This work was supported from the European Regional Development Fund-Project SINGING PLANT (No. CZ.02.1.01/0.0/0.0/16_026/0008446). This project has received funding through the MSCA4Ukraine project (ID 1233580), which is funded by the European Union. We are grateful to Lenka Sochurkova for the graphical design of Figure 1.

1. Medium preparation
<ol>
	<li>Prepare the cultivation medium by mixing 0.75 g of gelling agent with 50 mL of sterile deionized water in a glass bottle to achieve a 1.5% (w/v) concentration for one Petri plate (120 x 120 x 17 mm). Gently shake the mixture and then heat it in a microwave until boiling to dissolve the gelling agent (the solution becomes clear).</li>
	<li>Allow the medium to cool down to 58-60 &#176;C before proceeding to the next steps. All subsequent steps must be performed under sterile conditions within a laminar-flow hood to prevent contamination.</li>
	<li>Use Petri plates with light-tight edges to avoid excessive actinic light reflection and high background autofluorescence during the measurements. For this, apply black adhesive tape (or other means available) to cover all the sides of the empty Petri plate (Figure 2).</li>
	<li>Perform sterilization of the plate(s) after tape application by irradiation with germicide UV lamp for 20-30 min.</li>
	<li>If the experiment involves chemical treatment (i.e., abiotic/biotic stressors, plant hormones and/or growth regulators, etc.), add the appropriate amount of corresponding chemical directly to the medium. Be aware of a chosen chemical stability being added to the media (for example, if the chemical is not thermostable, add to the medium when it is cooled down right before pouring it into plate). Mix the medium thoroughly by shaking to ensure an even distribution of the chemical.</li>
	<li>Avoid illumination of plates to UV light after the media is poured (would lead to oxygen radical production that might interfere with the experiment).</li>
	<li>Pour the prepared medium into the square Petri plate(s) and allow the medium to solidify at room temperature.</li>
</ol>
2. Seed surface sterilization and plant growth conditions
<ol>
	<li>Get the required amount of Arabidopsis thaliana Col-0 seeds from the stock balk (10-20 mg) and add it to a 2 mL microcentrifuge tube. 
	NOTE: No specific modifications are necessary while working with different Arabidopsis lines (ecotype/mutant lines). Revision of the sawing grid and measuring steps should be performed for other plant species taking into account difference in seed size, germination rate and seedling size.</li>
	<li>Surface-sterilize Arabidopsis seeds by adding 70% ethanol to the tube for 2 min. Gently shake the tubes during sterilization.</li>
	<li>Remove ethanol by pipetting carefully, taking care not to lose any seeds. Wash the seeds by adding sterile water to the tube for 5 min to remove any residual ethanol (gently shake the tubes during the washing period).</li>
	<li>Let the seeds sediment to the bottom of the tube by gravity, remove the remaining water.</li>
	<li>Rinse the seeds again 2x with sterile water as described in step 2.3 to ensure they are free from any ethanol traits. Let the seeds sediment to the bottom of the tube by gravity, remove the remaining water.</li>
	<li>Add an equal volume of sterile water to the tube containing the seeds to create a seed-water suspension.</li>
	<li>Use a sowing grid to evenly distribute seed-water suspension of the given genotype on the selected areas of the medium plate (Figure 2 and Supplementary Figure 1). Distribute the seeds (approximately 30-40) in a row in each area using a wide pipette tip.</li>
	<li>Let the water dry in the seed areas for about 30 min, keeping the plate(s) open in a laminar flow hood to prevent contamination. Seal the plate with micropore tape and wrap it with aluminum foil.</li>
	<li>Stratify the seeds for 3 days at 4 &#176;C in darkness to (depending on the ecotype used) overcome the seed dormancy and/or to promote uniform germination.</li>
	<li>Transfer the plate with stratified seeds to white light (150 &#181;mol/m2/s) for 1 h to induce germination (unwrap the foil only for the light treatment).</li>
	<li>After the light treatment, wrap the plate with aluminum foil to protect the seeds from light and place it in a vertical position in a growth chamber and cultivate for 4 days in the dark at 21 &#730;C.</li>
</ol>
3. Chlorophyll fluorescence measurement and analysis
<ol>
	<li>Turn on the iReenCAM system and ensure that the system is ready and properly configured in the automatically initiated PS server software (e.g., if there is enough storage space for the experiment data, if the fluorescence camera is connected to PC, etc.).</li>
	<li>Activate the Scheduler software to create the experimental plan for the measurement by clicking Experiments &#62; New Experiment. Provide a descriptive name for the experiment and fill in the details (description).</li>
	<li>Set the required actions for the experiment by clicking Add Action which will lead to the schedule of experimental actions. 
	NOTE: The word action here means performing a complete experiment (i.e., including all the steps necessary to perform one plate measurement).</li>
	<li>Specify the conditions for a single round measurement (i.e., the length of light/darkness period).</li>
	<li>By clicking Generate List define the time intervals between measuring rounds. Choose the time when the round will start and finish (4 h in total) and the intervals between the rounds (in the current setup one round every 2 min).</li>
	<li>Click Generate and ensure that the time frame and intervals between light impulses are correct by checking the list generated on the left side of the screen.</li>
	<li>Choose the measuring protocol (Supplementary Figure 2). Save all modifications to the database for future reference.</li>
	<li>Just before the measurement starts, use green light of low intensity (see Table of Materials) inside the dark room and adjust the level of the shelf inside the measuring chamber or perform other preparational steps before removing the foil from the Petri plate. Then switch off the light and transfer the plates into the measuring chamber in the complete darkness.</li>
	<li>Carefully remove the aluminum foil covering the Petri plate containing the 4-day-old seedlings. Place the Petri plate horizontally inside the device measuring chamber. Inside the chamber, induce actinic light pulses, and perform imaging according to the experiment plan (actions) set in steps 3.1-3.7. 
	NOTE: It is critical to avoid any illumination of the plates with etiolated seedlings before placing them into the measuring chamber. The manipulation with the plate with etiolated seedlings must be performed in the dark room/chamber (for a possible experimental setup see Supplementary Figure 3).</li>
</ol>
4. Data extraction and analysis
<ol>
	<li>After completing the measurement, open the corresponding experiment in the analyzer software.</li>
	<li>To analyze the fluorescence of the seedlings, generate two types of masks- a rough (tray) mask that covers the area where the seedlings are located, and a precise (plant) mask that covers only the tissue of interest (typically cotyledons).</li>
	<li>Generate a tray mask by clicking the Create New Tray Type option. Assign the appropriate genotype names to the respective areas on the plate image.</li>
	<li>For assigning genotype, choose an image number at Round and then click Load Image in the upper part of the screen. The image of the plate will be shown on the screen. By clicking on any of the set of buttons that represent different shapes drawing tools (Supplementary Figure 4), enter New Shape Mode that allows to draw the area of interest on the plate image. Choose the necessary areas (e.g., different genotypes on the plate) and provide their appropriate naming.</li>
	<li>Click Esc to exit the shape mode. Save the generated tray mask (after providing a name for it) by clicking Store Tray Type.</li>
	<li>Go back a step and apply the mask that was generated in previous steps by selecting its name from the Change by Tray Type option.</li>
	<li>To generate plant mask with high accuracy, use the image acquired after 180 min of measurement (round 91) to set the minimum threshold value for fluorescence signal intensity, enabling background noise subtraction. For this, remove the tick from Auto Threshold and set a Manual Threshold at 0 (Supplementary Figure 4).</li>
	<li>Click Preview to ensure that the tray mask covers all necessary areas (genotypes) on the plate image. For this choose round 91 and click Refresh Preview.</li>
	<li>Enter the Run menu by clicking Run. Run the analysis exclusively for round 91 by putting a tick only on round 91. Then choose the output path and click Start Analysis.</li>
	<li>After the analysis is finished the Finish menu will open automatically. Pick the executed round (it will be the only one) from experiments and click Switch Analyzer to Analyzed Data to export the data for this specific time point (round 91).</li>
	<li>Extract the .xsel file from the exported archive, as this file contains the essential plant mask information by clicking Open Export Part icon.</li>
	<li>Re-open the experiment by clicking on Open Local Analysis Part icon. Enter the Mask Builder menu again, click Load Image, choose round 1 and then Load from File in the upper right corner of the screen and load the previously extracted .xsel file. The image of the plate will be shown with the tray mask applied.</li>
	<li>Save the mask by clicking Store Tray Type and apply it by selecting its name in Change by Tray Type option.</li>
	<li>Generate the plant mask by adjusting the fluorescence signal intensity threshold. Increase the value of Manual Threshold till the mask generated in the Preview menu fits perfectly to the ROI (e.g., cotyledons) in each of the genotypes (Supplementary Figure 4). Check if the mask fits to all rounds of the measurements by scrolling throughout the rounds (checking the proper mask positioning at round 1, 61 and 121 should be enough) in the Preview menu.</li>
	<li>Perform the analysis for all measuring rounds and export data. 
	NOTE: The output file includes chlorophyll fluorescence values of a given genotype for each time point, enabling the construction of charts of choice and facilitating further statistical evaluation.</li>
</ol>

High-Resolution Imaging of Chlorophyll Biosynthesis in &lt;em&gt;Arabidopsis&lt;/em&gt; Seedling

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	<li>Arsovski, A. A., Galstyan, A., Guseman, J. M., Nemhauser, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photomorphogenesis.">Photomorphogenesis.</a> Arabidopsis Book. 10, e0147 (2012).</li><li>Reinbothe, S., Reinbothe, C., Apel, K., Lebedev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+chlorophyll+biosynthesis--the+challenge+to+survive+photooxidation.">Evolution of chlorophyll biosynthesis--the challenge to survive photooxidation.</a> Cell. 86 (5), 703-705 (1996).</li><li>Heyes, D. J., 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=Photocatalysis+as+the+'master+switch'+of+photomorphogenesis+in+early+plant+development.">Photocatalysis as the 'master switch' of photomorphogenesis in early plant development.</a> Nat Plants. 7 (3), 268-276 (2021).</li><li>Hu, X. Y., Tanaka, A., Tanaka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+extraction+methods+that+prevent+the+artifactual+conversion+of+chlorophyll+to+chlorophyllide+during+pigment+isolation+from+leaf+samples.">Simple extraction methods that prevent the artifactual conversion of chlorophyll to chlorophyllide during pigment isolation from leaf samples.</a> Plant Methods. 9 (1), 19 (2013).</li><li>Chazaux, M., Schiphorst, C., Lazzari, G., Caffarri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+estimation+of+chlorophyll+a,+b+and+carotenoid+content+by+deconvolution+of+the+absorption+spectrum+and+new+simultaneous+equations+for+chl+determination.">Precise estimation of chlorophyll a, b and carotenoid content by deconvolution of the absorption spectrum and new simultaneous equations for chl determination.</a> Plant J. 109 (6), 1630-1648 (2022).</li><li>Marr, I. 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S., Grimm, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+function+of+tetrapyrrole+biosynthesis+in+plants+and+algae.">Regulation and function of tetrapyrrole biosynthesis in plants and algae.</a> Biochim Biophys Acta. 1847 (9), 968-985 (2015).</li><li>Pipitone, 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+multifaceted+analysis+reveals+two+distinct+phases+of+chloroplast+biogenesis+during+de-etiolation+in+arabidopsis.">A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in arabidopsis.</a> eLife. 10, e62709 (2021).</li><li>Stirbet, A. 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Z.B. and K.P. are employees of the PSI and Martin Trtilek is CEO and owner of the PSI company producing the iReenCAM. All these authors were involved in the development of the instrument as previously described7.

Critical steps of the protocol and troubleshooting - no light and take care of the mask 
As highlighted directly in the protocol description above, avoiding even the trace amounts of light both during cultivation of etiolated plants seedlings or just before starting the protocol is of critical importance11. In our setup, we use a dedicated dark chamber located in the walk-in phytotron and separated from the rest of the phytotron with light-tight rotating door (Supplem...

De-etiolation represents the most dramatic phase in the plant life cycle, characterized by a number of morphological changes and complete rearrangement of plant metabolism (from hetero- to auto-tropic)1. Chlorophyll biosynthesis is a hallmark of light-induced de-etiolation in plants and a very dynamic process. Formation of chlorophyll from dark-produced precursor protochlorophyllide must be tightly coordinated to avoid damage due to reactive byproducts2. The protochlorophyllide reduction to chlorophyllide is catalyzed by light-dependent protochlorophyllide oxidoreductases (PORs), unique enzymes activated directly by light. The reaction is very fast, taking place in the order of ms to s3, leading to recognizable chlorophyll accumulation within minutes after etiolated seedling irradiation4,5,6. More time (from hours to days) is required for chloroplast biogenesis to establish a fully functional photosynthetic apparatus3.
Various methods exist to analyze chlorophyll content, including high-performance liquid chromatography (HPLC) or spectrophotometry. Usually, these techniques demand the destruction of plant tissue4,5,6, restricting the determination of changes in chlorophyll levels over time. Methods allowing non-invasive chlorophyll kinetics establishment may open a whole new perspective to study plants in diverse aspects ranging from fundamental research questions, such as analyzing the process of chlorophyll synthesis in time and space, to more practical applications, such as assessment of stress tolerance or effect of biostimulants on the chlorophyll kinetics. Considering this, we introduced a system for monitoring chlorophyll formation, iReenCAM7. It incorporates a CCD camera, emission filters, light sources, and a pipeline for automated fluorescence analysis (Figure 1). The main feature of the developed device is high spatial and temporal resolution, outperforming in the parameters used in current approaches, and sufficient sensitivity and specificity when compared with standard analytical methods7.
The non-invasive procedure described here requires minimum reagents and comprises simple steps, allowing to obtain a chlorophyll kinetics profile in living Arabidopsis seedlings during very early stages of de-etiolation. The protocol can be useful for the study of highly dynamic process of chlorophyl synthesis influenced by number of factors, both exogenous (salt, drought, biostimulants, heavy metals, etc.) and endogenous (typically associated with changes in the gene activity) in origin without a need to detach any plant tissue, thus avoiding additional stress.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-benzylaminopurine</td>
			<td>Duchefa Biochemie</td>
			<td>B0904.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Merck</td>
			<td>Z691577</td>
			<td></td>
		</tr>
		<tr>
			<td>Arabidopsis thaliana Col-0 seeds</td>
			<td>NASC collection</td>
			<td>N1092</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultivation chamber</td>
			<td>PSI</td>
			<td></td>
			<td>custom made</td>
		</tr>
		<tr>
			<td>Dimethilsulfoxid</td>
			<td>Thermo Fisher Scientific</td>
			<td>042780.AK</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf single-channeled, variable (100-1000 &#956;L)</td>
			<td>Merck</td>
			<td>EP3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelrite</td>
			<td>Duchefa Biochemie</td>
			<td>G1101</td>
			<td></td>
		</tr>
		<tr>
			<td>iReenCAM device</td>
			<td>PSI</td>
			<td></td>
			<td>custom made/prototype</td>
		</tr>
		<tr>
			<td>Laboratory bottles, with caps (Duran), 100mL</td>
			<td>Merck</td>
			<td>Z305170-10EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar-flow box</td>
			<td>UniGreenScheme</td>
			<td>ITEM-31156</td>
			<td></td>
		</tr>
		<tr>
			<td>Linerless Rubber Splicing Tape, 19 mm width, black, Scotch</td>
			<td>3M Science. Applied to Life</td>
			<td>7000006085</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube, 2 mL with lid, PPT, BRAND</td>
			<td>Merck</td>
			<td>BR780546-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore tape</td>
			<td>3M Science. Applied to Life</td>
			<td>7100225115</td>
			<td></td>
		</tr>
		<tr>
			<td>Osram lumilux green l18w/66</td>
			<td>Ovalamp</td>
			<td>200008833</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri plates - Greiner dishes, square, 120 x 120 x17mm, vented</td>
			<td>Merck</td>
			<td>Z617679-240EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips, 1000 &#956;L, Axygen</td>
			<td>Merck</td>
			<td>AXYT1000B</td>
			<td></td>
		</tr>
		<tr>
			<td>The Plant Screen Data Analyzer software</td>
			<td>PSI</td>
			<td></td>
			<td>delivered as a part of the iReenCAM</td>
		</tr>
	</tbody>
</table>

non-invasive assay for chlorophyll biosynthesis kinetics determination during early stages of arabidopsis de-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Author Spotlight: Non-Invasive High-Resolution Measurement of Chlorophyll Synthesis During De-Etiolation

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll synthesis is a highly dynamic process. Therefore, the ability to measure it with high time and spatial resolution is necessary. We are proposing a highly accurate method that allows us to trace chlorophyll synthesis in vivo during the first hours of de-etiolation.Normally, chlorophyll is measured by high performance liquid chromatography or spectrophotometry. Also, new optical methods are being proposed for chlorophyll measurement. Chlorophyll synthesis is a very fast process that is triggered by minute traces of light in order of seconds.Recent approaches are mostly invasive, require tissue disintegration, making it difficult to accurately quantify the highly dynamic process of chlorophyll biosynthesis as sufficient time resolution, We were able to determine chlorophyll kinetics in living etiolated seedlings after the first pulse of light, and during the next four hours with high time and spatial resolution. The high precision and statistical robustness of our approach allowed us to study the effects of various factors during individual stages of de-etiolation. It is a non-invasive method with high accuracy and spatial resolution, opening new opportunities for further research, including studies of the possible impact of stress or biostimulants.Thanks to its relatively high throughput, it can also be used in forward genetics to clarify the highly complex process of plant de-etiolation.

The typical output obtained using the newly developed procedure in the 4-day-old de-etiolated Arabidopsis seedlings of wild-type (WT), ecotype Columbia-0 (Col-0) is shown in Figure 3. Under control conditions (DMSO-supplemented MS media), the chlorophyll biosynthetic curve starts with an initial burst of the chlorophyll synthesis, in which the protochlorphyllide pool synthesized during the scotomorphogenic phase of the growth, is quickly converted to chlorophyll owing to the light-i...

Watch this Scientific Journal Video about Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation at JoVE.com

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

non-invasive-assay-for-chlorophyll-biosynthesis-kinetics

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JoVE Journal

Biology

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Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

449 Views.  Masaryk University.  Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution. 

High-Resolution Imaging of Chlorophyll Biosynthesis in Arabidopsis Seedling

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