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

Summary

Many plant species change the positioning of chloroplasts to optimize light absorption. This protocol describes how to use a straightforward, home-built instrument to investigate chloroplast movement in Arabidopsis thaliana leaves using changes in the transmission of light through a leaf as a proxy.

Abstract

Chloroplast movement in leaves has been shown to help minimize photoinhibition and increase growth under certain conditions. Much can be learned about chloroplast movement by studying the chloroplast positioning in leaves using e.g., confocal fluorescence microscopy, but access to this type of microscopy is limited. This protocol describes a method that uses the changes in leaf transmission as a proxy for chloroplast movement. If chloroplasts are spread out in order to maximize light interception, the transmission will be low. If chloroplasts move towards the anticlinal cell walls to avoid light, the transmission will be higher. This protocol describes how to use a straightforward, home-built instrument to expose leaves to different blue light intensities and quantify the dynamic changes in leaf transmission. This approach allows researchers to quantitatively describe chloroplast movement in different species and mutants, study the effects of chemicals and environmental factors on it, or screen for novel mutants e.g., to identify missing components in the process that leads from light perception to the movement of chloroplasts.

Introduction

Light is essential for photosynthesis, plant growth, and development. It is one of the most dynamic abiotic factors as light intensities not only change over the course of a season or day, but also rapidly and in unpredictable ways depending on the cloud cover. At the leaf level, light intensities are also influenced by the density and nature of the surrounding vegetation and the plant's own canopy. One important mechanism that allows plants to optimize light interception under variable light conditions is the ability of chloroplasts to move in response to blue light stimuli^1,2. Under low light conditions, chloroplasts spread out perpendicular to the light (along the periclinal cell walls) in a so-called accumulation response, maximizing light interception and hence photosynthesis. Under high light conditions, chloroplasts move towards the anticlinal cell wall in a so-called avoidance response, minimizing light interception and the danger of photoinhibition. In many species, chloroplasts also assume a specific dark position, which is distinct from the accumulation and avoidance positions and often intermediary between those two^3,4. Various studies have demonstrated that chloroplast movement is not only important for the short-term stress tolerance of leaves^5,6,7, but also for the growth and reproductive success of plants, especially under variable light conditions^8,9.

Chloroplast movement is readily observed in real time in certain live specimens (e.g., algae or thin-leafed plants like Elodea) using light microscopy¹. Studying chloroplast movement in most leaves, however, requires a pretreatment to induce chloroplast movement, chemical fixation, and preparation of cross sections before viewing the samples under a light microscope¹⁰. With the introduction of confocal laser microscopy, it also became possible to image the 3D arrangement of chloroplasts in intact or fixed leaves^4,11,12. These imaging techniques greatly aid the understanding of chloroplast movement by providing important qualitative information. Quantifying chloroplast positioning (e.g., as a percentage of chloroplasts in the periclinal or anticlinal positions in these images or the percentage of area covered by chloroplasts per total cell surface) is also possible but quite time-consuming, especially if conducted at the intervals necessary to capture rapid changes in positioning^10,8. The simplest way to show whether dark-adapted leaves of a certain species or mutants are capable of chloroplast movement into the avoidance response is by covering most of the area of a leaf to keep the chloroplasts in the dark while exposing a strip of the leaf to high light. After a minimum of 20 min of high light exposure, the chloroplasts in the exposed area will have moved into the avoidance position, and the exposed strip will be visibly lighter in color than the rest of the leaf. This is true for wild type A. thaliana but not for some of the chloroplast movement mutants described in more detail later on¹³. This method and modifications of it (e.g., reversing what parts of the leaf are exposed, changing light intensities) are useful for screening large numbers of mutants and to identify null mutants that lack either the ability to exhibit an avoidance or accumulation response or both. However, it does not provide information about the dynamic changes in chloroplast movement.

In contrast, the method described here allows for the quantification of chloroplast movement in intact leaves using changes in light transmission through a leaf as a proxy for overall chloroplast movement: under conditions when chloroplasts are spread out in the mesophyll cells in the accumulation response, less light is transmitted through the leaf than when many chloroplasts are in an avoidance response, positioning themselves along the anticlinal cell walls. Hence, changes in transmission can be used as a proxy for the overall chloroplast movement in leaves¹⁴. The details of the instrument are described elsewhere (see Supplementary File), but basically, the instrument uses blue light to trigger chloroplast movement and measures how much red light is transmitted through that leaf at set intervals. More recently, a modification of this system has been described, which uses a modified 96-well microplate reader, a blue LED, a computer, and a temperature-controlled incubator¹⁵.

The option to use a combination of methods, including the optical assessment of leaves for screening, followed by measuring dynamic changes in transmission and the use of microscopy, has greatly aided our understanding of both the underlying mechanisms and the physiological/ecological significance of chloroplast movement. For example, it led to the discovery and characterization of various mutants, which are impaired in specific aspects of their movements. For example, A. thaliana phot 1 mutants lack the ability to accumulate their chloroplasts in low light, while phot 2 mutants lack the ability to perform an avoidance reaction. These phenotypes are due to an impairment in two respective blue light receptors^16,17,18. In contrast, chup1 mutants lack the ability to form proper actin filaments around the chloroplasts which are essential to move the chloroplasts into the desired position within a cell^11,19. In addition to mutant studies, researchers have assessed the effects of various inhibitors on chloroplast movement to elucidate the mechanistic aspects of the process. For example, chemicals such as H₂O₂ and various antioxidants were used to investigate the effects of this signaling molecule on chloroplast movement²⁰. Various inhibitors were used to elucidate the role of calcium in chloroplast movement²¹. In addition to helping to uncover the mechanisms of chloroplast movement, these methods can be used to compare chloroplast movement in various species or mutants grown in different conditions in an attempt to understand the ecological and evolutionary context of this behavior. For example, it has been shown that the extent of the effects of various mutations in the chloroplast movement pathway are dependent on the growth conditions^7,9, and that sun-adapted plants do not seem to move their chloroplasts much. In contrast, movement is very important for shade plants^10,22,23.

This methods paper, focused on the model plant A. thaliana, describes how to use a transmission device which is an updated version of a previously developed instrument⁹. While this instrument is not commercially available, people with a basic understanding of electronics or the help of engineering or physics colleagues and students will be able to build the instrument using affordable parts and following the detailed instructions (see Supplementary File). The open-source platform used to build the instrument has extensive web support and a community forum which offers help should problems arise²⁴.

The protocol focuses on how to use the instrument to determine changes in leaf transmission in a standard exploratory run that exposes a leaf to a wide range of light conditions and captures the dark, accumulation, and avoidance reactions of A. thaliana. These runs can be modified depending on the goal of the experiment and can be used with most plant species. The paper provides examples of transmission data of A. thaliana wildtype and several mutants and shows how to further analyze the data.

Protocol

1. Preparing leaves for a run

1. Place 8 A. thaliana plants in the dark overnight (> 6 h works for most species) to ensure that the chloroplasts move into their dark position. All replicas start with comparable transmission values.
2. Alternatively, place 8 complete leaves into a Petri dish with a moist filter paper at the bottom, close the Petri dish, and wrap it with aluminum foil.

2. Testing if the transmission device works

1. Connect the transmission device to a stable power source and press the power switch (ON/OFF button) of the device to reset the instrument (Figure 1A,B).
2. Connect the iPad to a stable power source, press the Home Screen button to activate the screen, and enter the passcode to log in.
3. Press the Settings icon, press the Display & Brightness icon, press Auto-Lock, and select Never by pressing this option to ensure that the screen stays on permanently. Otherwise the program will stop running when the screen goes to sleep. Press the Home Screen button to return to the main screen.
4. Double press the Home Screen button to see which applications are open, and close all of them by swiping them towards the top of the screen. Press the Home Screen button to return to the main screen.
   1. Find the LeafSensor app on the main screen or by swiping left or right. Press the icon of the app to open it (see Supplementary File). A green screen with text and white fields will appear to enter the information.
   2. Ensure the word Connected is seen in the lower part of the screen as it indicates 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.
5. Fill in the first 4 fields on the app page to name the experiment and to set up the conditions for a brief test run without leaves and with the leaf clips open:
   1. Name the experiment (use 8 or fewer uppercase letters or numbers) e.g., by typing TEST into the field named Expt Name.
   2. Choose how many different blue light intensities will be used in the experiment, e.g., by typing 3 into the field named # Light Intensities.
   3. Choose the blue light intensities for this run (choose an integer between 0 and 3000 and separate each number from the next with a comma; see Supplementary File on how to convert these numbers into actual blue light intensities of LEDs) e.g., by typing 0,100,1000 into the field named Blue Intensities.
   4. Choose the length of time each blue light intensity will shine onto the leaf (separate each number from the next with a comma) e.g., by typing 2,2,2 into the field named Blue Duration (minutes).
6. Press Start Experiment in the middle section of the screen. In the lower part of the screen 8 hyphens and the message Starting Experiment will appear.
   1. Ensure that no light is being emitted from the LEDs for the first two minutes, then weak blue light is being emitted, and after 2 min the blue light intensity is increasing.
   2. Ensure that intense red light is being emitted from the LEDs once a minute for the measurements.
      ​NOTE: As the experiment is running, numbers will appear on the app page for each of the eight sensors, and data will be updated once a minute. Ensure the output numbers from the photodiodes are around 1000-1023 (if the room is dark). An update on the lower left shows how many measurements were done so far e.g., Done 1 of 6 measurements (one measurement each minute).
   3. When the experiment is done, check for the appearance of the Experiment finished on the lower left of the app page. Now the instrument is ready for a run with leaves.
7. Press twice the Home Screen button, swipe out of the app and open it again. Press the ON/OFF button on the instrument to reset it.

3. Setting up leaves in the leaf clips

NOTE: This step has to be done in the dark with a green light source (e.g., place a green filter in front of a light bulb) to avoid inducing chloroplast movement. Alternatively, use very low white light and an extended dark period in the leaf clips. Remember, one part of the leaf clip holds the LED (larger opening), while the other holds the phototransistor (Figure 1C).

1. If the plants are dark-adapted entire, pick 8 leaves wide enough to cover the LEDs. Otherwise, remove the leaves from the Petri dish. Prepare 8 strips of filter paper about the length of a leaf clip and with a hole punched out at the top to not cover the LED.
   1. Moisten the filter paper and place it onto the leaf clip part that holds the LED. Repeat this for each of the eight leaf clips.
2. Place each leaf on the top of the wet filter paper of its leaf clip. Ensure the correct leaf side is facing towards the LED (usually experiments are done with the adaxial leaf surface facing the LED).
   1. Avoid placing the midribs of the leaf on top of the LEDs, and for more consistent results, place similar parts of each leaf (e.g., the widest section of the leaf) over the LED.
   2. Place the other leaf clip part with the phototransistor on top. Use a rubber band to hold the two leaf clip parts together if needed (Figure 1C,D).
3. Place each leaf clip into its 'boat' and fill the reservoirs with water using a pipette. Ensure the leaf or at least the filter paper touches the water to avoid dehydration of the leaves during the run (Figure 1D).

4. Conducting a run

NOTE: For a standard exploratory run, start out with 4 h of darkness (0 µmol photon m^-2 s^-1), followed by 7 h of low blue light (2 µmol photon m^-2 s^-1), followed by 60 min each of 5, 10, 30, 40, 50, 60, 90, 100 µmol photon m^-2 s^-1 of blue light. This will induce the leaves to exhibit their dark transmission, induce chloroplast movement into the maximum accumulation, and show different degrees of avoidance response.

1. Set up the LeafSensor app on the iPad using the steps described below.
   1. Type EXPLORA1 into the field named Expt Name.
   2. Type 10 into the field named # Light Intensities.
   3. Type 0,1,60,160,550,750,950,1150,1350,1950 into the field named Blue intensities.
   4. Type 240,420,60,60,60,60,60,60,60,60 into the field named Blue Duration.
2. Press Start Experiment. After the first min, the output values (usually between 990 and 820) will appear on the screen. If the values are far off, check if the leaves were placed correctly into the leaf clips.
3. When the run is done, ensure that the message Experiment Finished appears at the bottom left of the screen. Data will be saved automatically.
   1. Put the screen in the upright position (not horizontal). Two new options will appear on the screen, namely Save and Utilities.
   2. Press Utilities and a list of saved files will appear. Select the file of interest, in this case, EXPLORA1.
   3. Below the list of files , look for Selected Expt: EXPLORA1. Press Email, enter an email address, and the data file will be automatically attached to the message. Press Send.
      NOTE: If there is a long delay until the files arrive, restart the app and send the file again.
4. If a run was aborted, but data up to that point are of interest, press Save before selecting Utilities. After several runs, clean up the memory space: Press Utilities, select one file at a time, swipe left next to file, and press Delete to remove file.
5. Press Back to run another experiment or if done, press the Home Screen button, swipe to the main screen, press Settings, press Display & Brightness, press Auto-Lock, and press 2 Minutes.

5. Data analysis

1. Download the file EXPLORA1 from the email, add extension .csv to the file, double-click on the file. Data will be sorted into a spreadsheet with the data for the eight different sensors sorted into separate columns. The last column shows the time (in seconds) at which the data were collected. Delete the first row under the headings (Sensor1-8) if it contains nonsensical data.
2. Set up a master data sheet that will contain the results for each sensor in a separate sheet and that converts the output values into % transmission values using the equations obtained from the calibration of each leaf clip and sensor (see Supplementary File).
   1. Copy each data set into a separate data sheet (e.g., column A contains the time; column C contains the data of Sensor1).
   2. Set up column B so that it converts time from seconds to minutes. Set up column D so that it contains the formula to convert voltage to % transmission using the equation from the calibration.
   3. Set up a similar data sheet for each sensor and remember the formula used to convert the voltage output into % transmission values may be different for each sensor.
3. Plot % transmission (T) against time, min (Figure 2).
4. Save the data sheet under a new name so that the master data sheet can be reused.
5. To further analyze data (Figure 2), calculate ΔT (%) accumulation (e.g., change of T at maximum accumulation compared to T at dark), ΔT (%) avoidance (e.g., change of T at maximum avoidance compared to T at dark), or dT/dt (%/h) (e.g., change in T during the fastest part of the accumulation or avoidance reaction). For further details see ⁸.

Results

The different parts of the transmission device are shown in Figure 1. The microcontroller is the control unit of the device and controls the light conditions that the leaves, secured in black leaf clips, are experiencing, and stores the light transmission data it receives (Figure 1A,B). A close-up of the control unit of the instrument shows the ON/OFF button, the SD card for data storage capability, the Bluetooth shield (which sends the data to ...

Discussion

The device is extremely easy to use but it is of crucial importance to calibrate each leaf clip set-up of the transmission device independently since the positioning of the LEDs and phototransistors may slightly vary from leaf clip to leaf clip. Ensure the LEDs and phototransistors are inserted stably and re-check the calibration if the data seem off. Avoid getting water onto the device. The leaves in the leaf clips are placed into 'boats' filled with water to avoid water stress. Place these boats e.g., into a lo...

Materials

Name	Company	Catalog Number	Comments
Aluminum foil
Dark adapted leaves
Filter paper
iPad with LeafSensor app installed (see Supplemental Info)
Pipette	Any
Petri dish	Any
Transmission device (see Supplemental info)
Water