The Effect of UV Radiation on Lettuce Growth

[Challengedstudent]

Oh I see, what i did was i brought the control plants in to the house with artificial light (they weren't in the dark). I guess you could say that the other 2 groups of plants got artificial light as well with uv light too. All three plant groups though got about 10 hours of sunlight per day. So, should i say in my procedure that you need to expose the uv radiated plants to 12 hours of regular light as well to keep conditions quite the same? To explain my project's conclusion, should I still talk about photomorphogenesis and the other factors? I took a picture of the plants on the second week since i started my project (when there was signs of life) and a week after that, when they grew bigger, i took a picture as well. I'm now waiting for the end of my project which will be the end of January to take a final picture that'll I'll then observe to see the chlorophyll content. Oh, i called the uv strip company and they said that the strips would be pointless to my experiment as they don't measure uv intensity. I'm still going to talk to them tomorrow though.

[SciB]

Hi,

That's good. You treated the the control plants the same as the UV plants with the exception of the UV lamp, so you can compare them.

I'm surprised at the company who makes the UV test strips saying they don't measure intensity. What do they measure? Maybe they meant that they are not sensitive enough for what you want. Do call them again however.

The other thing i want you to do is call the company that makes the UV lamp. Not the company that makes the fixture--the lamp itself. if you look on the lamp, it should give a name like Philips or Panasonic. Google this and see if you can find a phone number. What I want you to find out is the spectrum of the lamp--how much light of EACH wavelength it produces. This is important because it will tell you how much other light the plants got in addition to the UV. You can also ask them if there's a way to estimate the UV intensity at a particular distance from their lamp.

Why don't you try doing the ImageJ analysis with some practice photos and see if you can get consistent readings using photos from the same plant. The density readings should all be pretty close if the method is working properly. I will try it too on some other type of plant.

Good luck! You have made really good progress and we are proud of your work!

Best wishes,

Sybee

[Challengedstudent]

Thank You. Actually, I called the uv strip company again and it turns out that they meant that the uv strips measure uv dosage, the thing that you wanted me to find out. I'm pretty sure I expose the uv strips to the same amount of time i expose my plants and to measure the dosage of sunlight i put it out the whole day but I may be wrong. When I do get the reading though, would that be the amount of uv radiation the plants receive per day? Oh also, I read about the lamp and it also exposes UV-A Radiation. I'll do more research on it but i remember reading about a certain process that repairs proteins and nucleotide bases when exposed to UV-A. I downloaded the ImageJ but am unaware of exactly how to use it. May you please help give me instructions and maybe I can attempt to do it? I am assuming that the option/ example shown from the link you sent me is measuring chlorophyll content since at the end it says 82% of the leaf surface was dark green. Please correct me if I'm wrong

[SciB]

Hi Tony,
Yes the accumulated UV ‘dose’ over the whole time period is what you need to know. And remember—these strips measure the entire UV spectrum, not just UV-B. The strips will tell you the amount of energy in millijoules received per square centimeter of surface. You can measure the amount of UV the plants get from sunlight and compare it to the amount from the lamp. From these two numbers you can calculate the extra amount of UV you gave your test plants.
Try to get the full spectrum of your UV lamp from the company that makes it. You need to know how much light of each wavelength the lamp delivers. It may be primarily UV, but there will be light of longer wavelengths also in the spectrum. When you present the results of your study you will show a picture of this spectrum so your audience can see exactly which wavelengths and how much light you gave the plants. And, yes, plants do have biological mechanisms for repairing damage to their DNA from UV. Humans also have these repair systems. UV can still be dangerous, however, so be sure to follow ALL the safety instructions for using your UV lamp. Wear UV-resistant safety glasses at all times around UV light sources!
I’m still trying to figure out the best way to do the Image J analysis of the leaf color to test whether your UV exposure caused any change to the chlorophyll content. Go ahead and download Image J [http://rsbweb.nih.gov/ij/download.html] if you haven’t already done so and play around with the program to learn how to use it. There are versions for PC, Mac, Linux etc. and you probably already have Java on your computer so you don’t need to download the bundle that includes Java. Just make sure you have the current Java version installed on your computer. If you haven’t updated it in a long time, you might want to do that. There is an Image J video tutorial for beginners that you can start with: http://rsbweb.nih.gov/ij/docs/examples/
I am going to take photos of some leaves, open them in Image J and try making density measurements. I’ll get back to you when I feel like it is working properly. Are you using a phone camera or digital camera? Try to get one to use that has at least 10 MPi resolution.
Good luck,
Sybee

[Challengedstudent]

Oh ok thank you.. I've downloaded it and tested the sample leaf out, following the example but I didn't get any results that I am aware would be useful to me. I am using an iPhone but I do have a camera if it is necessary. I tried looking around the lamp for the name of the company but all I got was Zilla. The lamp is called Zilla Desert UV T8 Terrarium Strip Light Fixture
.... If it says Phillips.. Do I call that company and they might know about that certain product?

[SciB]

Hi again,

I don't know if your UV treatment caused a change in the chlorophyll that could be seen as a change in the hue or intensity of the leaf color. Maybe it didn't cause any change, but it is still worth measuring to be able to say that scientifically. The sample leaf is just for measuring surface area. That is not what you need. You want to know the relative color density of the leaves. Check the part of the tutorial where they talk about measuring image density. That's what you want. I'll get back to you on it as soon as I've had a chance to take some pictures and try it. Use whichever camera has the greatest pixel number--at least 10 MPi.

Thanks for giving me the name of the lamp. I did a search and found this site that has some information about the Zilla UVB lamp: http://www.uvguide.co.uk/phototherapyphosphor-tests.htm
They show spectra for a variety of reptile lamps including the Zilla but not the full spectrum--only from 270 to 400 nm. You could try calling the company that makes the fixture and ask them who the manufacturer of the lamp is and if they can send you the FULL spectral output of the lamp, not just the UV part. Philips makes a lot of different UV lamps and they may make this one. You could try going to their website and getting the spectrum or call them.

The spectrum is not absolutely necessary for your report, but it is important. If you don't know ALL the light the lamp is putting out then you can't really say that any effects you see are due to UV. The UV test strips will tell you how much UV but not how much other light.

Good luck,

Sybee

[Challengedstudent]

I'm just assuming right now that the uv lamp has a wavelength of 275-400 nm. So, can this fact support my idea of photomorphogenesis because I researched that this is normally triggered when exposed to UVB radiation at 295-300 nm. If I were to print out the uv spectrum of the lamp, would I use it to support cases such as this and other scientific facts that back up the idea that a small amount of radiation doesn't harm plants, if this is the case

[SciB]

You can't assume that the emission of the lamp ends at 400 nm. In fact I am sure it does not. The company describes it as a FULL spectrum lamp which means it emits light at wavelengths from about 300 to 600 nm. It has been modified to produce a bit more UVB than most lamps. That's the only difference.

If you give plants an extra 12 hours of light a day, they will grow faster than plants not receiving that much light but it has nothing to do with UV. The plants can do photosynthesis for more hours per day, so they grow more. What color is the light from the Zilla lamp? UVB is blue-violet. I think the Zilla light probably looks like sunshine.

You cannot just show the UV part of the lamp spectrum because someone will ask you specifically what the rest of the spectrum looks like. What other evidence do you have that photomorphogenesis took place rather than just normal growth from photosynthesis?

[Challengedstudent]

So would you say my project has no results? When I weigh the plants and if they have a large fresh weight + if I see that the uv radiated plants have high chlorophyll content then that should prove that photomorphogenesis occurred.. The terrarium light was light blue..what should I do to support my results though and for any result

[SciB]

If your plants grew larger with the extra light and showed a higher chlorophyll content (darker green) then, yes, you can conclude that photomorphogenesis occurred. You just cannot say that the photomorphogenesis was only caused by UVB because the lamp you used produced light of other wavelengths. If the Zilla lamp had emitted ONLY UVB, then you could have claimed that UVB was the cause.

The lamp spectrum would show you what percentage of the light output was in the UVB region. When you test the light with the UV strips, they will tell you how much UV the plants got compared to the amount they got in sunlight. If the lamp puts out much more UVB than sunlight then you have a stronger argument that UVB is the cause.

I will do the color density test on some leaves today and get back to you about how to use Image J to do that.

Congratulations! You have learned a lot about how to do a scientific study and you are almost done.

Cheers,

Sybee

[SciB]

I tried using Image J to do density measurements on some lettuce leaves and it appears to be working ok. What kind of lettuce did you grow? I have some green leaf lettuce in my garden and that is where I got the lettuce leaves for the test. I attached a photo that I used so you could see what my leaf looked like. I used my phone camera which is 13 MPi and takes great pix. I put the camera on a ringstand under a fluorescent worklight and allowed it to autofocus before I took the picture. You HAVE to keep the lighting the same or you won’t be able to compare one leaf to another. I saved the image as a jpeg.

Here are the steps for using Image J to measure color density. Open the program on your computer and drag the image file onto the area of the Image J box above the toolbar. In order to take accurate measurements, you need to convert the image from color to grey so click on Image on the Image J toolbar then Type, then select 8-bit. This will convert your image to grayscale [NOTE: At the end, when you are closing the image file and it asks if you want to save the changes, be sure to say NO. If you say yes, you will lose the color image.]

Now click on Image again and then on Color---Edit LUT. Select Invert. This will change dark to light and vice versa. The reason you do this is so that the density numbers are higher for dark areas than light areas.

Now look at the toolbar that has all the icons and make sure the rectangular box is selected. Bring the cursor down to the image and holding down the left button drag a square box about 500 pixels wide by 500 pixels high. It does not have to be exactly this size but it is very important that you use the EXACT SAME SIZE BOX for each measurement, otherwise you cannot compare them. Now you can position the box anywhere in the image by placing the cursor inside the box, holding down the left button and dragging the box to where you want it. I put the box in six different places on the leaf so that I could see if there was much variation in density over the surface of the leaf (there was not). I avoided putting the box on the central vein because that is so light.

Now you have the box where you want it, go up to the toolbar and select Analyze---Measure. Now you will see a box that says Results with columns for Area, Mean Density, Max Density and Min Density. The area is the surface area within the box you drew in pixels squared. The mean density is in arbitrary units. I took six readings per leaf just to be sure there wasn’t a lot of variation from place to place, but you could just do one or two large boxes to get the average density reading. I also compared the mean density for two different leaves from the same plant and they were close but not identical, which is what I expected.

So you can set up your camera, take photos of leaves from all the plants [being careful to record which photo goes with which plant!], upload them to your computer then do the density measurements. You will want to average all the results for each group then compare the UV-treated groups to the control. Do you know how to use Student’s t-test? That is the statistical method you use to compare two means to prove that they are the same or different at a confidence interval of 95%. I hope you have had some basic statistics training in school because you need to use statistics to prove whether the added light you gave the plants really did cause a significant change. Try you-tube for some basic statistics videos to learn the methods. We can help you with the statistical analysis too.

Good luck,

Sybee

[Challengedstudent]

Thank you very much,
I will now try measuring the chlorophyll content. I grew romaine lettuce. If the mean density is high does that mean the chlorophyll content is high? Also, I am pretty sure I know how to find the means and I am aware of doing a student t test on excel. I have a question on that though. From my understanding, you will get a probability reading in the end. If the probability is higher, does that mean there is no significant changes amongst the means ( the mean density of the exposed and non exposed plants)

[Challengedstudent]

Hi sybee,
I have just finished testing out the leaves with imagej. For the 2 hour leaf I got an area of 202500, a mean of 100.5 and intden, I am assuming that is the max density of 20347895 and the same number for the rawintden... For the 0 hour I got a density of 17270527 and for the 12 hour I got a density of 26756981. May you Please tell me if these are the results I am supposed to get and what they mean ( when I present these results to the judges what do I say) oh, also when I did put the box in different areas I did get different numbers.. For the 4 areas I put it in, should I just find the mean of the 4 density results for each type of leaf? With these results, I am assuming chlorophyll contents were higher as the number if hours increased. So this can support the fact that photomorphogenesis occurred
Thanks

[SciB]

I'm a little confused by your results. First-- are you dragging the box around to do the measurements? As i said, the box in which the measurement is made has to be EXACTLY the same size for EACH measurement.

Ignore the surface area readings. They will be the same in every case if you used the same size box.

The reading you want is the 2nd one--MEAN. That is the average density of the image within the area of the box and will depend on the amount of chlorophyll that is present. You said first you got 100.5 for avg density and then you are telling me you got numbers like 26756981 for density, but the density cannot be that high. You must be taking the area readings.

There is no such thing as a reading that you are 'supposed' to get. The program will read the density of the images you use and you will have to make your conclusion from this data. The data is what it is.

Get back to me with your MEAN DENSITY readings of the control, the 2 hr UV and the 12 hr UV and let's see what they tell us. Remember also that you need to do measurements on every plant in each group and average them to get the values for comparison with the control. Did you do that?

Sybee

[Challengedstudent]

Oh, I see. I was looking at the wrong numbers. For the control, I got a mean of 85.287. For the 2 hour exposure, I got a mean of 100.483 and for the 12 hour exposure, I got a mean of 132.133. Would I need to do the student t test to figure out whether these means are similar or not? Yes, I took different leaves from each group and measured them. Should I find the mean of the different mean values I got for each group first?

[SciB]

Excellent! It looks like there is a trend towards greater density of chlorophyll with the extra light.

As I told you--do measurements on leaves from EVERY plant in a group then average them.

Do you know how to use Excel to get the mean [average] of a group of numbers? If you don't, try to find someone to teach you or learn it yourself by watching a youtube video. It is something you will need to learn for college, so the sooner you do the better.

The other calculation you need to do is the standard deviation [s.d. for short]. Do you know how to do that? You can do it in Excel. The s.d. is a statistical value that shows you how close the values are to the mean. The smaller the s.d., the better.

Once you have the mean densities and standard deviations, you need to graph them as a bar chart for each group. If you don't know how to use Excel for graphing, then get someone to show you or go to youtube and watch a video.

You are still planning to weigh the plants as we discussed in a previous post, right? Again, you should take the average for each group, get the s.d. and plot the results as a bar chart. Let me know what you find out.

Good job!

Sybee

[Challengedstudent]

Thank you for all your help. I looked up on youtube on how to calculate standard deviation on excel and will go ahead and do it. I did take a picture of the spectrum of the lamp I am currently using and I will try sending it to you as soon as I can. So with a greater density (in arbitrary units) there will be greater chlorophyll content and more chlorophyll proves that photomorphogenesis occurred?

[SciB]

You are certainly welcome. I am glad you succeeded in completing this difficult project. We're proud of you!

And, yes, the increased color density in the leaf is due to increased chlorophyll which was the result of the increased light that you gave those plants.

Did you get a chance to weigh the plants? The increase in chlorophyll should be correlated with an increase in fresh weight of the irradiated plants. That and the UV intensity measurements from the test strips are the last data you need to wrap up your study.

I hope you see that if you want to study the effects of UV specifically, then you need to have a light source that emits ONLY UV. That would have allowed you to say that it was UV light and not just light in general that was having an effect.

We look forward to working with you again. Next time talk to us BEFORE you begin your experiments. If you had told me what you were planning, I would have advised you to use a UV source--not a full-spectrum lamp.

Best wishes,

Sybee

[Challengedstudent]

Yeah. When the judges ask what would I have done differently, I would say that I should have used a light source that only emitted uv radiation. Though I got the results that I needed, I can't say that they were solely due to uv radiation. I am planning to weigh the plants when I am completely done radiating the plants (this week). Should I use a scale that weights in milligrams or ounces? Also, now that I have the conclusion that a low dose of uv radiation stimulates plant growth (which is about the amount of uv radiation we will receive as the ozone layer depletes) what should I say for the application? I don't think it's safe to say that our plants will get healthier. Please let me know.
Thank You

[SciB]

Hey, you're almost done!! Let's celebrate...

Well, not quite yet. OK. You need to use an electronic lab scale that weighs to a sensitivity of 0.001 g or 1 mg. Scientists in this country use the metric system for mass and volume, so no ounces.

You should be very careful about your conclusions. The one thing science fair judges look at hard is whether your data support your conclusions. If you say UV caused the irradiated plants to grow more than the controls, they are going to ask you how you know that. If you say because I used a lamp that produced UVB, they are going to say--Is that the ONLY light it produced? Then you're stuck.

I think the best thing for you to do is say up front that you chose the wrong lamp because it was advertised as a UVB emitter and you wrongly concluded that UVB was the ONLY light it emitted. Scientists make mistakes all the time. They just correct them before publishing their results, which in your case you can't do. Do some online research. If you can prove by using published data from other labs that UVB is the primary or only cause of photomorphogenesis, then you have an argument to say that the growth you measured in your irradiated plants WAS due to UVB and not simply to giving them more light. If photomorphogenesis occurs from light of other wavelengths as well as UV, then you cannot say that UV was the cause. All you can say is that giving plants more light caused them to grow faster and synthesize more chlorophyll.

You haven't said anything about measuring the UV dose with the test strips. You don't have to do this, but it would help your argument if you could actually show how much more UV the irradiated plants got. Can you borrow a UV dosimeter from someone? If you have an Agricultural Extension Service or Plant Science department nearby, you could try calling them and ask them if they would have a meter you could borrow to measure the UV dose rate. Tell them you are a student doing a science fair project on plants and UV and need to measure UV in sunlight compared to a lamp. They may be able to help you.

I'll be waiting to hear about the weight comparison.

Sybee

[Challengedstudent]

Ok. Photomorphogenesis is caused by many different photoreceptors though. The photoreceptor that I am mainly talking about is UVR8, a photoreceptor that reacts to UVB specifically.If there are photoreceptors that may have caused this that react to UVA, can that still support my conclusion/project because we are also exposed to UVA. I still haven't received the strips but i'll inform you about my results once I got them. I have the picture of the spectrum of the uv lamp, but I am unaware of how to send it to for I can't copy and paste the picture into the text box. Obviously, it emits uvb but it also emits some visible light as well. In order to check this, should I look up the electromagnetic spectrum and there, it'll tell which color means what type of light?

[SciB]

Just use the Upload Attachment option below the text box. You might have to scroll down a little if you don't see. Just click it, browse to wherever the image is stored and open it. The click Upload.

Can you send me the links to the reference papers you are using to make statements about photomorphogenesis [let's call it 'PMG' so we don't have to write out that long word!]? Do ALL plants have UVR8? Is that the MAIN receptor for PMG?

On the picture of the spectrum of your UV lamp, you just need to indicate which wavelengths are UV and which are visible light. Also, you need a spectrum of sunlight for comparison. Try to find a similar image so that the wavelengths line up as well as you can.

You might want to call the UV test strip company and remind them that you are on a deadline and need those strips NOW!

Good luck!

Sybee

[Challengedstudent]

These are some of the references I used for my project

http://www.photobiology.info/Jenkins.html
http://www.gla.ac.uk/researchinstitutes ... sestouv-8/

[SciB]

I read the Jenkins paper and the other one and they don’t specifically mention an increased growth rate as a result of UVB acting on its receptor UVR8; but they did say this: “…transcriptome analyses of uvr8 mutant plants in comparison to wild-type revealed that UVR8 regulates the expression of at least 100 genes in response to UV-B treatment (Brown et al., 2005, Favory et al., 2009).”
And that suggests to me that UVB might be able to promote growth since it regulates expression of so many different genes.

The main function of UVR8 seems to be to protect plants against the damaging effects of UVB and that makes sense because, although UVB is a very small part of the whole spectrum of sunlight, it is the most energetic part. “The UVR8 amino acid sequence is highly conserved among plant species, including in non-vascular plants such as mosses and green algae (Rizzini et al., 2011). Although studies to date have been limited to just a few species, it is likely that the protein evolved to help early photosynthetic plants survive exposure to UV-B wavelengths in sunlight.”

I think the change in chlorophyll content as reflected in the leaf color density measurements may be the one direct UVB-UVR8 response that you have caused with the lamp: “…several genes regulated by UVR8 encode chloroplast proteins, consistent with evidence that UVR8 helps to maintain photosynthetic competence (Davey et al., 2012).”

Once you complete the weight measurements, determine the UV intensities with the strips and compare the sunlight vs Zilla lamp spectra, you will have done a really good science project. I’m glad I was able to help. I wish I could set up a huge lab so that students like you and others could come and have all the equipment and supplies and mentoring they would need to do really in-depth science projects. We could even start our own peer-reviewed, science-fair journal! Well…maybe someday.

Good luck finishing up and preparing your presentation.

Sybee

[Challengedstudent]

Thank You for all the help. That plan sounds good to me. If you guys ever open a science lab, I'll gladly join. So I should mainly talk about the UVR8 and explain how it protects the plants from damage by encoding certain proteins? I'll inform you about my project after the science fair . Oh.. So for my application, should I change it since I got different results? Should I say that as the ozone layer depletes, most of our plants such as lettuce won't get negatively affected.

[SciB]

I would not say that. I would emphasize that plants have remarkable capabilities to resist and repair damage from UVB. Your lettuce was protecting itself through UVR8 and other crop plants may do the same. They have been adapting to changes in UV intensity for a billion years.

Remember--it's a dose effect. Most plants can adapt to gradual changes in UV, but more rapid changes such as might occur if the ozone layer were to disappear would harm them. UV is not the only effect. The carbon dioxide levels in the atmosphere have been increasing steadily since the 1700s and now the average global temperature is rising. These all affect plants.

Congratulations. You've done a great job and learned first hand how to do meaningful scientific research. I hope you continue to think about nature and the environment and plan some good experiments for your next science project.

Best wishes,

Sybee

[Challengedstudent]

Thanks, so my application would sound something like this. As plants evolved throughout time, they started developing adaptations against uvb such as the protein uvr8. This protects it against the harmful affects against the uv radiation. So, as the ozone layer depletes, our lettuce and other crops won't get harmed from uv radiation but this doesn't mean they won't get harmed at all. Other factors such as global warming may negatively affect them. But, my project raises alertness that people don't have to be quite alarmed as the ozone layer depletes because the plants wont' really get harmed.

Does this sound ok?

[SciB]

Yes, basically your assessment is correct, but I would add two qualifying conditions. First there is a limit to HOW MUCH UV a plant such as lettuce can protect itself from and second, you cannot say that ALL plants will behave the same way. What about the phytoplankton in the oceans? These single-celled, plant-like organisms have chlorophyll, do photosynthesis and provide more than half the oxygen we breathe on this planet http://en.wikipedia.org/wiki/Phytoplankton. Can they protect themselves from increased UV?

What I am telling you is to beware of making assumptions such as that ALL plants developed UV adaptations over time. Do you KNOW that ALL crop plants can adapt to increases in UV or only some? Also, you cannot assume that all plants that do have a protein like UVR8 have the same capability to resist UV.

One thing you could point out is that UV resistance would be a good candidate for genetic engineering in crop plants. If a plant lacked the UVR8 gene, for example, a genetic engineer could add it or extra copies could be added to make the plant more resistant to UV if the ozone layer was greatly depleted and UV levels got much higher.

Living things are exceptionally complex and if you try to make simple statements and conclusions about all of them you will usually be wrong. Also, keep in mind that science is constantly changing and evolving. The 'facts' we talk about today are for the most part our BEST GUESSES based on the information we have now. This can change in a heartbeat. You are just learning science and my best advice would be to always question 'facts', be skeptical and recognize that science is not a 'done deal', but a work in progress.

Best wishes,

Sybee

[Challengedstudent]

Oh ok I see. So now I could also say that my project shows that the protein uvr8 is a defense mechanism against certain amounts of UVB and so, genetic engineerers could add this protein into other plants that lack this in order to ensure they won't be negatively affected as well. I just got the uv strips and will give you the measurements as soon as possible

[SciB]

Someone is going to ask you, then--Well, which plants have UVR8 (or some other UV-responsive protein) and which don't? How will you answer them?

When I said that you can't make a blanket statement, i meant for you to go online and look up what percentage of plants DO have UVR8 or its equivalent. The GMO idea was mine but it's not based on any knowledge I have about plants that lack UV receptors. YOU will have to search for this information in a scientific journals database such as PubMed before you can say that inserting the UVR8 gene or extra copies of it would be useful.

Check phytoplankton. Do they have a UV receptor? They are single cells. Would putting the UVR8 gene into them make them more resistant to the harmful effects of UV?

Do let me know the results from your UV test strips. I'm not sure how long you will have to expose the strips, but i guess you can tell by just looking at them because they change color as the light acts on them. You just want to stop the test before the whole strip changes color completely. Be sure to record the times when you begin and end the exposure so you can calculate the dose per hour. I don't know what the units are but I would expect them to be some unit of energy like microjoules per square centimeter. with this value, you can calculate a dose rate by dividing it by the number of hours of exposure.

Sybee