TESTING FOR CARBOHYDRATES FROM DIFFERENT PLANT PARTS INTRODUCTION
The whole process of photosynthesis leads to the formation of monosaccharides which will then may polymerize into different forms of carbohydrates like the most common forms sucrose and starch. Starch is known to be a carbohydrate polymer of tens to hundreds of thousands of glucose monomers that are joined together by glycosidic bonds (Ophardt, 2003), while sucrose is a disaccharide or a molecule made up of two molecules of the monosaccharide glucose also joined together by glycosidic bonds (McMurry, 2011). The control of distribution of these substances, starch and sucrose, within the plant body depends on two mechanisms intrinsic to the plant itself. First mechanism is carbon allocation which is the regulation and the distribution of fixed carbon into various metabolic pathways (Friend, Coleman and Isebrands, 1994) and among plant organs. This happens through the synthesis of storage compounds that will be translocated at night when production of such substances is limited or ceased, utilization of the photosynthates to support the metabolic activity of the photosynthetic cell and the transport of these molecules to various tissues that are not capable of photosynthesis themselves. The second mechanism is partitioning, which refers to the differential distribution of the photosynthates within the plan and is dependent on t he the total biomass of the tissue t issue that composes the organ and the rate in which the tissues use the photosynthates per unit of its biomass. Generally, all plant tissues compete for the available carbohydrates that are being translocated but the ones nearest the source tissues and have the greatest connection to the source have an advantage over those who do not (Taiz and Zeiger, 2006). This exercise primarily aims to determine the presence of the photosynthates starch and sucrose in the different tissues that composes the different organs of a whole plant organism. Furthermore, the exercise aims to correlate the presence of starch in a particular area and the presence of the other photosynthate, sucrose. Also, the experiment through the aid of pre-conceived knowledge on photosynthate distribution throughout the plant body, aims to determine some factors that affects the concentration of the photosynthates in one organ relative to the other.
MATERIALS AND METHODS
This experiment that generally tests for the presence of carbohydrates in different plant parts was divided into two parts. For the first part, the presence of starch in the different organs of the plant, Iresene herbstii , was determined. Meanwhile, the second part tested for the presence of re ducing sugars
such as glucose, fructose, and galactose, in the same plant species. The two parts of the experiment both used five grams each of the leaves, roots and stem of I. herbstii . These plant organs were macerated separately using a mortar and pestle and then distilled water was added in each pestle, 1 ml for the starch test and 2 ml for the reducing sugar test. After maceration, the
liquid extracts were retrieved and placed in individual microcentrifuge tubes that are properly labeled. The microcentrifuge tubes were then placed in the centrifugation machine set to 5000 rpm and centrifuged for five minutes. After centrifugation, a 1000ul micropipette was used to retrieve the supernatant from the microcentrifuge tubes. For the starch test, 200 ul of the supernatants were dispensed into the different wells of a spotting plate and then iodine solution was subsequently added. Changes that followed the addition of iodine solution were carefully observed and noted. Meanwhile, for the test for reducing sugars, 1000 ul of each supernatant were put into individual test tubes and then 3 ml of Benedict’s solution was subsequently added. After addition, the test tubes were placed in a boiling water bath and the formation of a precipitate and its characteristics were noted. RESULTS
Figure 1. Iresine herbstii (whole plant). Plant used in the experiment wherein the roots, stem and leaves were macerated and was tested using iodine test and benedict’s test to determine the presence of starch and reduc ing sugars- glucose and fructose (Picture obtained at: http://plantsarethestrangestpeople.blogspot.com).
Figure 2. The leaf, roots, and stem of I. herbstii , from left to right respectively. The color of these organs of the
plant used was purple which indicate the high presence of anthocyanin pigments.
Iresine herbtii was choosen to be the source of the extracts from different organs because of its soft
leaves, stem and roots which helped in faster maceration and extraction of the crude extract. From figure 1 and figure 2, the color of the organs is purplish which indicate the high presence of anthocyanin pigments in the organ tissues. This high concentration of anthocyanin can be a cause or source of wrong results in this experiment because of it being a higher concentration than the starch or glucose that is being tested in the experiment.
Leaf
stem
root
Figure 3. Test for presence of starch from mixture of iodine solution with the extracts of Iresine herbstii
leaf, stem, and stem extracts (from left to right). No blue-black or purple-black precipitate was observed among the three extracts. Table 1. Test for Starch Extract
Result (after addition of iodine solution)
Leaf
(+) black solution with precipitate
Stem
(—) no precipitate or black solution
Root
(—) no precipitate or black solution
Figure 3 and table 1 indicate that the extract from the leaf only attained a positive result which is a black solution and a presence of a precipitate upon the addition of the iodine solution. This indicate that a large amount of starch is present in the leaf which is actually true because some of the triose-phosphate produced in photosynthesis is converted into starch and stored in vacuoles of the chloroplast.
Stems
leaves
roots
Figure 4. Test for reducing sugars from mixture of Benedict’s solution with the extract from Iresine herbstii stems, leaves and roots extracts. After heating, varying intensities of green solution and
presence and color of the precipitate were observed.
Table 2. Test for Reducing Sugars Extract
Color of solution
Color of precipitate
Stem
light green
green precipitate
Leaves
dark green
Reddish brown precipitate
Roots
bluish green
Yellow orange precipitate
The test for reducing sugars using the benedict’s solution indicates the presence of glucose or fructose in the extract. The observation was done after the addition of benedict’s solution and heating. Our
result showed that the extract from stem had the least concentration of reducing sugars glucose and fructose because of its green precipitate. It was found out that the leaves have the highest concentration of glucose or fructose because of the reddish brown precipitate observed after heating . Discussion
In plants, the main products of photosynthesis are monosaccharides that eventually polymerize into different forms of carbohydrates commonly in the form of sucrose and starch. Starch is a polymer made by plants to store energy composed of thousands of glucose monomers that are joined together by glycosidic bond. Sucrose on the other hand, functions as the primary transport sugar and plays a central role on plant growth and development (Winter & Huber, 2000). In this experiment, plant organs
namely the leaf, the stem and the roots, were used to test for starch and sucrose content found in plant tissues. For the first part of the experiment, iodine test was conducted in order to test for starch in different plant extracts obtained from 5g of leaves, stem, and roots. Each organ was washed thoroughly with water to remove adhering particles. After obtaining the extracts from the different plant organs, each were centrifuged for 5 minutes at 5000 rpm to further acquire purer extracts. Iodine served an indicator, which turns blue-black or purple in the presence of starch in the supernatants obtained after centrifugation. From the results obtained in the e xperiment, only the leaf showed a positive re sult to the test by turning into a dark purplish color from primarily being light purple extract due to the presence of anthocyanin. On the other hand, the stem and the root retained their original colors, which were light purple and brown respectively, even after the addition of iodine. With respect the result of the iodine test, it is evident that the leaves possess the highest starch content among the other plant organs mainly due to the fact that leaves are the ones primarily involved during photosynthesis. The triose phosphate produce during photosynthesis is converted into starch and are then stored in the chloroplast. Since roots are found underneath the soil, they do not really play a role in the food making of the plants. The stem on the other hand could also contain starch but of minimal amount. The second part of the experiment dealt with the test for reducing sugars, glucose or fructose. The same process was conducted in this part only that the supernatants were subjected to Benedict’s
test for reducing sugars. The supernatants of the different plants organs were placed in separate test tubes then 3mL of Benedict’s solution was added to each. The test tubes were then boiled to heat in
water bath until a notable change in color was observed. For this particular test, a reddish brown color indicates a positive result. From the data obtained in the experiment, it was observed that the leaves had the highest concentration of reducing sugars due to the red/brown precipitate evident after heating. The stems and roots on the other hand obtained green and yellow precipitates respectively indicating that no or little amount of reducing sugars are found in the plant organs. This result again due to the fact that leaves are ones mainly incorporated in the process of photosynthesis, thus, possess a high amount of reducing sugars as compared to the stem and roots. REFERENCES
Friend, A.L. Coleman, M.D. and J.G. Isebrands. (1994). Carbon allocation to root and shoot systems of woody plants. Biology of Adventitious Root Formation. NY: Plenum Press th
McMurry, J. (2011). Organic Chemistry . 8 Edition. USA: Cengage Learning Ophardt, C.E. (2003). Starch. http://www.elmhurst.edu/~chm/vchembook/547starch.html Retrieved 11 November 2014 Taiz, L. and E. Zeiger. (2006). Plant Physiology. Fourth Edition. Massachusetts: Sinauer Associates, Inc., Publishers Reference: http://www.ncbi.nlm.nih.gov/pubmed/11005202
