Name: using changes in leaf transmission to investigate chloroplast movement in arabidopsis thaliana
Uploaded: 2021-07-14T15:00:00
Description: Watch this Scientific Journal Video about Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana at JoVE.com

Funding was provided by a Fiske Award and a Wellesley College Faculty Award.

1. Preparing leaves for a run
<ol>
	<li>Place 8 A. thaliana plants in the dark overnight (&#62; 6 h works for most species) to ensure that the chloroplasts move into their dark position. All replicas start with comparable transmission values.</li>
	<li>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.</li>
</ol>
2. Testing if the transmission device works
<ol>
	<li>Connect the transmi...

<ol>
	<li>Senn, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Die Gestalts- und Lagever&#228;nderung der Pflanzenchromatophoren. , (1908).</li><li>Zurzycki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+chloroplast+displacements+on+the+optical+properties+of+leaves.">The influence of chloroplast displacements on the optical properties of leaves.</a> Acta Societatis Botanicorum Poloniae. 30, 503-527 (1961).</li><li>Wada, M., Kagawa, T., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+movement.">Chloroplast movement.</a> Annual Review of Plant Biology. 54, 455-468 (2003).</li><li>Wada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+movement.">Chloroplast movement.</a> Plant Science. 210, 177-182 (2013).</li><li>Kasahara, M., Kagawa, T., Oikawa, K., Suetsugu, N., Miyao, M., Wada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+avoidance+movement+reduces+photodamage+in+plants.">Chloroplast avoidance movement reduces photodamage in plants.</a> Nature. 420, 829-832 (2002).</li><li>Davis, P. A., Hangarter, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+movement+provides+photoprotection+to+plants+by+redistributing+PSII+damage+within+leaves.">Chloroplast movement provides photoprotection to plants by redistributing PSII damage within leaves.</a> Photosynthesis Research. 112, 153-161 (2012).</li><li>Howard, M. 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M., Bae, A., Pirani, Z., Van, N., K&#246;niger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+chloroplast+movement+reduces+growth+and+delays+reproduction+of+Arabidopsis+thaliana+in+natural+and+controlled+conditions.">Impairment of chloroplast movement reduces growth and delays reproduction of Arabidopsis thaliana in natural and controlled conditions.</a> American Journal of Botany. 107 (9), 1309-1318 (2020).</li><li>Trojan, A., Gabry&#347;, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+distribution+in+Arabidopsis+thaliana+(L.)+depends+on+light+conditions+during+growth.">Chloroplast distribution in Arabidopsis thaliana (L.) depends on light conditions during growth.</a> Plant Physiology. 111, 419-425 (1996).</li><li>Oikawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+unusual+positioning+is+essential+for+proper+chloroplast+positioning.">Chloroplast unusual positioning is essential for proper chloroplast positioning.</a> The Plant Cell. 15, 2805-2815 (2003).</li><li>K&#246;niger, M., Bollinger, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+movement+behavior+varies+widely+among+species+and+does+not+correlate+with+high+light+stress+tolerance.">Chloroplast movement behavior varies widely among species and does not correlate with high light stress tolerance.</a> Planta. 236, 411-426 (2012).</li><li>Kagawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+NPL1:+a+phototropin+homolog+controlling+the+chloroplast+high-light+avoidance+response.">Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response.</a> Science. 291, 2138-2141 (2001).</li><li>Berg, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+low-cost+microcontroller-based+photometric+instrument+for+monitoring+chloroplast+movement.">A simple low-cost microcontroller-based photometric instrument for monitoring chloroplast movement.</a> Photosynthesis Research. 87, 303-311 (2006).</li><li>Johansson, H., Zeidler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+chloroplast+movement+analysis.">Automatic chloroplast movement analysis.</a> Molecular Biology. 1398, 29-35 (2016).</li><li>Briggs, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phototropin+family+of+photoreceptors.">The phototropin family of photoreceptors.</a> Plant Cell. 13, 993-997 (2001).</li><li>Jarillo, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototropin-related+NPL1+controls+chloroplast+relocation+induced+by+blue+light.">Phototropin-related NPL1 controls chloroplast relocation induced by blue light.</a> Nature. 410, 952-954 (2001).</li><li>Sakai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+nph1+and+npl1:+blue+light+receptors+that+mediate+both+phototropism+and+chloroplast+relocation.">Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation.</a> Proceedings of the National Academy of Sciences, USA. 98 (12), 6969-6974 (2001).</li><li>Wada, M., Kong, S. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-mediated+movement+of+chloroplasts.">Actin-mediated movement of chloroplasts.</a> Journal of Cell Science. 131, 1-8 (2018).</li><li>Wen, F., Xing, D., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+is+involved+in+high+blue+light-induced+chloroplast+avoidance+movements+in+Arabidopsis.">Hydrogen peroxide is involved in high blue light-induced chloroplast avoidance movements in Arabidopsis.</a> Journal of Experimental Botany. 59 (10), 2891-2901 (2008).</li><li>Tlalka, M., Fricker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+calcium+in+blue-light+dependent+chloroplast+movement+in+Lemna+trisulca+L.">The role of calcium in blue-light dependent chloroplast movement in Lemna trisulca L.</a> The Plant Journal. 20, 461-473 (1999).</li><li>K&#246;niger, M., Bollinger, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+movement+behavior+varies+widely+among+species+and+does+not+correlate+with+high+light+stress+tolerance.">Chloroplast movement behavior varies widely among species and does not correlate with high light stress tolerance.</a> Planta. 236, 411-426 (2012).</li><li>Higa, T., Wada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+avoidance+movement+is+not+functional+in+plants+grown+under+strong+sunlight.">Chloroplast avoidance movement is not functional in plants grown under strong sunlight.</a> Plant, Cell and Environment. 39, 871-882 (2016).</li><li>. Arduino.cc Available from: <a target="_blank" href="https://www.arduino.cc">https://www.arduino.cc</a> (2021)</li><li>K&#246;niger, M., Delamaide, J. A., Marlow, E. D., Harris, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+thaliana+leaves+with+altered+chloroplast+numbers+and+chloroplast+movement+exhibit+impaired+adjustments+to+both+low+and+high+light.">C. thaliana leaves with altered chloroplast numbers and chloroplast movement exhibit impaired adjustments to both low and high light.</a> Journal of Experimental Botany. 59, 2285-2297 (2008).</li></ol>

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 &#39;boats&#39; filled with water to avoid water stress. Place these boats e.g., into a lo...

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&#39;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 stimuli1,2. 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 two3,4. Various studies have demonstrated that chloroplast movement is not only important for the short-term stress tolerance of leaves5,6,7, but also for the growth and reproductive success of plants, especially under variable light conditions8,9.
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 leaves4,11,12. 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 positioning10,8. 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.&#160;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 receptors16,17,18. 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 cell11,19. 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 conditions7,9, and that sun-adapted plants do not seem to move their chloroplasts much. In contrast, movement is very important for shade plants10,22,23.
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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dark adapted leaves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iPad with LeafSensor app installed (see Supplemental Info)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission device (see Supplemental info)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

using changes in leaf transmission to investigate chloroplast movement in arabidopsis thaliana

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.

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 ...

Watch this Scientific Journal Video about Using Changes in Leaf Transmission to Investigate Chloroplast Movement in Arabidopsis thaliana at JoVE.com

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

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.

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

Chloroplast movement in leaves has been shown to help minimize photoinhibition and increase growth under certain conditions. Much can be learned about ...

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.

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