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

Imagerie à haute résolution de la biosynthèse de la chlorophylle chez les plantules d’Arabidopsis

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.

Pleins feux sur l’auteur : Mesure non invasive à haute résolution de la synthèse de la chlorophylle pendant la désétiolation

Test non invasif pour la détermination de la cinétique de biosynthèse de la chlorophylle au cours des premiers stades de la désétiolement d’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.

Mesure et analyse de la fluorescence de la chlorophylle chez les semis d’Arabidopsis à l’aide du système iReenCAM

Pour commencer, mettez le système iReenCAM sous tension et assurez-vous qu’il est correctement configuré dans le logiciel serveur PS lancé automatiquement. Activez le logiciel de planification, puis cliquez sur Expériences, puis sur Nouvelle expérience, pour créer le plan expérimental de la mesure. Donnez un nom descriptif à l’expérience et remplissez les détails.Cliquez sur Ajouter une action pour planifier la mesure d’une plaque et vérifier les dates de la mesure. Cliquez sur Générer une liste pour définir les intervalles de temps entre les rondes de mesure. Choisissez l’heure de début et de fin de chaque tour et les intervalles entre les tours.Après avoir cliqué sur générer, vérifiez la période et les intervalles entre les impulsions lumineuses de la liste générée sur le côté gauche de l’écran. Choisissez le protocole de mesure et enregistrez toutes les modifications dans la base de données pour référence future. Juste avant la mesure, retirez soigneusement le papier d’aluminium recouvrant la plaque de Petri contenant les plants d’arabidopsis âgés de quatre jours.Placez la plaque horizontalement à l’intérieur de la chambre de mesure. En fonction de la date et de l’heure préalablement définies pour la mesure, des impulsions lumineuses actiniques sont induites automatiquement dans la chambre. Vérifiez si le premier tour est effectué en temps voulu dans le planificateur.Le tour effectué doit avoir le statut OK et les autres doivent avoir En attente. L’imagerie fluorescente des plantules pour les mesures de chlorophylle sera générée automatiquement à la suite de chaque tour effectué.

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 ...

Recherche

Biologie

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

Arabidopsis thaliana Stérilisation et culture de la surface des semences en chambre de croissance

Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

Extraction et analyse des données de fluorescence de la chlorophylle chez les semis d’Arabidopsis