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

シロイヌナズナ実生のクロロフィル生合成の高分解能イメージング

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.

著者スポットライト:脱エチオレーション中のクロロフィル合成の非侵襲的高分解能測定

シロイヌナズナ脱etiolationの初期段階におけるクロロフィル生合成速度測定のための非侵襲的アッセイ

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.

iReenCAMシステムを用いた シロイヌナズ ナ苗木のクロロフィル蛍光測定と分析

まず、iReenCAMシステムの電源を入れ、自動的に起動するPSサーバーソフトウェアでシステムが正しく設定されていることを確認します。スケジューラーソフトウェアをアクティブにし、実験、新規実験の順にクリックして、測定の実験計画を作成します。実験のわかりやすい名前を入力し、詳細を入力します。[アクションの追加] をクリックして、1 プレートの測定をスケジュールし、測定の日付を確認します。「リストを生成」(Generate List) をクリックして、測定ラウンド間の時間間隔を定義します。各ラウンドの開始時間と終了時間、およびラウンド間の間隔を選択します。[生成]をクリックした後、画面の左側に生成されたリストから、時間枠と光インパルスの間隔を確認します。測定プロトコルを選択し、後で参照できるようにすべての変更をデータベースに保存します。測定の直前に、生後4日のシロイヌナズナの苗が入ったペトリプレートを覆っているアルミホイルを慎重に取り除きます。プレートを測定チャンバー内に水平に置きます。測定用に事前に設定された日時に基づいて、活性光パルスがチャンバー内に自動的に誘導されます。スケジューラで最初のラウンドが期限内に実行されるかどうかを確認します。実行されたラウンドのステータスはOK」で、他のラウンドは「保留中」です。クロロフィル測定のための苗木の蛍光イメージングは、実施された各ラウンドの結果として自動的に生成されます。

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 ...

生物学

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

シロイヌナズナ 成長チャンバーでの種子表面滅菌と栽培

Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

シロイヌナズナ苗におけるクロロフィル蛍光のデータ抽出と解析

iReenCAMシステムを用いた シロイヌナズ ナ苗木のクロロフィル蛍光測定と分析

iReenCAMシステムを用いたシロイヌナズナ苗木のクロロフィル蛍光測定と分析