Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana

This method measures changes in light transmission of leaves as a proxy for chloroplast movement which influences light absorption of leaves to different degrees depending on species, environment, and mutations. This affordable home-built device makes it possible to quantify the dynamic changes in chloroplast movement in a straightforward and semi-automated fashion. The data complement microscopy studies.

It's important to closely follow the instructions and treat the instrument with care. To begin, connect the transmission device to a stable power source and press the power switch of the device to reset the instrument. Next, connect the iPad to a stable power source.

Press the home screen button to activate the screen and enter the passcode to log in. Press the settings icon, followed by the display and brightness icon. Next, press the auto-lock and select never to ensure that the screen stays on permanently so that the program will run without any interruption.

Then press the home screen button to return to the main screen. After closing all the applications, find and open the LeafSensor app and enter the information in the green screen that shows text and white fields. Ensure that the word connected is seen in the lower part of the screen, indicating that the app is communicating with the transmission device.

If the message Adafruit NOT Found appears, check that the device is plugged in and press the start button on the device again. Fill in the first four fields on the app page to set up the conditions for a brief test run with open leaf clips without any leaves. Start with naming the experiment into the field named experiment name, then choose how many different blue light intensities will be used in the experiment by typing the number into the field named number of light intensities, then type the blue light intensities to be used by selecting an integer between 0 and 3, 000 and separate each intensity from the next with a comma into the field named blue intensities.

Type the length of time for each blue light intensity into the field named blue duration. Press Start Experiment in the middle section of the screen. In the lower part of the screen, eight hyphens with the Start Experiment message will appear.

Let the experiment run til the first data appears. After ensuring no light is emitted from the LED for the first two minutes, observe the emission of weak blue light for two minutes and then the emission of strong blue light for another two minutes. Also observe intense red light emitting briefly from the LED once a minute throughout this sequence.

Check for the Finished Experiment status indicated by the message Experiment Finished on the lower left of the app page, then press the home screen button twice. Swipe out of the app and open it again. Reset the instrument by pressing the on/off button to prepare the instrument for a run with leaves.

In the dark, using only very low white or green light, pick one wide leaf from the dark-adapted plant to cover the LED. Prepare the filter paper strip of about the length of a leaf clip. Then punch a hole at the top of the filter paper strip to prevent covering the LED.

Place a moistened filter paper on the leaf clip part that holds the LED. Remove the dark-adapted leaf clip from the Petri dish and then place the leaf on the top of the wet filter paper of its leaf clip, ensuring the adaxial leaf surface is facing the LED. Then place the other leaf clip part with a photo transistor on the top.

If needed, use a rubber band to hold two leaf clip parts together. Place the leaf clip in the respective boat and using a pipette, fill the reservoirs with water, ensuring the leaf or at least the filter paper touches the water to avoid dehydration of the leaves during the run. Set up the LeafSensor app on the iPad and type EXPLORA1 into the field named experiment name.

Type 10 into the field named number of light intensities. Then enter the numbers into the field named blue intensities and blue duration. Press Start Experiment.

After the first minute, observe the appeared output values. If the values are far off, check if the leaves were placed correctly into the leaf clips. When the run is completed, the data will be saved automatically.

After rotating the screen in the upright position, two options, save and utilities, will appear on the screen. Press Utilities and then from the list of the saved files, select the EXPLORA1 file. Below the list of files, EXPLORA1 will now appear in selected experiment.

Next, press Email. Enter an email address and the EXPLORA1 file will be automatically attached to the message and then press Send. The percent transmission data were plotted against time which show that the transmission of Arabidopsis thaliana decreased at low light intensities, while an avoidance response was induced when the light intensities further increased.

The average percent transmission values of the wild-type and mutant Arabidopsis thaliana leaves during 19-hour long runs are shown. When the leaves were exposed to low blue light in both wild-type and phot2 mutants, the initial fast decrease in transmission was followed by a slow decrease, indicating that the chloroplasts were moving into the accumulation response. Compared to wild-type, phot1 showed a reduced accumulation response.

When the blue light intensity is increased stepwise after 11 hours, the percent transmission increases and the chloroplasts move into the avoidance response. The degrees of change in the percent transmission relative to the dark value, also called Delta T, were dependent on the exact blue light intensities. The negative values indicate that the leaves showed an accumulation response, while positive values indicate an avoidance response.

The speed of change in transmission is calculated as the slope of change in transmission when the light is increased. In the representative example, the transmission speed changed during the initial responses when avoidance was triggered as the blue light intensity was increased. When investigating the transmission changes in an uncharacterized mutant or species, confirm how the transmission values and chloroplast positions correlate with a microscopy study.