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,....

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....

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

List of materials used in this article

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