Experiments on Mutant Plants

11th Grade Biology

The Effect of UV Light on Arabidopsis CHS Mutants

(The picture on the left is of two of the four total pots of

Arabidopsis plants that my partner and I used for our experiment. The one on the left has wild type plants, and the one on the right has plants with the CHS mutant. They are the controlled plants, meaning they are growing under normal conditions.)

Introduction:

CHS, otherwise known as chalcone synthase, is an enzyme (a biological catalyst, which is the increase in the rate of a chemical reaction) that appears a lot in higher plants. It is a mutation that allows plants to absorb large amounts of lethal Ultraviolet-B radiation. The pigment analysis of flavonoids, which are a major component of the mutation CHS, show an increment in accumulation of UV absorbing molecules in UV T1 that increases the capability of leaves to block radiation from UV-B rays, and this is the reason for this mutant’s higher resistance to the radiation. UV-B rays are anywhere between 290-320 nanometers long. For this experiment, my partner and I researched how an Arabidopsis with the added mutant CHS reacts under normal light versus how it acts under a wide spectrum UV light source. We tested to see if the CHS mutation would actually protect the plant from harmful sources of UV radiation, and if protection extends beyond strictly UV-B rays, so that we could have a better overall understanding of how CHS works and use it to our advantage.

For this experiment, we compared the growth of Arabidopsis plants when exposed to different forms of light, specifically between normal light and wide spectrum UV radiation. We placed two pots of Arabidopsis under a wide spectrum UV light, one of which was the wild type (normal), and the other which was our mutated plant, with a CHS gene. Two more pots of Arabidopsis were placed under a normal light, again with one wild plant and one mutated plant. We chose this treatment for our plants because it the most common treatment for CHS affected Arabidopsis. Due to changes in the atmosphere, different (potentially stronger) levels of radiation will be emitted, and thus we may be obliged to mutate plants so that they can continue to grow through the harmful levels of UV radiation in their natural environments. After learning how this gene works, it can be adapted to be used in farmland and widespread as a solution for drastic environmental changes. Our overall hypothesis for this project was that the mutant plants would grow better under UV light than the wild types.

Materials:

30 wild-type Arabidopsis seeds
30 CHS mutant seeds
2 mL 0.1% agarose (for cold treatment)
2 2mL mictrotubes
4 small pots (2" square)
Soil w/ fertilizer
Water
Fluorescent lights
UV lights (which we shared with the rest of our class)
Large Tray w/holes (which we shared with the rest of our class)
Humidity dome (which we shared with the rest of our class)

Methods:

The very first thing I did was put thirty wild-type and thirty CHS mutant seeds into two respective 2mL micrtotubes. I then added 1mL of 0.1% agarose to each tube and the seeds were refrigerated for three days. After taking our seeds out of the refrigerator, we planted the seeds evenly into the four different pots (wild type seeds were planted in pots labeled "wild type," and mutant seeds were planted in pots labeled "mutant CHS"). The two different seeds, mutant and wild type, and the experimental condition were the independent variables of this experiment; in other words, they are the variables that cannot be controlled. However, the size, shape, color, and health (among other aspects) of the plants are all dependent variables; they are variables that can be controlled! They can be directly affected based on the type of soil we use to plant the seeds in, the amount of water we give them, and how strong the plants' light source is. These dependent variables were the ones we were most interested in observing, because they would give us the most insight into coming up with an answer to our research paper. We would begin capturing data the moment that we could see our plants growing, and from this moment on we would capture data every school day at the beginning of class. We would jot down the average rosette width (or if it was there, bolt height) for each plant, write individual observations, and end by comparing three pairs of plants: wildtype controlled vs wildtype experimental, mutant controlled vs mutant experimental, and wildtype experimental vs. mutant experimental. Controlled means it is under the normal fluorescent light; in other words, it is growing under normal conditions. We water the plants every other day, and we do this by placing our pots in a tub of water and leaving them there for five minutes (there are holes in the bottom of the pots so that the soil can take in water).

So far, our treatment is going well. We've observed that the wildtype plants are growing much faster and taller/longer than the mutants are, which leads us to believe that CHS must have some control over Arabidopsis growth genes. Besides this, everything seems to be going fairly well; all the plants are growing regardless of how much compared to others. The plants that are being put under the wide spectrum UV-light do appear to be leaning to the side instead of up like their counterparts that are under the fluorescent light.

Results:

We tested the Arabidopsis plants over a course of thirty one days. We recorded rosette diameters for the first eighteen days, and bolt heights for the remainder of the days. The graph showing rosette diamater comparisons over a course of eighteen days can be found here: http://tinyurl.com/rosettediametercomparisons. The graph showing bolt height comparisons over a course of the final five days of testing can be found here: http://tinyurl.com/boltheightcomparisons.

Figure 1 shows two Arabidopsis plant pots that my partner and I were testing under controlled conditions for the experiment. The one on the left has the mutant gene CHS, and the one on the right has normal, wild type plants. This picture was taken on December 5, 2014, the final day of testing.)

Figure 2 shows two Arabidopsis plant pots that my partner and I were testing under experimental conditions for the experiment. The one on the left has the mutant gene CHS, and the one on the right has normal, wild type plants. This picture was takn on December 5, 2014, the final day of testing.)

The following comparisons were written on December 5, 2014, the final day of testing:

Wild Type Controlled vs. Mutant Controlled (Effect of Mutation): There are many more leaves in the wild type, and the bolts are longer, straighter, and more erect. The colors look the same too.
Wild Type Controlled vs. Wild Type Experimental (Effect of Treatment): There are more leaves in the controlled,and there are also more bolts (which are, on average, longer than the ones in the mutant.
Wild Type Experimental vs. Mutant Experimental (Effect of Treatment on Mutation): There are leaves and bolts growing out of the wild type, and there is barely anything in the mutant plant

The graphs in the document on the right correspond to the three different things being compared above. If you cannot view the document, click here:

http://tinyurl.com/arabidopsisexperimentgraphs

Conclusion:

Wild Type Controlled vs. Mutant Controlled (Effect of Mutation):

The mutant CHS decreases the growth rate of Arabidopsis. When put under controlled conditions, the wild type’s rosette diameter reached 15 mm after 7 days. The mutant took over twice as long to reach a rosette diameter of 15 mm than the wild type, at eight extra days. We think that this is a significant difference and is likely not due to chance. The CHS mutant decreased the growth rate of Arabidopsis when put under controlled conditions, which in our experiment was putting them under fluorescent lights and watering them every two days. This leads to the conclusion that having a CHS mutant will lessen the growth of an Arabidopsis plant in comparison to a wild type. This might be due to the fact that since CHS is so well at absorbing UV-B light, it might need an excess amount of it to grow well.

Wild Type Controlled vs. Wild Type Experimental (Effect of Treatment):

The experimental conditions used in our treatment, which was setting plants under a wide spectrum UV lamp, did not cause a significant effect to wild type plants which were put under normal conditions (a fluorescent light). When put under controlled conditions, both the wild type and mutant rosette diameters reached 25 mm after eleven days. We think that this is due to chance. Compared to the mutants that were not able to protect themselves from the wide spectrum UV light, the wild type Arabidopsis must have protection against the radiation from normal, fluorescent light as well as UV light.

Wild Type Experimental vs. Mutant Experimental (Effect of the Treatment on the Mutant):

The mutant CHS decreases the growth rate of Arabidopsis when put under a wide UV spectrum lamp. Under UV spectral light, the wild type’s rosette diameter reached 15 mm within eight days, compared to the mutant, which reached 15 mm after 14 days. The mutant took almost twice as long as the wild type, at six extra days. We think that this is a significant difference and is likely not due to chance. The CHS mutant decreased the growth rate of Arabidopsis when put under a wide spectrum UV lamp because CHS is specific to the absorption of UV-B radiation. The lamp that we used was a wide spectrum, meaning it also radiated UV-A and UV-C light. This leads to the conclusion that having a CHS mutant will only protect plants against specific UV-B rays, and we came to this conclusion because our experimental mutant plants grew less than the experimental wild type plants.

These finding are important in a global context because it means adding CHS mutants to plants might not in fact be a good idea. Before doing this experiment, we believed that CHS would help protect plants against all UV radiation, but, according to our results, it is evident that the mutant not only stunts growth in general, but it most definitely does not protect against wide spectrum UV light. Our research suggests it can protect against UV-B rays specifically, but it appears as though that is the extent. For this reason, it would not be a good idea to add this mutant to crops in order to help them survive, for there is both UV-B and UV-A radiation emmited from the sun.

For the next experiment, it might have been a good idea to make sure that we were planting enough seeds in our pots. The main reason for this is because some of our pots did not have very many plants growing (at least two plants died in each of our mutant pots). I would also make sure to water them more regularly, as Dorothy and I would occasionally forgot to on the days in which we were supposed to.

A future study I would conduct is testing Arabidopsis plants with a CHS mutant under strictly UV-B light. All of our research taught us that the CHS gene helps protect plants from UV-B radiation (by absorbing it), and I think it would be beneficial to conduct a specialized study on this in order to test its relevance and potentially confirm it.

If you would like to view my raw data collection for the Arabidopsis project, click here:

http://tinyurl.com/arabidopsisdatacollection

If you would like to view the pictures of plants that my partner and I took for this project, click here:

http://tinyurl.com/arabidopsisimages

Works Cited:

"PREP." Would You like to Help Discover the Function of a Gene? The Partnership for Research and Education in Plants, n.d. Web. 16 Dec. 2014.