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

Imágenes de alta resolución de la biosíntesis de clorofila en plántulas de 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.

Autor destacado: Medición no invasiva de alta resolución de la síntesis de clorofila durante la desetiolación

Ensayo no invasivo para la determinación cinética de la biosíntesis de clorofila durante las primeras etapas de la desetiolación de 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.

Medición y análisis de fluorescencia de clorofila en plántulas de Arabidopsis utilizando el sistema iReenCAM

Para comenzar, encienda el sistema iReenCAM y asegúrese de que el sistema esté configurado correctamente en el software del servidor PS iniciado automáticamente. Active el software del programador y, a continuación, haga clic en Experimentos, seguido de Nuevo experimento, para crear el plan experimental para la medición. Proporcione un nombre descriptivo para el experimento y rellene los detalles.Haga clic en Agregar acción para programar la medición de una placa y verificar las fechas de la medición. Haga clic en Generar lista para definir los intervalos de tiempo entre rondas de medición. Elija la hora de inicio y finalización de cada ronda y los intervalos entre las rondas.Después de hacer clic en generar, revise el período de tiempo y los intervalos entre los impulsos de luz de la lista generada en el lado izquierdo de la pantalla. Elija el protocolo de medición y guarde todas las modificaciones en la base de datos para futuras consultas. Justo antes de la medición, retire con cuidado el papel de aluminio que cubre la placa de Petri que contiene las plántulas de arabidopsis de cuatro días de edad.Coloque la placa horizontalmente dentro de la cámara de medición. En función de la fecha y la hora previamente establecidas para la medición, se inducen automáticamente pulsos de luz actínica en la cámara. Compruebe si la primera ronda se realiza a su debido tiempo en el programador.La ronda realizada debe tener el estado OK" y otras tienen Pendiente. Las imágenes fluorescentes de las plántulas para las mediciones de clorofila se generarán automáticamente como resultado de cada ronda realizada.

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 ...

Investigación

Biología

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

Arabidopsis thaliana Esterilización de la superficie de la semilla y cultivo en cámara de crecimiento

Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

Extracción y análisis de datos de fluorescencia de clorofila en plántulas de Arabidopsis