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

Hochauflösende Bildgebung der Chlorophyll-Biosynthese in Arabidopsis-Keimlingen

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.

Autor im Rampenlicht: Nicht-invasive hochauflösende Messung der Chlorophyllsynthese während der De-Etiolation

Nicht-invasiver Assay zur Bestimmung der Chlorophyll-Biosynthese-Kinetik in frühen Stadien der 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.

Chlorophyllfluoreszenzmessung und -analyse in Arabidopsis-Sämlingen mit dem iReenCAM-System

Schalten Sie zunächst das iReenCAM-System ein und stellen Sie sicher, dass das System in der automatisch initiierten PS-Server-Software korrekt konfiguriert ist. Aktivieren Sie die Scheduler-Software und klicken Sie dann auf Experimente und dann auf Neues Experiment, um den Versuchsplan für die Messung zu erstellen. Geben Sie einen beschreibenden Namen für das Experiment an, und geben Sie die Details ein.Klicken Sie auf Aktion hinzufügen, um die Ein-Platten-Messung zu planen und die Daten für die Messung zu überprüfen. Klicken Sie auf Liste generieren, um die Zeitintervalle zwischen den Messrunden zu definieren. Wählen Sie die Start- und Endzeit für jede Runde und die Intervalle zwischen den Runden.Nachdem Sie auf Generieren geklickt haben, überprüfen Sie den Zeitrahmen und die Intervalle zwischen den Lichtimpulsen in der generierten Liste auf der linken Seite des Bildschirms. Wählen Sie das Messprotokoll und speichern Sie alle Änderungen in der Datenbank, um später darauf zurückgreifen zu können. Entfernen Sie kurz vor der Messung vorsichtig die Aluminiumfolie, die die Petriplatte mit den vier Tage alten Arabidopsis-Sämlingen bedeckt.Legen Sie die Platte horizontal in die Messkammer. Basierend auf dem zuvor für die Messung eingestellten Datum und der Uhrzeit werden in der Kammer automatisch aktinische Lichtimpulse induziert. Überprüfen Sie, ob die erste Runde rechtzeitig im Scheduler durchgeführt wird.Die durchgeführte Runde sollte den Status "OK" haben und andere haben "Ausstehend". Die Fluoreszenzbildgebung von Keimlingen für Chlorophyllmessungen wird automatisch als Ergebnis jeder durchgeführten Runde generiert.

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 ...

Forschung

Biologie

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

Arabidopsis thaliana Sterilisation und Kultivierung der Saatgutoberfläche in der Wachstumskammer

Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

Datenextraktion und Analyse der Chlorophyllfluoreszenz in Arabidopsis-Sämlingen