arabidopsis thaliana seed surface sterilization and cultivation in growth chamber

High-Resolution Imaging of Chlorophyll Biosynthesis in Arabidopsis Seedling

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.

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

Arabidopsis thaliana Sterilisering og dyrkning af frøoverflade i vækstkammer

Til at begynde med tilsættes de nødvendige mængder arabidopsis thaliana Columbia nul frø i et to milliliter mikrocentrifugerør. Til overfladesterilisering af frø tilsættes 70% ethanol til røret i to minutter og rystes forsigtigt røret. Efter fjernelse af ethanol vaskes frøene med sterilt vand i fem minutter.Fjern vandet fra røret og lad frøene sedimentere til bunden af røret ved tyngdekraften, før du fjerner det resterende vand. For at skabe en frøvandssuspension tilsættes et lige stort volumen sterilt vand til røret indeholdende frøene. Brug en bred pipettespids til at fordele ca. 30 til 40 frø jævnt i træk i hvert område på den mellemstore plade efter et sågitter.Lad vandet tørre i frøene i 30 minutter. Forsegl pladen med mikroporetape og pakk den ind i aluminiumsfolie. Stratificere frøene i tre dage ved fire grader celsius i mørket for at overvinde frødvale og fremme ensartet spiring.Pak derefter folien ud af pladen og overfør pladen med stratificerede frø til hvidt lys i en time for at fremkalde spiring. Pak pladen med aluminiumsfolie efter lysbehandlingen for at beskytte frø mod lys. Placer pladen lodret i et vækstkammer, og dyrk den i fire dage i mørke ved 21 grader Celsius.

Research

JoVE Journal

Biology

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

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

Klorofylfluorescensmåling og analyse i arabidopsis-frøplanter ved hjælp af iReenCAM-systemet

Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

Dataekstraktion og analyse af klorofylfluorescens i arabidopsisplanter