Evaluating Cell Fractionation Procedures and Cytochemical Tests Evaluating Cell Fractionation Procedures and Cytochemical Tests Joshua

W. Zollman
1

From the Undergraduate Department of Biology: The Pennsylvania State University

To whom correspondence should be addressed: Joshua W. Zollman, 342 Atherton Hall, University Park, PA 16802, USA. Tel.:(610) 220 2134; E-mail: jzollman@psu.edu

INTRODUCTION Plastids are a major type of organelle found in plant cells. A variety of different kinds of plastids exist in plant cells, including chloroplasts and amyloplasts (2). These plastids serve crucial biochemical roles in plant cells, including photosynthesis and the storage of starch and sugars (1). Chloroplasts are active in the conversion of light energy into sugar and are generally found in green plant tissues where photosynthesis occurs (2). On the other hand, amyloplasts function as reservoirs of starches that are used for long-term storage. Amyloplasts are often found in tubers and seed endosperm (2). Plastids also exhibit significant capacity to interconvert (4). For example, the proplastids of tobacco cells have been found to develop into amyloplasts when grown in the presence of the plant hormone cytokinin (5). Additionally, chloroplasts can develop into amyloplasts and vice versa depending on the presence of light (4). The ability for chloroplasts and amyloplasts to interconvert leads to possible agricultural applications of which plant physiology research groups are currently investigating (1). However, in order to determine the factors that lead to the differentiation and interconversion of these plastids, the plastids must be effectively separated from each other as well as from other cellular components such as nuclei and mitochondria (1). There exist a variety of methods for isolating specific organelles from a cell, including differential centrifugation (3). Differential centrifugation allows for the separation of cell parts into fractions based on size, shape, and density, and is thus a potentially useful method for the separation of nuclei, amyloplasts, chloroplasts, and mitochondria (1, 3). After organelle separation via differential centrifugation has been performed, cytochemical tests then can be administered to

help determine the identities and quantities of cell parts present in each fraction (1). There are several features of nuclei, amyloplasts, and chloroplasts that should theoretically allow for their separation via differential centrifugation. Their varying densities and relative sizes should cause them to separate in different fractions (1). For example, intact nuclei have an average density of 1.32g/cm-3, while chloroplasts have a density of 1.21-1.24g/cm-3 (3). A cell homogenate can be prepared and centrifuged at a relatively low speed (and consequently subjected to relative low centrifugal force) allowing for the denser cell components to pellet. The supernatant can then be removed, resuspended, and centrifuged at successively higher speeds (1, 3). There are several characteristics of nuclei, amyloplasts, and chloroplasts that allow for cytochemical tests to be effectively administered. Methylene Blue stains nucleic acids blue, while Lugols iodine stains starch purple (1). Because nuclei contain nucleic acids, the nuclei should stain blue when subjected to Methylene Blue. Lugols iodine should likewise stain amyloplasts purple, due to their high starch content. Chloroplasts contain the green pigment chlorophyll and thus should appear green when unstained (2). Since it is necessary to isolate chloroplasts and amyloplasts in order to study their interconversion, it crucial that effective separation techniques are utilized in the laboratory. Accordingly, this studys purpose is to determine the extent that cytochemical tests and cell fractionation are useful for isolating chloroplasts and amyloplasts (1). Using both pea seed and spinach leaf homogenate, I set out with the expectation that cell fractionation and cytochemical tests would be highly effective in the separation of cell parts. Based on the data

Evaluating Cell Fractionation Procedures and Cytochemical Tests available, I anticipated finding a high abundance of nuclei in the first fraction (P1) due to its relatively high density, and less abundance in successive fractions. Conversely, intact chloroplasts and amyloplasts have lower densities than nuclei (3), so I expected them to pellet in later fractions. Because amyloplasts tend to be found in storage tissues, such as seeds (2), I expected to find a higher abundance of amyloplasts in pea seed homogenate than spinach leaf homogenate. On the other hand, since chloroplasts tend to be highly abundant in green tissue (2), I expected to find a higher concentration of chloroplasts than amyloplasts in the spinach leaf cell homogenate. EXPERIMENTAL PROCEDURES Partially processed pea and spinach homogenates were prepared by the method of Burpee (2009). Note that three groups in the class carried out the procedure for the pea cell homogenate while the three other groups carried out the procedure for the spinach leaf cell homogenate. 2 In the next phase of the experiment the cell homogenate was centrifuged at 100g for 10 minutes using a Sorvall Superspeed RC-2B centrifuge. The supernatant was then removed using a plastic pipet and the pellet (P1) was separated and resuspended in 5 ml CRS buffer. The supernatant was then centrifuged at 800g for 10 minutes. The resulting supernatant was then removed and the pellet (P2) resuspended in 5 ml CRS buffer as was P1. The resulting supernatant was then centrifuged again at 10,000g for 10 minutes and subsequently removed (S3). The pellet was again resuspended in 5 ml of CRS buffer. Each pellet was resuspended in the CRS buffer with a gentle flick on the test tube, with the exception of P3, which was shaken vigorously. Following the procedure in the Lab Manual (1), the microscopic and cytochemical detection of the organelles was carried out with a compound light microscope at 100x and 400x magnification and by use of Methylene Blue solution and Lugols iodine solution. 3 slides of each of P1, P2, P3, and S3 were prepared. One drop of Lugols iodine was added to each of the four slides of one set. This procedure was then repeated with Methylene Blue instead of Lugols iodine. Qualitative and quantitative observations were then recorded at either 100x or 400x magnification. All observations were recorded using 400x magnification except for P3 with Methylene blue, S3 with Lugols iodine, and S3 with no stain, which were observed at 100x magnification. It should be noted that the slides of P1 were prepared and observed while P2 was in the centrifuge (10 minutes) and the slides of P2 were prepared and observed while P3 was in the centrifuge (10 minutes). RESULTS After individual group data was aggregated, the class agreed upon two tables of consensus data. A plus-minus (+-) scheme was used to describe the relative abundance of organelles in each view. The purpose of the (+-) scheme is to easily represent the approximate number of organelles present in each field of view since the exact number of organelles could not always be counted. In this scheme, +++ denotes a non-countable quantity of organelles, ++ denotes a countable quantity of organelles, + denotes few organelles present, +(-) denotes less than 10 organelles present, and denotes the absence of organelles. This scheme was standardized among members of each group as well as each group of the six groups in the lab. Variations in the amount of CRS buffer added to each pellet were crudely accounted for when groups data were aggregated. The magnification under which each pellet was viewed was also crudely taken into account when recording the relative abundance of each organelle. The criteria for identifying each organelle type were as follows: Amyloplasts should to appear as large purple circles when subjected to Lugols iodine solution (1). Nuclei should to appear relative small and stain purple when subjected to Methylene blue. In the unstained slides, chloroplasts should appear as large, green to yellow bodies (2). Consensus data for the pea cell homogenate (Figure 1) showed high abundance (+++) of nuclei, amyloplasts and chloroplasts in P1. P2 showed a countable quantity of nuclei, amyloplasts, and chloroplasts, while few (+) nuclei and amyloplasts were present in P3 and chloroplasts were completely absent (-). Less than 10 (+-) nuclei and amyloplasts were reported in S3 while chloroplasts were again absent (-).

Evaluating Cell Fractionation Procedures and Cytochemical Tests Consensus data for the spinach leaf cell (Figure 2) homogenate showed similar trends to the pea cell homogenate, however isolation of amyloplasts and chloroplasts in P2 and P3 was more successful. Countable quantities (++) of nuclei, amyloplasts and chloroplasts were each found in P1. Relative abundance of nuclei remained constant in P2, while relative abundance of chloroplasts and amyloplasts increased (+++), unlike the P2 results of the pea cell homogenate. P3 demonstrated countable quantities of nuclei and amyloplasts (++) but a non-countable quantity of chloroplasts (+++). S3 was found to contain few (+) of each nuclei, chloroplasts and amyloplasts. Both the pea cell homogenate and the spinach cell homogenate demonstrated a decreasing trend of nuclei from lower to higher fractions (P1S3). However, the pea cell homogenate also demonstrated a similar decreasing trend of relative abundance of amyloplasts and chloroplasts. The spinach cell homogenate did not exhibit this trend because higher abundances of chloroplasts and amyloplasts were found in P2 and P3. DISCUSSION The results, for the most part, did not demonstrate the effective separation of nuclei, chloroplasts, and amyloplasts. This calls into question the effectiveness of differential centrifugation and cytochemical testing for this kind of study. As expected, in the case of both cell types, a large number of nuclei pelleted in early cell fractions compared with few in later fractions. However, due to their low densities, it was not expected that a large number of chloroplasts and amyloplasts would appear in early fractions. Contrary to this expectation, results from both pea and spinach cell homogenates showed significant presence of these organelles in P1. In addition, in the case of the pea cell homogenate, the abundance of every organelle decreased with each successive round of centrifugation. These findings suggest that the centrifugation step was not effective in separating organelles by density. Since the abundance of every organelle decreased, it appears as though each successive fraction was simply more diluted than the last. A more likely explanation is that the pellet suspension step exaggerated the presence of organelles in early fractions. Additionally, If the pellets were not resuspended properly in the CRS buffer, this could cause the suspensions to be enriched with a mixture of organelles instead of one particular type. There are a variety of possible errors that could have caused this: Improper separation of the supernatant from the pellet could have altered the results, causing cell parts from earlier fractions to appear in later ones. The fact that P3 was shaken vigorously instead of gently flicked like all of the other fractions could have altered the number of organelles present in P3. Along with this, it is possible than some organelles were stuck on the side or bottom of the test tube, causing them to appear in later fractions than expected. Despite the possible errors in the resuspension step, the results of the spinach leaf cell homogenate suggest that amyloplasts and chloroplasts were successfully separated in fractions P2 and P3. The increase in abundance of both amyloplasts and chloroplasts from P1 to P2 suggests that the organelles were successfully separated by density. P3 also exhibits high relative abundance of chloroplasts (+++), although the relative abundance of amyloplasts decreased from P2 to P3. These findings also confirm the hypothesis that more chloroplasts would be found in the spinach leaf cell homogenate than the pea seed cell homogenate. However, it is worth noting that this finding could have easily been coincidental. While the cytochemical tests seemed to be effective, there were instances when organelles were hard to distinguish. Lugols iodine often seemed to stain all of the cell parts a strong brown color, making it hard to differentiate cell parts. Perhaps, next time more care should be taken to ensure that only a small amount of Lugols iodine solution is applied to each slide. Variations of the procedure carried out between groups performing the experiment also could have altered results significantly. Some groups added only 1ml of CRS Buffer to their pellets, whereas other groups added 5ml. The groups that added higher amount of buffer would have a more diluted solution to view under the microscope, causing less organelles to appear when viewed under the microscope. In addition, variance in the magnification used could cause results to vary significantly between groups. For example, a view at 100x magnification would

Evaluating Cell Fractionation Procedures and Cytochemical Tests likely cause far more organelles to appear visible than a view at 400x would. The class did its best to account for differences in both the amount of CRS buffer used and the magnification used when viewing slides. However, in order to reduce groupto-group variation, and consequently make results more accurate, I would suggest standardizing the procedure. This could reduce the potential for inaccurate results dramatically. Another way to increase the reliability of the results would be to repeat the study numerous times and average the results. There are also other centrifugation techniques that could lead to more successful isolation of cellular components. For example, standard isopycnic sucrose gradient centrifugation has been found to separate intact nuclei from other organelles highly effectively (3). Removing nuclei from the cell would aid in isolating pure preparations of other organelles. Other techniques for the separation of chloroplasts could also be explored, such as the procedure of Mills et al. (1980), which allows for isolation chloroplasts in less than 5 minutes (6). Additionally, procedures that do not require centrifugation could be used, such as the procedure of Cookburn et al. (1968), in which spinach chloroplasts can be separated in pyrophosphate media (7). Both of these procedures would allow for pure preparations of these chloroplasts to be isolated. There are important agricultural applications associated with the interconversion of amyloplasts and chloroplasts (1). However, the interconversion of chloroplasts and amyloplasts cannot realistically be studied without effective techniques for isolating these plastids from whole cells. This study demonstrated that the differential centrifugation and cytochemical testing are not necessarily effective methods for the isolation of nuclei, amyloplasts, and chloroplasts from plant cells.

Evaluating Cell Fractionation Procedures and Cytochemical Tests REFERENCES

1. Department of Biology. (2012, Fall). Bio 230W Laboratory Manual: Cell Fractionation. University Park: The Pennsylvania State University. 2. Pyke, K. A., (1999) Plastid Division and Development. The Plant Cell 4, 549-556 3. Quail, P. H., (1979) Plant Cell Fractionation. Annual Review of Plant Physiology. 30, 425-484 4. Gillott, M.A., Erdos, G., and Buetow, D.E., 1991. Light-induced chloroplast differentiation in soybean cells in suspension culture: Ultrastructural changes during the bleaching and greening cycles. Plant Physiology, 96, 962. 5. Miyazawa, Y., Sakai, A., Miyagishima, S., Takano, H., Kawano, S., and Kuroiwa, T., 1999. Auxin and cytokinin have opposite effects on amyloplast development and expression of starch synthesis genes in cultured bright yellow-2 tobacco cells. Plant Physiology, 121, 461. 6. Mills, W. R., Joy, K.W., 1980. A Rapid Method for Isolation of Purified Physiologically Active Chloroplasts, Used to Study the Intracellular Distribution of Amino Acids in Pea Leaves. Planta, 146, 75-83. 7. Cockburn, W., Walker, D.A., Baldry, C.W., 1968. The Isolation of Spinach Chloroplasts in Pyrophosphate Media. Plant Physiol. 43, 1415-1418. FOOTNOTES
1

This lab was completed as part of the Biology 230W class in fall 2012. See reference 1 for lab manual citation. 2 My lab group carried out this and all following parts of the procedure. We used the pea seed cell homogenate. FIGURE LEGENDS FIGURE 1. Shows class consensus results of the number of organelles in each fraction for both pea seed cell homogenate and spinach leaf cell homogenate. +++ denotes a noncountable quantity of organelles, ++ denotes a countable quantity of organelles, + denotes few organelles present, +(-) denotes less than 10 organelles present, and denotes the absence of organelles.

Evaluating Cell Fractionation Procedures and Cytochemical Tests Figure 1 Class Consensus Data of Pea Seed and Spinach Lead Homogenates