-3, C-4 and CAM plants kept in varying light conditions. This research should be used as a resource informative only in the areas tested. It should be noted that these results were for the specific type of C-3, C-4, CAM plants tested, and are not an accurate generalization of all plants in these families. Also, there is the possibility that other carbohydrates and enzymes not mentioned are present. The constraints of our tests may have disallowed for their finding. There was also a constraint on time. Time was extremely limited for the complexity and mass number of replications and trials our experiment required. 20 hours of lab time was grossly insufficient. Another possible cause of error is that we did not run an action spectrum. An action spectrum would have allowed us to gauge the photosynthetic efficiency more directly and more accurately. The fact that it was not included was not an oversight. The plant samples that we chose to work with did not yield the amount of stock solution required to run the action spectrum. Unfortunately, that also goes back to the time issue. Given only the time that we had, we were unable to obtain and set up the extra samples that would have been necessary to run this experiment properly.

A formal conclusion cannot be drawn from our data due to the lack of an accurate method for measuring the photosynthetic efficiency. However, the trend in our data and the prior research seems to support our initial predictions. We are confident that further testing would show this, however, since this information is not currently available (due to the absence of testing the action spectrum), this deduction is not viable.

The implication of the information gained through this study is such that it provides the understanding of the effects that the specialized variations on the standard carbon fixation pathway has on photosynthetic efficiency and the associated carbohydrate and protein yields. This information would be appropriately used in determining the most efficient lighting conditions for each plant or in order to achieve a desired carbohydrate and enzyme output.

Freeman, Scott. 2002. The Calvin Cycle. Biological Science: 147-151.

Keeley JE, and Rundel PW. 2003. Evolution of CAM and C-4 carbon-concentrating mechanisms. 
	International Journal of Plant Sciences 164(3): S55-S77

Krha, Maleszewski, Wilterding, Sayed, and Luckie. 2004. Cell Physiology Studies. 
	LBS 145 Spring Laboratory Manual: 39-72.

Poorter, Hendrik and Navas, Marie L. 2003. Plant growth and competition at 
	elevated CO2 : on winners, losers and functional groups. Tansley Review: 
	185.