Investigation 11: Plant Transpiration

On April 15th, the lab group consisting of Vikram, Vinay, Shreyan and myself were tasked with observing plant stomata and recording our data to derive some sort of meaning from it. In plants, stomata are the small openings on the underside of a leaf that open and close to allow gas exchange to occur with the plant's surrounding environment. This is an extremely important role played by the stoma because it helps leaves take in water from the roots through a process called transpiration. Transpiration is the way a plant uses the cohesion of water molecules to pull water up from the roots as more water vapor is evaporated out of the leaves. The stomata also open and close depending on environmental factors, but we are not investigating the opening and closing of stomata in our experiment.

Transpiration in action

Now how will we count the number of stomata on the bottom of the leaf? We could manually count each one using a microscope, but that would take forever and the stomata are green so they blend into the leaf and are difficult to spot. The lab group decided that we would use clear nail polish and apply it to the bottom of the leaf, to get impressions of the stomata. Then we would use tape to remove the nail polish from the bottom of the leaf with the impressions still intact, and apply the tape to a microscope slide in order to view the stomata. Then, because we have a specific field of vision using a microscope, we would calculate how big the area under the microscope was and then extrapolate that data to make an estimate on how many stomata cover the leaf. Then we could use this data to make hypotheses about the relationship between plants, the number of stomata they had and their environment.

The group performed the procedure outlined above using three different types of leaves: ivy, grass and ginkgo. Using the nail polish, we painted a small section of the leaf with it and waited for it to dry. After it was dried, we carefully applied tape to a section of nail polish and delicately pulled it off of the plant so as not to disturb the polish. Then we looked at the slides that we had prepared under a microscope at different magnifications.

Here is the ivy leaf we observed at 400X magnification:

Here is the blade of grass we observed at 400X magnification:

Here is the ginkgo leaf we observed at 100X magnification:

Next, we had to calculate the field of view that the microscope gave us for each leaf at each different magnification so that we could determine the stomata to surface area ratio for each leaf. To do this, we started by putting a plastic ruler under the microscope at the lowest magnification, 40X, and saw how large the field of vision was measure on the ruler. We discovered that at 40X magnification, the microscope's vision had a diameter of 5 millimeters. Using this data, and our knowledge that the diameter of the field of vision was inversely proportional to the magnification of the microscope. Therefore, to find the diameter of the field at 100X magnification, we had to multiply the original 40X diameter by 40/100 or 0.4, and to find 400X magnification, we had to multiply the original by 0.1.

After finding the diameters of the microscope's field of vision, we wanted to find the surface area of the field, so we used our knowledge of the formula for the surface area of a circle, which is A = pi*r^2, where r is the radius. Also, we know that the diameter is twice as long as the radius, so the formula we will use will look more like A = pi*(d/2)^2, where d is the diameter of the field of vision.

Finally, using the number of stomata per leaf and the area of the field of vision, we determined the stomata to surface area ratio for each plant. This calculation is in stomata per millimeter squared.

Here are our calculations:

Diameter at 40X: 5mm
Diameter at 100X: 5mm * 0.4 = 2mm
Diameter at 400X: 5mm * 0.1 = 0.5mm

Area at 100X: pi * (1mm)^2 = 3.14mm^2
Area at 400X: pi * (0.5mm)^2 = 0.785mm^2


And here is a graph of our findings:

Leaf	Number of stomata counted in FOV	Are of the FOV (mm^2)	Stomata to surface area ratio (stomata/mm^2)
Ivy	30	0.785	38.2
Grass	10	0.785	12.7
Ginkgo	26	3.14	8.3

We found that Ginkgo leaves have the lowest stomata to surface area ratio of the 3 plants that we tested with only 8.3 stomata per millimeter squared, and Ivy had the highest ratio, with a whopping 38.2 stomata per millimeter. Now the question that the lab group pondered was why there was such a large difference in the number of stomata that each plant had.

We hypothesized that the ginkgo has a low number of stomata because of its environment. Used to be planted in full sun and in warmer climates, the ginkgo would lose a lot of water due to transpiration if it had many stomata. Therefore it only has a few so that it can bring water up the trunk from the roots, but not too many as to create an inefficiency by losing a lot of water. We also believe that the ivy has so many stomata because it is a vine plant that grows upwards and in the shade. Ivies, when given the proper support, can grow up the side of a building and reach 30 meters above the ground. Therefore, to create enough of a pulling force on the water in the roots to get it to the leaves 30m upwards, there must be many stomata to transpire rapidly. Also, because ivies grow in the shade, they dont have to worry about losing a lot of water due to evaporation and are able to have so many stomata. Finally, the blade of grass we looked at had a somewhat low amount of stomata per millimeter squared, about 12.8 which is slightly higher than that of the ginkgo. Unlike the ivy, grass does not grow very tall so it doesn't have to have a lot of stomata to bring water to the leaf. Also, grass grows in the sun, so like the ginkgo, it is in danger of losing a lot of water to heat, so it can't have too many stomata otherwise it will die.

In conclusion, this lab has shown us that there is a strong correlation between the number of stomata that a plant has on its leaves and its environment. The three plants we tested had differing stomata counts due to heat and height, but other plants also have different stomata counts because of humidity or scarcity of water. A plant in an extremely humid climate would have many stomata so that it could transpire as much as possible even though the concentration gradient of water to air is radically different than plants in a desert climate. Also, plants like cacti must conserve water because it is so scarce in the desert, so it cannot have many stomata.

Investigation 3: BLASTing Keratin

Introduction
As a follow up to our group's work at determining the lineage of an ancient reptilian bird, which was completed last week, this week each member of the lab group was tasked with BLASTing a human gene coding for some sort of protein and then discovering the similarity of that gene to genes of other closely related organisms. For my experiment, I decided to BLAST human keratin, an important gene for human structures like hair and nails. My initial prediction for this gene was that it would be closely related to other keratin genes of mammals because other mammals, from dogs to rhinos, have keratin based structures like hair. I did not think that other organisms outside of the mammal family would have this gene because snakes or fish or worms do not have hair or nails as mammals do.

Procedure
After picking my gene of interest, I began by going onto the BLAST Entrez Gene website to search for the particular gene that I would use for this investigation. I entered "human keratin" into the search box at the top of the webpage and pressed enter. From there I was taken to a results page on which many different types of human keratin were displayed, shown below.

I was not sure which keratin gene to pick because I did not know that humans had that many types of keratin in their bodies. In the end I decided to choose Keratin 18 for two reasons. First and foremost, it was top of the search list so perhaps it is more keratin-y than other keratin genes. Secondly, and most importantly, 18 is my lucky number so I thought I would have a better chance of producing a good experiment with the number 18.

Clicking on the Keratin 18 link, I was taken to the homepage for keratin 18 with many different pieces of information. this is shown below. I needed to figure out the actual nucleotide sequence of keratin so that I could BLAST it, so I scrolled down to the section labeled "mRNA and Proteins" and clicked on the first link as the lab manual instructs.

From there I was taken to another page full of information, but I clicked the link labeled "FASTA" to find the actual nucleotide sequence. The link too me to the sequence, which is pictured below once again.

I then copied and pasted the sequence into the BLAST search page, labeled it "Human Keratin", and selected the option to check the gene against both similar and dissimilar genes from other organisms. I did this so that I could gather more results and have a more well-rounded report. From there I BLASTed the gene and waited for my results to come back.

Results
After BLASTing my gene, I was met with a long page of results from my search, and I have included a screenshot of the top few results.

Looking at these results, I see that my predictions were, for the most part, quite true. as expected, the Keratin 18 gene is extremely similar to other Homo sapien genes, but it is also strikingly similar to Keratin 18 genes of different organisms such as the Gorilla gorilla, the Western gorilla, or the Pan trogolodytes, the common Chimpanzee.

Other results that I received, such as similarity to the Papio anubis or the Macaca mullata, were also quite predictable because both of these organisms are primates, being the Olive Baboon and the Rhesus Macaque respectively. In fact, all of the results that I obtained that were displayed on the first age of results, or the first 100 results, were all similar primates. Therefore, one can obviously conclude that humans are descended from primates, but also that Keratin 18 is an extremely common form of Keratin found in many different primates.One can also conclude that the gene for Keratin 18 is extremely unique to primates because it is not found in other organisms such as dogs or rhinos, as I had previously hypothesized.

Perhaps I did not see any matches for organisms other than primates simply because there were so many matches to Keratin 18 that they could not be displayed in the 100 results that I was given. If BLAST gave more results, I bet that I could find a similar gene in a non-primate, but perhaps this gene would be more dissimilar to Keratin 18 in humans than it is similar.

Thoughts
I think that it would be beneficial to an analysis of the BLAST tool that we have used for the past two weeks, as well as to the concept of evolution as a whole, to answer a question posed by Mr. Wong in his assignment. The question is: "Does the use of DNA in the study of evolutionary relationships mean that other characteristics are unimportant in such studies?"

I do not think that DNA should be the be-all end-all when it comes to evolutionary relationships. I think that many different factors must be taken into account, such as morphology and environmental factors. I think that, although DNA can provide substantial support for relationships between many organisms, it is not foolproof. For example, there could be an organism on an island in the middle of the Pacific that evolves a certain gene that is beneficial to survival. On another island in the middle of the Atlantic, another organism develops an extremely similar gene because it is faced with the same environmental conditions as the organism in the Pacific. Though these genes might be extremely closely related, even up to 99%, it is possible for two genes in two separate organisms that are completely unrelated to develop similarly. Though the odds of this occurring are so infinitesimally slim, it is still possible.

Investigation 3: Comparing DNA Sequences to Understand Evolutionary Relationships with BLAST

Introduction
Recently, a team of scientists have discovered a fossil specimen in China after extensive digging. The scientists have sent the fossil to Mr. Wong's Period 7 AP Biology Class to try and place the species on a cladogram to graph its evolutionary relationships. Luckily for our team, small samples of DNA were salavged from the long dead species, unfortunately only 4 small sequences could be saved. The first approach to figuring out where the fossil lies on the tree of life undertaken by Christos, my fellow researcher, and myself was to use morphology to make a hypothesis of where the fossil lies in the evolutionary tree of life. then we will use gene sequence analysis to compare the genes of the organism that were salvaged in the Basic Local Alignment Search Tool, or BLAST, with genes of other, modern organisms. BLAST contains the DNA sequences of many different organisms so we will be able to pinpoint the location that the species lies on the cladogram.

Procedure:
The first step in our procedure is to create a hypothesis of where the species would lie on the evolutionary cladogram based solely on morphology. Looking at the morphology of the fossil, Christos and I agreed that the organism was bony and therefore also has a backbone. The organism also has eyes on each side of its head, so it does not have binocular vision like humans do. Also, the head shape of the organism is reminiscent of a lizard, so perhaps the organism is closely related to reptiles. The long tail that tapers to a point, further supporting evidence that the creature is related to reptiles. The specimen has long legs and short stubby arms, so we can assume that the creature was bipedal, unlike other reptiles. Along with the dark patches along the top of the organism, which may be some sort of feather or proto-feather, the body shape and long legs shows that perhaps the creature is also related to birds. Unfortunately, the researchers cannot see the internal organs of the fossil because soft tissue does not fossilize, so we cannot make any more guesses about the structures of the organism. The organism is picture below.

I believe that the organism belongs in its position on the cladogram below because it does not have fur, yet it is also closely related to reptiles because its appearance is similar to that of reptiles. Also, the creature is obviously a vertebrate, so it must go after the vertebrate branch of the cladogram.

The next step in our procedure is to use the BLAST database and compare the similarity of the genes that we salvaged from the specimen's fossil. To do this, we downloaded the files from Canvas onto a laptop, and then using the "Saved Strategies" link on the homepage of BLAST, which is located at http://blast.ncbi.nlm.nih.gov/Blast.cgi, we uploaded the downloaded files onto the website. Then, after waiting for a few seconds for the website to recognize the file and input all of the information it needed to do a BLAST search, we pressed the "View" button on the webpage. This took us to the official BLAST search page, but the search parameters were all filled out by the "Saved Strategy" that we downloaded. Pressing the "BLAST" button at the bottom of the page activated the search engine and then we waited as the file we uploaded was compared against hundreds of thousands, if not millions, of genes sequenced by different Genome projects

Below are pictures of the 4 different gene sequences' top five matches in the BLAST database. These are genes in other organisms that are similar or even exactly the same as the genes found in the fossil. The more similar the gene is to the gene of another organism, the more closely related the two organisms are.

BLAST of Gene 1

BLAST of Gene 2

BLAST of Gene 3

BLAST of Gene 4

For the first gene tested, the top result for similarity with the new species is the species Gallus gallus, which is a red junglefowl, or a tropical chicken. This agrees with my previous assumption that the new organism is reptilian because chickens and all aves are descended from reptiles. The similarity in genes is 99%.

For the second gene tested, the most similar result from the BLAST was a gene in Drosophilia melanogaster, which is the common fruit fly. This seems to go against my earlier hypothesis that the organism is a vertebrate and related to reptiles because an insect like the fruit fly has no internal bones and predates the evolution of reptiles by many millions of years. Perhaps the organism is more closely related to insects than Christos and I previously thought, but perhaps this is just a fluke and the organism only shares a single gene with the fruit fly. The other two genes will tell whether or not the organism is more reptilian or not. The similarity between the genes is only 92%.

For the third gene tested, the top result was a gene for Taeniopygia guttata ubiquitin-conjugating enzyme E2Q family member 1, which is quite the mouthful. This gene codes for a certain enzyme that is found in the zebra finch, which is another bird. This further supports the earlier hypothesis, as well as my thought that the species has some sort of proto-feather structures on its body. Furthermore, the relationship with the zebra finch distances the new organism from the insect genes it seems to have. The similarity between the genes is 95%.

The fourth and final gene tested was most similar to a gene found in Alligator sinensis mitochondria. Therefore it is a gene that is found in the mitochondrial DNA of the Chinese alligator. This is the final; piece of evidence linking this fossilized creature to reptiles and birds. Also, because it is closely related to the Chinese alligator with a gene similarity of 100% and the specimen was found in China, it must be some close ancestor of the Chinese alligator.

Conclusion
After looking at the BLAST results and discussing the outcomes, Christos and I came to a consensus on where this organism should lie on a cladogram of species, specifically the one we were given. We believe that the organism is a type of reptile that branched out to be the common ancestor for certain species of both birds and alligators. Because of the immense similarity to the Chinese alligator that we discovered, we believe that the organism is more closely related to alligators and crocodiles than birds, even though two of the genes discovered were bird genes with high reates of similarity. Keeping all of this in mind, we placed the organism on the cladogram below.

Our initial hypothesis taking into account simply morphology was somewhat accurate because we had placed the organism on the correct evolutionary branch, but we were not sure of how far down the branch the organism would be. Using both the morphological hypothesis we formed along with the accuracy of the BLAST database searches, we were able to determine to a finer degree where the organism should be placed on the above cladogram.