Farewell and Best Wishes
As this research project is now in the final stages of wrapping-up, we wish to thank everyone who participated in this inquiry; the students, mentors, teachers and others behind the scenes. We appreciate all of your efforts and contributions to this online learning community.

Scientific exploration is a process of discovery that can be fun! There are many unanswered questions about plants just waiting for new scientists to consider, investigate, and share.

We will be archiving groups and projects on December 17, after which time new posts will not be able to be added. Please come back and visit the PlantingScience Project Gallery anytime to view this project in the future. You can search the Gallery by key word, team name, topic, or school name.

Good bye for now.
Warm regards,
The PlantingScience team

Thanks for sending the poster!  It looks great.  Very professional layout.  

On the graphs, are they showing only the change in stem length or number of leaves on each day, not the total?  I am only guessing, but otherwise I can't see how the stem length could go up and then back down.  Or like in Fig 5, how can you have a stem length of >10 on day 20, but then go back to 0 on day 22?   

Thanks for the nice comments!  I have enjoyed working with you all.  If you make a poster or figures with your results, I would love to see it.  :)  Hope you are enjoying Thanksgiving!

Looks like you are in the final stages of your projects.
It’s great to see that teams from your school are wrapping up and posting conclusions. Enjoy the final stages of your project, and feel free to post any final comments or questions you have for your mentors.

Hey Sven, thank you so much for helping us on this arabidopsis journey. I do truly think I can plant now, and although it’s been a lot of work, I can definitely say that I had a radical time. Your input has really shaped our project, and your patience and knowledge have been critical for our success. Thank you so much!

Thank you so much for all of your help and feedback. You have been so responsive and nice. Our project is a lot better because of you.

Hi Sven, thank you so much for all your help throughout our project. Especially with the abscisic acid solution, that was really helpful. Once again thank you so much for all of your help!

Happy Monday!  Hope your plants are doing well.  Are you starting to see differences between the different genotypes or treatments? 

When you planted the seeds, how did you put them onto the pods?  Did you try to get a specific number of seeds per pod?  (I know the seeds are super tiny! I used to do a lot of research germinating individual Arabidopsis seeds.  I spent hours staring at seeds under a microscope so that I could put them on petri dishes in 10 rows of 10 seeds each per plate.)  I was just curious because I noticed that some pods appear to have a lot of seedlings and others have only a few or none.  

Hi! All of our plants except one in the low water tray has died, and then same for medium, but high water is still growing rapidly in most pods. Pictures coming soon.

Thanks for the photos!  Looks like you have some healthy looking Arabidopsis plants.  :)

Post some photos of your plants if you get a chance.  Would be cool to see your setup.  

We have now filled our methanol-abscisic acid solution up to a liter with water in order to dilute the high concentration. Is this amount of dilution safe enough for our plants?

So based on your diagram from the pdf, I understand that you are exposing Ler wt, aba1-1, and abi1-1 to high, medium, and low watering.  And for each of these three conditions you have a treatment of spraying with ABA or no spraying with ABA.  That sounds like a nicely balanced experiment. 

So you can see the differences between these genotypes under three different watering regimes and see if addition of ABA rescues any effects that are seen in the ABA-related mutants.  

Hi Sven, sorry for not updating you recently, this pdf should work, thank you for being patient with us!

This upload is just a rename of the previous upload, we realized that we should specify what we are sending in, this is our actual experimental proposal.

I still can't read the gdoc file, could you please upload it in another format like .doc or .pdf.  Google Docs can export either of these formats.  Thanks!

Here are the first 6 daily logs (including today) of our planting process: 

Day 1: Planted all seeds according to our diagram (one wt seed accidentally placed in abi1-1 pod and one abi1-1 seed accidentally placed in wt pod)

Day 2: Switched bins for flatter surfaces, no germination

Day 3: Germination of all seeds except for 5 of the pods

Day 4: Germination of all but one pod, added 100 more mL of water to each tub

Day 5: Third day of germination, 2 pods without germination (Medium water, ABA wt, and low water ABA, abi1-1)

Day 6: Refilled each tray to 400mL water, made ABA solution (50 mg ABA solution + 20mL methyl alcohol), sprayed this ABA solution on ABA plant group

We are planning on soaking the plants in water for 3 days a week, 2 days a week, and 1 day a week for high, medium and low water levels respectively. And yes, we will do the same length of soaking for ABA and no ABA plants. What do you think about 400 milliliters for a standard measure?

When you talked about how long to soak the plants, is that happening every day?  Every 2 days?  Once a week?  You could try to do the same length of soaking or watering with ABA and no ABA solution, but just decrease how many times you do it in a week.  Like how you can water stress your plants at home if you forget to water them one day.  

We are planning on soaking each plant type in water for different amounts of time. Low water would be 1 minute, medium water would be 2 minutes, and high water would be 4 minutes. Essentially doubling the amount of time each plant would be soaked. 

 

The aba1-1 and abi1-1 mutants would germinate better than the Ler wt because of either their lack of production of abscisic acid (aba1-1) or unresponsiveness (abi1-1).

In our document we have a diagram of our experiment, and have by default put that each seed variation (ex: aba1-1 with added abscisic acid and high water) would have 10 seeds tested each. This would account for possible germination failures but also comes out to 160 seeds total. Obviously we believe that many of these will die, but we are still unsure if limiting the number to 8 of each seed would make anything easier/more efficient.

We want to know the proper amount of methanol uses to dissolve ABA powder, does 5ml for 15mg sound right? Also should we add ABA before or after germination, the experiments we are looking at did it after 3 days of growth in a petry dish. Please get back to us on this ASAP. Thanks.

Sorry for the delayed response, it doesn't look like I have been getting all of the notifications.  
In response to your questions: (sorry if you already figured this out)

For ABA powder, it will depend on the final concentration that you want.  For your experiments you probably want to make a more concentrated ABA stock in methanol and then pipet a small amount of this  into the water that you add to the plants.  
A good stock concentration might be 10 mM (milli molar).  To calculate a molar concentration you need to know the molecular weight of the compound, in this case ABA.  That should be written on the tube somewhere and (I googled it) should be 264.32 (if it is different, use what the bottle says).  The molecular weight represents the number of grams in 1 mole of ABA.  So if we want a 10 mM stock, then we want 0.010 moles of ABA per Liter of solution.  Let’s say we want to mix up 10 mL of solution (which is 0.010 Liters).

So 264.32 grams/mole x 0.010 moles x 0.010 Liters = 0.02643 grams of ABA, which is 26.43 mg (milligrams).  So you would add 26.43 mg to a tube and then fill it to a total of 10 mL of Methanol.  If the amount to make is only 1 mL, then it would be 2.64 mg of ABA into a total volume of 1 mL.  Then you would have your 10 mM stock solution.  If you want a 100 mM solution in 1 mL of water, then you would add 26.43 mg of ABA powder into a total volume of 1 mL of methanol. For a 1 M solution in 1 mL of water, you would add 264.32 mg of ABA powder into a total volume of 1 mL of methanol.

Hope that makes sense.  Ask questions if it doesn’t and I can help.  You want to make your stock solution a high enough concentration so that you don’t add too much methanol into your ABA-water for the plants.  Try to keep the amount you add to less than 1 mL as a rough guide.

So after you have your stock solution (10 mM or 100 mM or 1M solution), then you can use that to add to your water to get your final concentration.  5 µM (micro molar) or 10 µM ABA should have a good effect on your plants, you can go higher, but 100 µM in the water is probably too high and may cause more stress for your plants.  Plant hormones like ABA work at very low concentrations!  You can go lower, 1 µM or 2µM ABA will still have a mild effect.  

If you start with a 10 mM solution and you want a 10 µM solution in water, you need to know how much water you are mixing up.  So if you mix up 100 mL of water with ABA (which you can add to your plants), then you would want dilute your stock to get 10µM.  The metric system makes it easy to change units because 1 M = 1,000 mM = 1,000,000 µM.  So we have 10 mM (10,000 µM) and we want 10 µM, so it will be a 1:1000 dilution.  So for 100 mL of solution, add 100 µl of your 10 mM stock solution.  If you want to make 1 Liter, add 1 mL of the 10 mM stock, or use a 100 mM stock and add 100 µL.  

I hope that helps! 

How are you planning to manipulate water level for the plants? 

That will be a fun experiment!  I have worked with both of those mutants in the lab.  

Wild-type

Ler is short for Landsberg erecta.  So we write the name Ler (the “er” is italic, but the “L” is not).  Since this is a wild-type (meaning it is a wild variety with no mutations), we usually write the name as: Ler wt.  Another common wild-type variety is Col wt (Columbia wild-type).  

Mutants

The first time I heard that I was working with “mutant seeds” I thought it sounded really strange, like something out of science fiction.  But when we say a plant is a “mutant” we just mean that a single gene (or multiple genes) are changed.  Usually, this means that the gene doesn’t function anymore, which is called a loss-of-function mutation.  Sometimes the gene is only changed a little and it still has some function.  There are also “dominant mutations” where a gene is mutated to be turned on all the time.  But both aba1-1 and abi1-1 mutants are loss-of-function mutations.

Mutant names

You will notice that I wrote aba1-1 and abi1-1 in lowercase and italics.  We use lowercase and italics to refer to loss-of-function mutant names.  When we refer to the gene name (the coding sequence on the DNA), we use uppercase italics.  So aba1-1 is a mutation in the ABA1 gene and abi1-1 is a mutation in the ABI1 gene. When we talk about the proteins, we use all uppercase with no italics (DNA (genes) are made into RNA (transcripts), which then get made into proteins: DNA =transcription⇒ RNA =translation⇒ protein).  

So the ABA1 gene codes for the ABA1 protein and the ABI1 gene codes for the ABI1 protein.   Sometimes they have other names, too.  You can look up information on a gene like it’s other names and functions by going to The Arabidopsis Information Resource (TAIR: https://www.arabidopsis.org/index.jsp) and typing the gene name into the search box at the top right of the screen.  Try it for ABA1 and ABI1. 

Your mutants 

If you look at the description on TAIR, you can see that ABA1 is the “first step of the biosynthesis of the abiotic stress hormone abscisic acid (ABA).”  That means that you need the ABA1 gene to make ABA.  So the aba1-1 mutant can’t make ABA.  But if you add ABA then it can have an ABA response because the plant can still sense the ABA.  

The ABI1 gene is involved in responding to ABA.  So the abi1-1 mutant is “insensitive to aba,” meaning that it can’t respond to ABA.  ABI1 is part of the receptor complex that senses ABA.  So why can abi1-1 mutants still respond slightly to ABA?  Because there are other similar genes like ABI2 that do something similar.  Often in plants (and animals, and people), there are multiple genes for the same or similar functions, so knocking out 1 gene may not have a huge effect.  If we only had 1 gene for every function, it might be easier to knock out a vital gene and kill the plant.  

My question for you

You mentioned that ABA is involved in stress response in plants.  ABA is also involved in seed germination.  ABA is high in dormant seeds and prevents seed from germinating.  So what effect do you think will the abi1-1 and the aba1-1 mutations have on seed germination?  Will the seeds germinate better or worse than wild-type? 

-We are comparing wild type LER with mutants ABA1-1 and ABI1-1

 

-Wild type LER produces/responds to abscisic acid by controlling the closing of stomata, development, and response to environmental stressors

 

-Mutant ABA1-1 will not respond to abscisic acid but will when sprayed with it. ABI1-1 will not respond to abscisic acid either, and will be effected only slightly when sprayed because of its resilience

-We will stress the mutant response by manipulating water levels for each type.

Hi Amy, David, and Juliana,

I’m your PlantingScience mentor, my name is Sven.  I’m a plant scientist.  I have studied how seeds decide to sprout (or not to sprout) and how roots detect water in the soil and respond to droughts.  I’m currently looking at how microorganisms in the soil can interact with the plant to improve things like drought tolerance and make the plant grow better.  

In my work, I also get to do fun things like build robots that take photos of plant roots at set timepoints and measure the growth over time under very controlled conditions.  Science requires controlling a lot of factors so that we are only changing one or two factors to see the effects they have.  

Outside of work, I like being outdoors, reading books, tinkering with electronics, and taking photographs of nature.

Tell me a little bit about each of you?  Your hobbies or interests?  Or just how your day is going today. :)

Sven

Hi Sven and Lindsey, my name is David and I’m a junior. This is going to be a radical time!

Hello Lindsey and Sven,

My name is Amy and I’m a sophomore. Looking forward to working with you guys!

Hi Sven and Lindsey, I’m Juliana and I’m a sophmore

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