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 steri...

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

<ol>
	<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. L., Suryana, N., Lukulay, P., Marr, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+chlorophyll-a+and+chlorophyll-b+by+simultaneous+multicomponent+spectrophotometry.">Determination of chlorophyll-a and chlorophyll-b by simultaneous multicomponent spectrophotometry.</a> Fresenius J Anal Chem. 352 (5), 456-460 (1995).</li><li>Balakhonova, V., 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=Ireencam:+Automated+imaging+system+for+kinetic+analysis+of+photosynthetic+pigment+biosynthesis+at+high+spatiotemporal+resolution+during+early+deetiolation.">Ireencam: Automated imaging system for kinetic analysis of photosynthetic pigment biosynthesis at high spatiotemporal resolution during early deetiolation.</a> Front Plant Sci. 14, 1093292 (2023).</li><li>Virgin, H. I., Kahn, A., Vonwettstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+chlorophyll+formation+in+relation+to+structural+changes+in+chloroplasts.">The physiology of chlorophyll formation in relation to structural changes in chloroplasts.</a> Photochem Photobiol. 2 (2), 83-91 (1963).</li><li>Reinbothe, C., 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=Chlorophyll+biosynthesis:+Spotlight+on+protochlorophyllide+reduction.">Chlorophyll biosynthesis: Spotlight on protochlorophyllide reduction.</a> Trends Plant Sci. 15 (11), 614-624 (2010).</li><li>Kobayashi, K., Masuda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+tetrapyrrole+biosynthesis+in+arabidopsis+thaliana.">Transcriptional regulation of tetrapyrrole biosynthesis in arabidopsis thaliana.</a> Front Plant Sci. 7, 1811 (2016).</li><li>Wang, Y., Folta, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+green+light+to+plant+growth+and+development.">Contributions of green light to plant growth and development.</a> Am J Bot. 100 (1), 70-78 (2013).</li><li>Brzezowski, P., Richter, A. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+relation+between+the+kautsky+effect+(chlorophyll+a+fluorescence+induction)+and+photosystem+ii:+Basics+and+applications+of+the+ojip+fluorescence+transient.">On the relation between the kautsky effect (chlorophyll a fluorescence induction) and photosystem ii: Basics and applications of the ojip fluorescence transient.</a> J Photochem Photobiol B-Biol. 104 (1-2), 236-257 (2011).</li><li>Kupper, H., 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=Analysis+of+ojip+chlorophyll+fluorescence+kinetics+and+q(a)+reoxidation+kinetics+by+direct+fast+imaging.">Analysis of ojip chlorophyll fluorescence kinetics and q(a) reoxidation kinetics by direct fast imaging.</a> Plant Physiol. 179 (2), 369-381 (2019).</li><li>Avin-Wittenberg, 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=Global+analysis+of+the+role+of+autophagy+in+cellular+metabolism+and+energy+homeostasis+in+arabidopsis+seedlings+under+carbon+starvation.">Global analysis of the role of autophagy in cellular metabolism and energy homeostasis in arabidopsis seedlings under carbon starvation.</a> Plant Cell. 27 (2), 306-322 (2015).</li><li>K&#243;sa, A., Preininger, &. #. 2. 0. 1. ;., B&#246;ddi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+deficiency+hinders+etioplast+development+in+stems+of+dark-grown+pea+(pisum+sativum)+shoot+cultures.">Nitrogen deficiency hinders etioplast development in stems of dark-grown pea (pisum sativum) shoot cultures.</a> Physiol Plant. 155 (3), 330-337 (2015).</li><li>Liang, Y., 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+nondestructive+method+to+estimate+the+chlorophyll+content+of+arabidopsis+seedlings.">A nondestructive method to estimate the chlorophyll content of arabidopsis seedlings.</a> Plant Met. 13 (1), 26 (2017).</li><li>P&#233;rez-Patricio, M., 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=Optical+method+for+estimating+the+chlorophyll+contents+in+plant+leaves.">Optical method for estimating the chlorophyll contents in plant leaves.</a> Sensors. 18 (2), 650 (2018).</li><li>Spyroglou, I., 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=Mixed+models+as+a+tool+for+comparing+groups+of+time+series+in+plant+sciences.">Mixed models as a tool for comparing groups of time series in plant sciences.</a> Plants (Basel). 10 (2), (2021).</li><li>Tanaka, R., Kobayashi, K., Masuda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrapyrrole+metabolism+in+arabidopsis+thaliana.">Tetrapyrrole metabolism in arabidopsis thaliana.</a> The Arabidopsis book / Am Soc Plant Biol. 9. 9, e0145 (2011).</li><li>De Wit, M., Galvao, V. C., Fankhauser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-mediated+hormonal+regulation+of+plant+growth+and+development.">Light-mediated hormonal regulation of plant growth and development.</a> Annu Rev Plant Biol. 67, 513-537 (2016).</li><li>Liu, X., Li, Y., Zhong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+light+and+plant+hormones+in+the+control+of+arabidopsis+seedling+chlorophyll+biosynthesis.">Interplay between light and plant hormones in the control of arabidopsis seedling chlorophyll biosynthesis.</a> Front Plant Sci. 8, 1433 (2017).</li></ol>

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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Biology

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

Chlorophyll Fluorescence Measurement and Analysis in Arabidopsis Seedlings Using the iReenCAM System

Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

423 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

Summary