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Module Two – Light Sensors and Plant Development

 Introduction

 Seedlings grown in darkness have a very specific look.  They have a long stem, tiny leaves and roots that do not elongate.  The seedling’s environment tells it that it is in soil, so it adopts a body plan and behaviors designed to help it seek light.  When seedlings find light they change their strategy.  Their stem grows slower, their leaves develop and they turn green as chlorophyll accumulates. The figure to the right shows this process, a processes called photomorphogenesis.

The process of photomorphogenesis, or light-mediated development.  The two seedlings are genetically identical, are the same age and are grown on the same media.  Why are they so different?  The answer lies in their light environment. Plants use light to change their architecture to best fit with the current conditions.

 Photomorphogenic development is controlled by different light sensors, specialized molecules that sense the light environment.  Arabidopsis has a number of light sensors.  Some control seedling bending, some control the plant’s clock, some control flowering and some have a major role in photomorphogenic development.

 Some of the plant photoreceptors have been well studied for over 50 years, others have yet to be discovered.  Of the best studied are the phytochromes.  Phytochromes sense red and far-red light. In Arabidopsis phytochrome B (phyB for short) interprets the red light signals from the environment.  The phyA receptor (phytochrome A) senses the invisible light that is off the end of the red part of the spectrum, a light quality called “far-red”.  Together, phyB and phyA are an important combination because they tell the plant about the relative amount of red and far-red light.  This information can advise the plant of time of day, season, or the distance to neighbors.  The other major photomorphogenic receptor is cryptochrome 1, or cry1 for short.  The cryptochrome 1 receptor controls stem elongation in response to the blue and UV section of the spectrum.

 The goal of module 2 is to use the R2R light system to illustrate how the various plant photoreceptors respond to different parts of the light spectrum to control seedling development.  In this study you will grow Arabidopsis thaliana seedlings on vertical agar plates under different qualities of light. You will grow different kinds of seedlings.  The first are normal or “wild type” seedlings.  The rest are mutants, seedlings with defects in the various light sensors.  Mutants for phyA, phyB and cry1 receptors have been included for this study. You will compare the growth of wild type and mutant seedlings under different colors of light.

 

Materials

 1.10 Square Petri dishes

2 Arabidopsis thaliana seeds- wild type (Col-0), phyA, phyB and cry1 mutants.

3. 3 g Powdered agar

1.      300 ml water (1 ¼ cups)

2.      Rubbing alcohol

3.      a small 10 x 10 cm (4 x 4 inches) piece of paper for each seed type

4.      Forceps (tweezers), a toothpick or a pin

5.      R2R Light Source

6.      A box 20 x 20 x 20 cm (see box design)

7.      Sharpie marker or other quality permanent marker

 

 Method

 Plate Preparation 

  1. Boil the water and mix in the agar.  Continue to boil for 5 minutes or until complete dissolved.  The solution should be clear.
  2. Remove from heat for 10 minutes. Allow the solution to cool, but not solidify.
  3. Carefully pour approximately 30 ml into each plate.  The measurement does not need to be accurate, it is essentially needs to be enough liquid to cover the bottom of the plate.
  4. Allow the plates to solidify.  Store them in the refrigerator, not the freezer.

 

Planting.

1.  Sprinkle a small amount of each seed type (maybe 20-30 seeds) onto a separate small piece of paper.  I like a post-it note because it doesn’t move around.  It is important to label each sheet clearly with pencil, but permanent marker is okay.

2.  Cover the seeds with a pool of rubbing alcohol, just enough to cover them.  This will remove surface contamination and will not affect germination.

3.  Allow the paper to dry.  This might take 5-10 minutes.

4.  While the paper is drying, use a permanent marker to label the squares on the dish. The standard dish is a 6x6 grid.  Draw a horizontal line across the plate between rows 2 and 3.  Label the boxes in row 3.   Label three as Col-0, then one each for phyA, phyB, and cry1.

4.  After the seeds are dry take a pointed object, like a pin, toothpick or tweezer prong and touch it into the agar on a plate (near the edge).  This is only to wet the tool.  Once it is wet you can pick up seeds just by touching them.

5. Select a single seed on the paper and transfer it to the agar plate. It is best to place them so that they are directly above a horizontal line on the plate, and place them into the square so that the seed type matches the label. Place six seeds per square.  This takes a lot of practice!  The first few are difficult, but soon you can do them very quickly.   Complete one line all the way across the plate.

6. Put the plate in the refrigerator for 2 days.

 

Light conditions.

  1. After two days in the cold, take the square plate and place it between two other plates with a rubber band so that it stands by itself.  The seeds should be in a horizontal line, perpendicular to the incoming light.
  2. Place the Petri dishes into the box you have prepared and place the light source on top.  Plug it in, follow the directions for setting the color and intensity, and then your experiment has started!
  3. Don’t forget the control!  You should have an identical set of seeds on a plate wrapped in at least two layers of aluminum foil.  This should be in the box with the rest of the plates.

 

Measurement

  1. There are several ways to assess seedling growth in response to light.  The most simple is to take a picture of the seedlings through the plate after 4 days (96 hours).  Alternatively, you can take a picture every 24 h for a number of days, or use a document scanner.  The scanner works best because it is a high resolution image.
  1. Once all images are captured you have to measure the seedlings.  There are several ways to do this.
    1. Simple.  Open your image files on a computer screen.  Measure the square on the Petri dish with a ruler, on the screen.  Record the value.  Measure the seedlings and record their height.  Just record from the top of the seedling to where the stem meets the root. Calculate the actual seedling height in millimeters.  The square of the Petri dish is 13 mm.
    2. Slightly More Complicated.  Download Image Tool 3.0 Software from the following website http://ddsdx.uthscsa.edu/dig/download.html.  It is free and simple.  Once installed, open an image an

 

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