Chlorophyll A And B Concentrations In Brassica Rapa

Published Date: 02 Nov 2017

Erin Campbell

Section ABE

Julia Ossler

Chlorophyll A and B Concentrations in Brassica rapa Plants, Relative to Differing Acidic Soil pH Levels.

Abstract

Acid rain has the ability to change the pH of soil in the areas it falls. This can kill plants that are heavily relied on in our food web. Thus animals can suffer as well as humans if acid rain persists to be a problem. In this lab my fellow teammates and I preformed an experiment on how differing acidic pH levels affect the chlorophyll concentration of dwarf Brassica rapa. We chose pH levels of 4,5, and 6 because the plant species we picked will still be able to live moderately, plus acid rain has not been recorded to be able to extremely change pH soil levels. In the beginning we planted the seeds in corresponding soil containers to which pH would be added. The acidic solution was not to be added till after germination, so as not to kill the seed before any growth. Next, each week we sprayed the plants with the pH corresponding to the container. After 3 weeks the plants would then be tested for chlorophyll concentration. We tested chlorophyll concentration in the plants with the altered soil once they matured because chlorophyll concentration has a direct link to whether or not a plant is doing well. In our experiment we found no growth of chloroplast in plants living in pH 4 soil, low chlorophyll concentration of pH 5 plants, and relatively normal/ moderate chlorophyll concentration of pH 6 plants. This helps to conclude that more acidic pH levels inhibit the uptake of nutrients in a plant through the vascular system. Thus with climate changes tending towards more extreme weather changes plants will have a harder time surviving. For the future some sort of root treatment must be used on plant species like B. rapa to help them absorb more nutrients when they are living in acidic condition.

Introduction

Acid rain has always been a fear of the human race for quite awhile. Of course most people see it as a terrifying rain that descends from the skies and burns holes through roofs, cars, and what not. In actuality, acid rain is created when SO2 and NO, released from plants and factories and plants as pollution, start to concentrate in the atmosphere. These two compounds come together with water molecules in the air and form an acidic solution. This is then released when it rains (umac.org, n.d.). What it does to some areas is rather devastating. In the United States, mainly on the east coast, there have been occurrences of acid rain destroying vegetation. Sometimes this destruction is seen in the foliage on plants or underground in the root system. This problem has brought me and my teammates together to form an experiment on a dwarf species, B. rapa, testing what the effects are to chlorophyll concentration when differing levels of a pH solution, resembling acid rain, is added to the soil of the germinating plant. We hope in the end that our findings may lead to knowing more information on how the altered pH affects the plant. We will be looking at what nutrients it inhibits as well as what the affect to photosynthesis there is. Once this information is known there will be able to be a forward movement to help protect plants from acid rain.

B. rapa is a biennial dicot, that prefers to grow during either spring, summer, and/or fall. The plant contains yellow flowers with green foliage. The seed is small and brown, allowing it to be easily dispersed. B. rapa has a very rapid growth rate and which implies the title "Wisconsin Fast Plant". Since, the plant grows mainly in warm condition B. rapa does not survive well in cold temperate environments. The plant also requires quite a bit of water, making it hard to survive in droughts. The soil in which B. rapa grows is varied. It can adapt to coarsely textured, fine, or medium textured soil. Also, the soil in which the B. rapa prefers the most has a fairly neutral pH, ranging from 8-5 (plants.usda,gov).

One mutation of the B. rapa plant is that of dwarfism. A dwarf gene is placed into the plant making it severely shorter than the average plant. The height of dwf1 B. rapa is around 7-10 cm compared to the original plant which is around .91 meters (plants.usda.gov). This lowered height capability can be used for space efficient farming. Since the plants are smaller more can be grown using less land. Specifically in certain B. rapa cultivars, the dwarf gene has reduced land coverage problems (Muangprom and Osborn 2004). Even in other plants such as the cucumber, the dwarf gene has been implemented to help reduce the size of the plant and allow for the same productivity with less land usage (Li, Yang, Pathak, He, and Weng 2011). In addition to the gene allowing for the plants to save space, the gene alters the internal contents of the plant as well. Since, the dwarf plant is smaller than the average sized plant there will be less cells and organelles, like chloroplast. Also, with a lower number of chloroplast an increase in chlorophyll concentration may occur, since the plants must still be photosynthetic. This could make the plants look greener than the original.

Chloroplasts are highly dependent on nutrient uptake in the roots of the B. rapa plant. This uptake is made possible by the numerous root hairs covering the roots. These root hairs allow for more surface area, thus taking in more water and suitable nutrients into the plant. The nutrients that are needed for the growth of the plant as well as for use in chloroplast formation are only allowed in if they can pass through small channels in the casparian strip. The casparian strip is a waxy layer in the endodermis of roots that keeps out unwanted and harmful minerals and ions. However, if none of those nutrients that are passable are available to get through to the vascular stele, chlorophyll concentration will begin to decline in number.

The vascular stele is the transportation system for all plants and it directly feeds to the chloroplast in plants to take carbohydrates down the plant as well as to drop off needed minerals, like phosphate to be used in photosynthesis. According to an observation in Finland, a rather larger storm on the cost caused an upwelling, which decreased the average water level on the coast of Finland. This caused the nutrients and minerals in the soil to be washed away and the enrichment of the soil plummeted. In effect of this circumstance, the chlorophyll concentration within the chloroplast found in the local plants decreased in number and concentration (Kuvaldina, Lips, and Liblik 2010).

Besides weather ailments acidic or alkali pH can also deplete the soil of nutrients by either not allowing the availability of the nutrients or by stripping them completely from the soil. A study by Smith, Fisher, and Argo, documented fertilizer content and pH substrate tests on nutrient growth in soils. The conclusive findings of the study showed that, as the pH became either more alkali or more acidic, the nutrient availability decreased in the soil. Some of these nutrients and minerals missing from inhibited in the soil were iron and manganese, which are much needed in the growth of a plant. (Smith, Fisher, and Argo 2004). All in all whether from weather or other oddities in soil, if pH is destroying or inhibiting nutrients and minerals for chlorophyll, then photosynthesis can not occur and plants will not be as productive.

Hypothesis: For the chlorophyll in B. rapa to be maintained and allotted for photosynthesis minerals and nutrients are required, but as pH of the soil tends to be more acidic those nutrients cannot be taken up and there is a decrease in chlorophyll concentration.

Methods

We created 3 different soil environments for the dwarf B. rapa seeds that were planted. In doing this we used a peat, clay, organic material, and perlite/vermiculite mixture. Also we added fertilizer pellets by spacing them out in the soil.

We then planted 6 dwarf B. rapa seeds in each container. Placing the seeds about 2 or 3 cm into the soil we covered them lightly with more soil mixture. We made sure to label each container with the pH (4,5,6) that we used on the soil.

Once planted, we placed each plant in the exact same environment setting. We made sure that each plant received the same amount of daylight, deionized water, oxygen flow, and food, if offered. These needed to be controlled, so they were not changed for any of the plants.

We kept the plants in the same environment, up until they grew into mature plants. This also allowed for more control, which would give us less varied results.

To make the three different pH solutions that were used to imitate acid rain, we used vinegar and diluted it with water. To check what pH the solution was we used pH strips. We kept testing and diluting till all of the 150-200 mL pH solutions were made at 4,5,and 6.

Once the seeds germinated, approximately after1 week, and sprouts were seen poking through the soil we began adding the pH mixtures. We sprayed 50 mL of each pH solution onto the sprouts in the container that corresponded to the pH level. It was continued for 3-4 weeks, until the plants matured.

When the plants were mature, the chlorophyll concentrations were calculated for our data. The different chlorophyll concentrations were our dependent variable.

To preform the chlorophyll concentration we each snipped a leaf off of one plant from each corresponding pH container. The leaf was then weighed to use in the final calculations.

Next we added the leaves to a test tube full of 5 mL of water and placed it in a hot water bath until the water in the test tube was boiling. Once the water was done boiling it was discarded. This softened the tissue of the leaf to make it easier to then release the chlorophyll.

Then we added 5 mL of ethanol to the test tube with the leaf inside. This solution needed to boil for 3 min. After the 3 min were over, we discarded the ethanol solution into a larger test tube and repeated this step 2 more times.

When all of the ethanol was collected we measured the total volume using a graduated cylinder. The volume was also used in the final calculations.

Then it was time to use the spectrometer. This machine calculated the absorbance of the chlorophyll pigments and helped find the concentrations in each leaf.

To do this we first set the spectrometer to the correct wavelength 663 nm and 645 nm and transmittance absorbance to 0 ppm. Then we filled a small test tube with water to use as a blank. This set the machine to absolute zero.

We then poured the ethanol solution from the leaves into a small test tube and placed it in the spectrometer machine. Once the absorbance level popped up we recorded it; this was done for each pH solution.

Once the absorbance values were collected we used this equation to find the chlorophyll A and B concentrations. ChA = (12.7 x abs663) – (2.69 x abs645) x (V/100) x W ChB = (22.9 x abs645) – (4.86 x abs663) x (V/100) x W

After using the formulas to find the concentrations of chlorophyll in each plant it was time to make tables and graph the data.

Results

Table 1: Absorbance at 663 nm and 645 nm of Chlorophyll Pigments in B. rapa at pH 4,5, and 6

pH level

Absorbance at 663nm (ppm)

Absorbance at 645 nm (ppm)

4

0

0

5

.137

.112

6

.602

.614

The absorbance for wavelengths 663 nm and 645 nm increases going down the columns in relation to the increasing pH. There is quite a large difference in the absorbance values for pH 5 and 6 as it jumps from .137 to .602 and from .112 to .614. At each wavelength it almost triples. Even more so, there is no record of any absorbance in the pH 4 plants at all.

Table 2: Chlorophyll A and B concentration in B. rapa at pH 4,5, and 6

pH Level

Chlorophyll A Concentration (g/L)

Chlorophyll B Concentration (g/L)

4

0

0

5

1.74

2.56

6

7.64

14.1

The concentration of both chlorophyll A and B increase rapidly as the pH increases for the soil environment of the B. rapa. At pH 4 there is no concentration whatsoever for both A and B. This is very similar to the absorbance findings in table 1; while there was a drastic change in pH 5 and 6, when looking at both chlorophyll types. For pH 5 and 6 the concentration value at A nearly doubled for B. Also the values for pH 5 were are close to multiplying by 7 to reach the pH 6 concentration values.

Table 3: Weight of Leaf and Final Ethanol Volume used for Chlorophyll A and B Concentration Calculations in B. rapa.

pH level

Weight (g)

Volume of Ethanol Solution (L)

4

0

0

5

.04

7.8

6

.139

10.6

As the pH level increases going down the left hand column, both the weight of the leaves collected and volume of the ethanol solution increase. The weight between all of the leaves collected does not differ too much compared to the volume. There is a huge jump from 0 mL to 7.8 mL and then to 10.6 mL. It does not seem like a drastic difference when you look at the weights of all the leaves and see 0 g to .04 g to .139 g.

Series 1 in blue is absorbance at 663 nm and series 2 in red is absorbance at 645 nm

In this graph, one can compare the relative absorbance values at the two different wavelengths. As seen above, there is a significantly higher absorbance for pH 6 compared to the others. For pH for absorption no values are even offered. The trend seen in the graph shows that as pH values rise, so does the absorption of the chlorophyll pigments. Furthermore, the graph show that relatively, there is not much difference in which wavelength amount the pigments in the chloroplast pick up.

Series 1 in blue is Chlorophyll A and series 2 in red is Chlorophyll B

The chlorophyll concentration graph for both A and B types are very similar to that of the absorbance graph. The same trend is seen: rising pH values lead to higher chlorophyll concentration. One can also conclude as well that in most plants that there is a higher concentration of type B chlorophyll compared to type B. For pH 6 the concentration of B almost doubles that of A.

Discussion

After all of the data was collected and calculated at the end of our experiment the trend we found was, that in accordance with absorbance, the concentration for both Chlorophyll A and B was lower as the pH decreased. Even in comparing weight and volume, 0 g,.04,.139 g and 0 mL,7.8 mL, 10.6 mL seen in table 3, all decrease with decreasing pH levels. Leaf size does relate to chlorophyll concentration; if a bigger leaf is produced it has more surface area, which allows for more chloroplast to built, mainly on the surface. Leaves are not as large or in the case of the plants with soil pH of 4 nonexistent. Next looking at absorbance in both table 1 and the first graph, in soil pH 4 there is no plant, so absorbance could only be recorded at 0. The plant with pH of 5 did contain leaves however rather small, in both wavelengths used to record the absorbency of the pigments they only registered as .137 ppm at 663 nm and .112 ppm at 645 nm. While the plant in pH 6 soils registered quite above both with .602 ppm at 663 nm and .614 ppm at 645 nm. These absorbencies are a direct link to the amount of chlorophyll. The pigments in chlorophyll absorb light frequencies and if there are more pigments, then the absorbency is higher. The next step in collecting data was calculating the actual chlorophyll A and B concentrations in the leaves chosen. This was done by plugging in weight of the leaf, volume of the ethanol solution, and absorbance figures at 663 nm and 645 nm. In looking at both table 1 and the second graph one can easily see that concentration of both pigments steadily increased with increasing pH number. B. rapa in pH 4 held no chlorophyll concentration, pH 5 contained 1.74 M and 2.56 M for A and B chlorophyll, pH 6 had a chlorophyll A and B concentration of 7.64 M and 14.1 M.

In analyzing the data, the trend seen is acceptable and was actually predicted before the experiment was conducted; pH has a major affect on soil and how the nutrients are allotted as well as whether or not they are available. In B. rapa the pH of the soil must be between 5-8. This would explain why the plants in pH of 5 and 6 survived while plant in pH 4 did not. In addition, even though the plants could survive the acidity of the soil still affects how fit the plant is. As the soil is more acidic it inhibits the nutrients more from entering the root hairs and traveling up the vascular system. When these nutrients are not available as frequently, the plant must build up enough nutrients to stabilize the chloroplast already formed instead of building more and more. This is why there is less concentration in the plants with lower pH. Even when the mutations are the dwarf, which allows for a higher concentration of chlorophyll than the average plant, there is still a negative affect.

All in all this data can come together to show that the roots are the key to nutrient uptake. Furthermore, that without those precious nutrients there is a significantly smaller amount of chloroplast, which would decrease the rate of photosynthesis for the plant. This may even cause the plant to become even more stunted in growth. Thus, acid rain is a problem and there will need to be a solution to it in the future, otherwise the earths vegetation will parish. I suggest working with the roots to help imbibe more nutrients then ever before. Or even playing with the genetics of the plant to allow for more root hairs to grow or to find a new process that will help with this problem. Maybe, nutrients will have to be taken in from another area other than the roots.

In conclusion, the chlorophyll concentration trend for both A and B chlorophyll increased in the dwarf mutation B. rapa for those with a higher pH number, when a pH solution increasing in acidity, resembling acid rain, was added to separate plant containers over a period of time. This showed that acid rain does affect chlorophyll formation and maintenance, relating to nutrient and mineral uptake by the plants roots. Thus, our hypothesis was supported.

However, there were some errors that occurred and could be fixed in future experiments. One major problem was waiting for the plants to germinate before spraying them with the acid rain solution. Even though the plants sprouted, except the seeds in pH 4, more would have grown which would have given more data. Also, the process for extracting chlorophyll form the leaf was faulty. The hot plates were not behaving correctly and would shut off their heat randomly. This caused fluctuations in the heating of the test tube, which could have affected the degrading of the cell walls in the beginning as well as the ability of ethanol to release chlorophyll properly. In addition, the spectrometer we used was very temperamental and would change the absorbency constant randomly. Even though this may have only changed our data slightly, it still makes our data less accurate. Furthermore, it would be more beneficial in gaining information if the pH process could also be compared between dwarf mutated and non-mutated B. rapa. This would help to show the concentration differences in both and which plant may survive better in the acidic conditions.

In the end of the lab a lot of work was preformed, data collected, graphs and tabled made, and plants killed. However, it was not all for nothing. We learned a lot in conducting our experiment. We learned of the negative affects of acid rain and what mechanisms are affected by having too acidic soil. In our findings it was shown that the purpose of the roots is the main part of the plant that takes up any and all nutrients. Since, the pH only affected the soil where the roots were it was, the cause of the lower concentrations must be directly related to the roots. We believe that if someone finds a way to maybe make the roots more attractive to what is imbibed then the acid will not have as great of an effect. Also, we learned about how to extract chlorophyll. It is a lot like extracting DNA, except it is a much faster process. The boiling water in the beginning breaks down the cell walls and most of the internal structures of the leaf. While the ethanol works to break apart the internal components even more so. It actually allows for the chlorophyll to be extracted. In addition, we recognized that the weight and volume must be used to find the concentration. They were both needed to conclude how much chlorophyll there was per section. To wrap it all up, we learned and experienced a lot in our lab, from obtaining previous research, collecting data, preforming a unique experiment, and eventually analyzing and forming conclusions from the data.