chlorophyll fluorescence measurement and analysis in arabidopsis seedlings using the ireencam system

Beeldvorming met hoge resolutie van de biosynthese van chlorofyl in Arabidopsis-zaailing

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.

Auteur in de schijnwerpers: niet-invasieve meting met hoge resolutie van chlorofylsynthese tijdens de-etiolatie

Niet-invasieve test voor bepaling van de kinetiek van chlorofylbiosynthese tijdens vroege stadia van de-etiolatie van Arabidopsis

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.

Meting en analyse van chlorofylfluorescentie in Arabidopsis-zaailingen met behulp van het iReenCAM-systeem

Schakel om te beginnen het iReenCAM-systeem in en zorg ervoor dat het systeem correct is geconfigureerd in de automatisch gestarte PS-serversoftware. Activeer de planningssoftware en klik vervolgens op Experimenten, gevolgd door Nieuw experiment, om het experimentele plan voor de meting te maken. Geef een beschrijvende naam op voor het experiment en vul de details in.Klik op Actie toevoegen om de meting van één plaat te plannen en de datums voor de meting te controleren. Klik op Lijst genereren om de tijdsintervallen tussen meetrondes te definiëren. Kies de start- en eindtijd voor elke ronde en de intervallen tussen de rondes.Nadat u op genereren hebt geklikt, bekijkt u het tijdsbestek en de intervallen tussen lichtimpulsen in de lijst die aan de linkerkant van het scherm wordt gegenereerd. Kies het meetprotocol en sla alle wijzigingen op in de database voor toekomstig gebruik. Verwijder vlak voor de meting voorzichtig de aluminiumfolie die de petriplaat met de vier dagen oude arabidopsis-zaailingen bedekt.Plaats de plaat horizontaal in de meetkamer. Op basis van de datum en tijd die eerder voor de meting zijn ingesteld, worden automatisch actinische lichtpulsen in de kamer geïnduceerd. Controleer of de eerste ronde op tijd wordt uitgevoerd in de planner.De uitgevoerde ronde moet de status OK hebben"en andere hebben In behandeling. Fluorescerende beeldvorming van zaailingen voor chlorofylmetingen wordt automatisch gegenereerd als resultaat van elke uitgevoerde ronde.

Research

JoVE Journal

Biology

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

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 ...

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

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Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

Gegevensextractie en analyse van chlorofylfluorescentie in Arabidopsis-zaailingen