AbstractPlants have numerous mechanisms for obtaining sugar; normally, in the absence of sunlight, plants will use starch granules for ATP production. This experiment was performed to test how plants take up sugars from their environment without sunlight. It was hypothesized that plants, when deprived of sunlight, and provided with no sugar, would rely solely on their starch granules. However, when provided with sugar, the plants should use sugar uptake mechanisms rather than their reserves. When the plants were provided with light, it was hypothesized that plants would photosynthesize normally, neither relying on starch nor outside sugar. To test this discs were cut out from 7-9 day old Brassica rapa plants. The discs were then placed in solutions that had different molarities of sucrose or glucose and half the solutions were subjected to constant light while the other half were placed in zero light. Starch amounts were measured using an IKI solution and a color scale. The data showed that sugar uptake increased linearly with molarity. These results also clearly suggest that the plants must have taken up the required sugars from their environment to undergo cellular respiration for ATP synthesis. On the other hand, the discs that were placed in the light were able to undergo photosynthesis and as a result, these plants were able to acquire the necessary sugars. Therefore, the results remain consistent with the hypothesis.

this highly branched molecule is called amylopectin. Starch consists of approximately 20-25% amylose, and approximately 75-80% amylopectin. These complex polysaccharides contain glucose and sucrose, which are the prime subjects of this experiment. By isolating plants in different environments, this experiment aims to investigate how plants use the starch reserves in their cells versus free glucose or sucrose in their immediate environment. Two of the main processes involved in energy production in plants are photosynthesis and cellular respiration. Photosynthesis is the process that plants use to obtain sugars by using CO2, H2O, and light energy (photons). There are two parts to photosynthesis: the light reactions and the Calvin cycle (dark reactions). In the “light reactions”, energy from incoming photons is used to reduce an electron accepter called NADP+ into NADPH. In the process, a pair of electrons and a hydrogen nucleus (H+) is added. Water is also split in the process, which is the source of the atmospheric O2 that is released. The light reactions also produce a small of ATP by phosphorylation. In the Calvin cycle, the sugars are produced. First, carbon dioxide is fixed from the plants’ environments, where the NADPH and ATP produced in the light reactions can reduce CO2 to carbohydrates. Note how this process creates little ATP for the rest of the cell to use. In addition, the result of the Calvin-Benson cycle is not glucose but G3P (glycerate 3-phosphate). Approximately 5 out of 6 of these intermediate sugars are recycled into the Calvin-Benson cycle to produce RuBP (ribulose 1, 5-bisphosphate), while the rest are condensed to hexose phosphates. These sugars can later become glucose, which is further processed to become energy for the organism. During cellular respiration, glucose that was either produced by photosynthesis or ingested is converted into ATP that the cell uses to carry out various cellular processes. Cellular respiration actually consists of three parts: glycolysis, the Krebs cycle, and oxidative phosphorylation. In short, both glycolysis and the Krebs cycle reduce NAD+ to NADH, which are used as electron carriers in oxidative phosphorylation. With substrate-level phosphorylation, these two processes

Sugar uptake and starch consumption in Brassica rapaSurya Bhupatiraju, Zaroug Jaleel, and Whitney Hagins*Department of Science, AP BiologyLexington High School, 251 Waltham Street, Lexington, MA 02421*Correspondence: whagins@sch.ci.lexington.ma.us

IntroductionAutotrophic organisms in the Plantae kingdom produce their own food by using sunlight as their energy source. However, what happens if this source of light is cut off? Where do the plants turn to in order to satiate their daily food requirements? All plants and animals have stores of starch, which consist of numerous glucose molecules glycosidically linked together to form chains of monosaccharides used to make polysaccharides. In plants, this polysaccharide is called starch. When glucose molecules are linked with - 1, 4 linkages, the resulting linear molecule is called amylose. When glucose molecules are linked with - 1, 6 linkages,

also produce small amounts of ATP. In addition, the Krebs cycle also produces CO2, which is a by-product of cellular respiration. Finally, in oxidative phosphorylation, the electrons from NADH are put through the electron transport chain, creating a proton gradient between the inter-membrane space and the mitochondrial matrix. By chemiosmosis, (H+) molecules pass through ATP synthase to produce ATP. For every glucose molecule that is “processed” by the cell, approximately 36-38 ATP molecules are produced from both substrate-level and oxidative phosphorylation. Plants carry out both of these processes, and therefore is an excellent model organism to examine these reactions. The plants that were used for this experiment were Wisconsin Fast PlantsTM. Specifically, the Rapid-Cycling Brassica rapa species was used, a common plant genetically modified to complete their life cycle within 21 days. This made it possible to sample leaves almost a week after planting them as there would be enough leaf tissue available for experimentation. This project seeks to discover whether small leaf discs will use free glucose from either their environment or their starch reserves under “light” or “dark” conditions. One way to induce this selective uptake is to sever leaf tissue from the plant itself. This was done by punching out holes from leaves using a straw, and putting leaves into separate groups. Each group had different concentrations of sucrose and glucose in this case. Additionally, some were in the dark, while others were under continuous light. These discs were kept in their respective environments for about 48 hours, at which point, they would first be boiled in alcohol and water (to eliminate chlorophyll), and then tested for starch content, by being dipped in an iodine-potassium-iodide (IKI) solution. The IKI solution then stains the amylopectin and amylose, which can be used to determine the relative abundance of starch. The topic of sugar uptake was studied in 1981 by David A. Reinhart and Robert J. Thomas, both of whom worked on an experiment involving the uptake of 14C-sucrose into plants. For their experiment, they treated mature leaves of the plant Polytrichum commune with 14C-sucrose. By using radioactive tracers in the sugar, they were able to trace the locations to which the sucrose was going; they called these depositories “sinks”. Their research showed that sucrose accumulated in the growing stem apex, young leaves, bud initials, and underground axes. Reinhart’s and Thomas’ project deals more with where the sucrose goes rather than what happened to starch granules in the chloroplasts; our

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project has a greater emphasis on the starch content in the plants, and its relationship to sucrose and glucose uptake. In another experiment, done in 1983, Robert A. Saftner and Roger E. Wyse probed the relationship between plant hormones and the amount of sugar uptake by sugar beet root tissue discs. Using several different plant hormones, either alone or in various combinations, a variety of results were discovered. Of particular interest is the fact that none of these hormones or any combinations of them had any notable effect on the passive mechanism of sucrose uptake. Saftner, Jaleh Daie, and Wyse performed another experiment in 1982 that has even more relevance to our project. They explored the possibility of active versus passive transport of sucrose in plant cells. They found that the active sucrose uptake showed a dependence on external sucrose; when the external concentration reached 20 M sucrose, the active transport mechanism appeared to approach saturation. With passive transport, the sucrose uptake rate in unplasmolyzed tissue showed a linear dependence with external sucrose concentration. Something interesting to note is that both of Wyse’s projects involve sugar beet root and they used discs cut out from the roots. It is also interesting to note that these projects were all done with sucrose, and not any other sugar. In 1978, Roger Wyse independently worked on a project where he dealt strictly with the uptake rates of sucrose, fructose, and glucose, and how two or more of the sugars could inhibit the uptake rates of other sugars. He found that while fructose and glucose displayed typical saturation kinetics, they both showed a markedly lower uptake rate than the uptake rate of sucrose, especially at higher concentrations. Additionally, it was shown that glucose uptake rates actually had a strong inhibitory effect on the uptake rates of sucrose and fructose, while the latter had little effect on the uptake rates of glucose. The sucrose uptake rates were largely linear for concentrations between 0.5 and 500 M. The other two both showed a “leveling off ” of the uptake rates as concentrations increased; fructose starts to level off at 6 M and glucose starts to level off at 0.75M. These results show that sucrose is actually more usable by plant cells than glucose or fructose, both of which are monosaccharides. Our decision to pursue this project stemmed mostly from a project done by Dr. Paul Williams using Brassica rapa. Williams’ variable of interest was the glucose in the plant when each plant is exposed to different amounts of light. His setup was similar to ours’, in that he also controlled the molarity of the sucrose and glucose solutions. However, he used screens to simulate varying degrees of light, and investigated how the sugar was taken up. Our hypothesis has multiple components. For the discs

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that are placed in the dark, it is hypothesized that the leaf discs will use more of their starch reserves as the concentration of sugar increases regardless of whether or not it is glucose or sucrose. Thus, as the sugar concentrations increase, the discs should appear darker. For the discs that are placed in the light, they should all photosynthesize normally, and the concentration of sugar in their immediate environment should not really have a significant effect on the starch consumption of the leaf discs. Finally, it is hypothesized that the discs that are placed in sucrose should follow similar, if not the exact same patterns as the ones placed in glucose.

Table 1: Trial 1

Glucose Light Dark

Sucrose Light Dark

Table 2: Trial 2

Glucose Light Dark

Sucrose Light Dark

Materials and MethodsBrassica rapa seeds were grown for 7-9 days under continuous fluorescent light. The variation in time was due to the amount of leaf tissue used. Plastic straws were used to punch out 96 leaf discs from the leaf tissue. Three leaf discs were placed into film canister lids, and the lids were arranged in groups of 8. Into 4 of the lids, 2mls of the following concentrations of glucose were added to each lid: 0M, 0.25M, 0.50M, and 0.75M. The same procedure was done for the sucrose solutions in the remaining 4 lids. Water saturated paper towels were placed on 4 different black plastic boxes with clear lids; half were labeled “light” and half “dark”. The two “dark” boxes were placed in a cardboard box, covered with clear plastic wrap, a black trash cover, and kept in the dark for 48 hours. The two “light” boxes were covered with clear plastic wrap and placed under fluorescent light for 48 hours. Leaf discs from each experimental condition were then placed into beakers containing 15mls of water. The beakers were boiled until the color from the chlorophyll was removed (generally >2 minutes). To ensure that all the chlorophyll was extracted from the leaf discs, they were then transferred to 10mls of ethanol and incubated for 10-15 minutes. Each leaf disc was then carefully stained in a 25% iodine solution (to test for the levels of starch), laid on plastic wrap and grouped by sugar, light/dark, and molar concentration. After a 10 minute incubation at room temperature, the color of the leaf discs were compared to the color scale.ResultsFor each experimental condition, a total of 5 trials were run, and the results were recorded. For all figures the numbers indicate the relative level of darkness of the leaf disc after being stained with iodine: a result of “10” indicates nearly complete darkness and a measure of “1” indicates that no staining was visible (leaf disc

was completely white). Our results indicate that all leaf discs that were placed in the light used low amounts of starch from their reserves. The only irregularity that was observed was that the discs that were placed in 0.25M glucose used more starch than the other “light” ones. Other than this concentration, the plants in the light had a darkness ranging from 8.133 to 8.533, with an average darkness of 8.40133. However, the dark plants exhibited a very distinct pattern of starch consumption. It was found that as the molarity of sugars surrounding the discs increased, the discs used more of the sugar, and consequently, less of their starch. During trial 1, no “light” data was available, due to procedural difficulties(Table 1). In our next trial, the chlorophyll wasn’t properly removed in many of the samples, thus making it difficult to measure how dark they were(Table 2). In trial 3 we discovered that if the plants were left to sit in ethanol, the chlorophyll would be lost more efficiently, and this led to improved results(Table 3). We performed 2 more trials, using this modified protocol, and got more robust data (Tables 4 and 5). The averages of all 5 trials is shown in Table 6.

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Glucose Light Dark

Sucrose Light Dark

Table 6: Calculated averages of all 5 experimental trials for all conditions of glucose or sucrose under “light” or “dark” environmentsDiscussionFor the leaf discs that were placed in the light, all of them used low amounts of starch from their reserves. This is because the plants were able to photosynthesize under the light, and there would be no need to use the starch reserves. However, the dark plants exhibited a very distinct pattern of starch consumption. It was found that as the molarity of sugars surrounding the discs increased, the discs used more of the sugar, and consequently, less of their starch. The reason for this is clear; with more sugar available, the leaf disc will be able to use more of the free sugars. What this also shows is that the plant can use its own starch reserves in conjunction with sugar uptake mechanisms when deprived of sunlight. Additionally, the results support the fact that as the molarity of the sugar concentration increases, the amount of starch used decreases nearly linearly. In fact, the correlation between darkness of leaf disc and the molarity of the glucose concentrations has a correlation coefficient of 0.985001, while the correlation between darkness of leaf discs and the sucrose concentrations has a correlation coefficient of 0.996817; these results strongly support a linear correlation. The experiment performed did not show any statistically significant difference between the uptake rates of sucrose versus glucose, although a subtle change was observed in the data after averages were taken. Experiments done by Dr. Roger Wyse showed that sucrose uptake rates were considerably faster than the uptake rates of both glucose and fructose. This would imply that if a leaf disc were isolated in a sucrose solution, the plant would take up sucrose faster, use less starch, and therefore be darker in color. On average, for

Glucose Light Dark

Sucrose Light Dark

Glucose Light Dark

Sucrose Light Dark

Glucose Light Dark

Sucrose Light Dark

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the discs exposed to the same molarities of sugar, there was more starch left in the plants exposed to sucrose. These results are consistent with previous research which showed that sucrose uptake rates were faster than glucose uptake rates, and therefore the disc would be able to use less of its starch granules. The data suggests that occasionally, plants will still use starch even when placed in 48 hours of continuous light. It should be expected that the plants exposed to light should have a darkness index close to 9 - 10. However, their darkness reached as low as 7.756. This suggests that plants may be forced to dip into their starch reserves when photosynthetic availability is low. What can be inferred is that the plants are constantly in an equilibrium of producing extra glucose and using starch from its reserves. Further experimentation may be done to show how and when plants use starch, even when under 48 hours of fluorescent light. Another idea for further experimentation is investigating how plants use both photosynthesis and sugar uptake in conjunction with one another to optimize their growth. It is important to acknowledge the existence of errors in the procedural and data collection processes. For example in the first trial, the “light” containers were not covered, and as a result, all the water evaporated. Therefore, a significant part of data was lost in the first trial. Another important problem that came up was with the issue of removing the chlorophyll. At first, the leaf discs were heated in an ethanol solution on a hot plate and it was found this method did not completely destroy the chlorophyll. After a while, the ethanol evaporates very quickly, and if the discs are left too long to boil, they would be singed to a crisp, and if not left long enough, they would retain their pigment. Therefore, it was decided that instead of letting the ethanol boil, it would be better to let the leaf discs sit in the ethanol solution for 10 minutes, which turned out to work quite well. Our later data is more complete, reflecting our new procedural changes. Based on the results that were obtained, this experiment affirms the hypothesis presented. When plants are provided with sugar and are kept in the light, both the plant’s starch granules and the free sugar outside the leaf are more or less distractions to the plant’s normal functions. However, when the plant is placed in the dark and without a source of sunlight, it will use the free sugar in its environment, and the higher the concentration of sugar, the less of its own starch it will use. With these results, it has been shown that plants have multiple mechanisms for maintaining their homeostasis and have a variety of options when in dire need of nutrients.

References“The Calvin cycle (The Nonlight Requiring Reactions).” The Connecticut River Homepage. UMass Amherst Biology Department, Jan. 2011.

Etxeberria, Ed, et al. Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells. Journal of Experimental Botany, 2005.

Government of Canada. Canadian Food Inspection Agency. The Biology of Brassica Rapa L. Nepean: Plant Health and Production Division, 1999.

Guan, Han Ping, and Harry W Janes. “Growth and Sugar Accumulation.” Light Regulation of Sink Metabolism in Tomato Fruit. Plant Physiology. July 1991.

Kamiya, Akio. Effects of Light on Growth and Glucose Consumption in Colorless Chlorella Mutant Cells. Kanagawa: JSPP, 1985.

Mah, C. S., and T. C. Hew. Sugar Uptake and Invertase Activity in Dendrobium Tissue. Blackwell, 1989. 1-2.

Reinhart, David A., and Robert J. Thomas. Sucrose Uptake and Transport in Conducting Cells of Polytrichum commune. American Bryological and Lichenological Society, 1981.

Saftner, Robert A., Jaleh Daie, and Roger E. Wyse. Sucrose Uptake and Compartmentalization in Sugar Beet Taproot Tissue. American Society of Plant Biologists, 1983.

Saftner, Robert A., and Roger E. Wyse. Effect of Plant Hormones on Sucrose Uptake by Sugar Beet Root Tissue Discs. American Society of Plant Biologists, 1984.

Williams, Paul. “Photosynthesis + Respiration.” 16 Jan. 2001. MS.

Williams, Paul, and Dan Lauffer. “Wisconsin Fast Plants.” Wisconsin Fast Plants. 5 Jan. 2011.

Wyse, Roger. Sucrose Uptake by Sugar Beet Tap Root Tissue. American Society of Plant Biologists, 1979.