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

Arabidopsis 묘목에서 엽록소 생합성의 고해상도 이미징

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.

저자 스포트라이트: De-etiolation 중 엽록소 합성의 비침습적 고분해능 측정

애기장대 탈에티올화의 초기 단계 동안 엽록소 생합성 역학 측정을 위한 비침습적 분석

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 시스템을 사용한 애기장대 묘목의 엽록소 형광 측정 및 분석

시작하려면 iReenCAM 시스템을 켜고 자동으로 시작된 PS 서버 소프트웨어에서 시스템이 올바르게 구성되었는지 확인하십시오. 스케줄러 소프트웨어를 활성화한 다음 Experiments(실험), New Experiment(새 실험)를 차례로 클릭하여 측정을 위한 실험 계획을 생성합니다. 실험에 대한 설명이 포함된 이름을 제공하고 세부 정보를 입력합니다.Add Action(작업 추가)을 클릭하여 한 플레이트 측정을 예약하고 측정 날짜를 확인합니다. 목록 생성(Generate List)을 클릭하여 라운드 측정 사이의 시간 간격을 정의합니다. 각 라운드의 시작 및 종료 시간과 라운드 사이의 간격을 선택합니다.생성을 클릭한 후 화면 왼쪽에 생성된 목록에서 조명 임펄스 사이의 시간 프레임과 간격을 검토합니다. 측정 프로토콜을 선택하고 나중에 참조할 수 있도록 모든 수정 사항을 데이터베이스에 저장합니다. 측정 직전에 4일 된 애기장대 묘목이 들어 있는 페트리 플레이트를 덮고 있는 알루미늄 호일을 조심스럽게 제거합니다.플레이트를 측정 챔버 내부에 수평으로 놓습니다. 측정을 위해 이전에 설정된 날짜와 시간에 따라 광선 펄스가 챔버에서 자동으로 유도됩니다. 스케줄러에서 첫 번째 라운드가 적시에 수행되는지 확인합니다.수행된 라운드는 OK" 상태여야 하며 다른 라운드는 보류 중이어야 합니다. 엽록소 측정을 위한 묘목의 형광 이미징은 수행된 각 라운드의 결과로 자동으로 생성됩니다.

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 ...

연구

생물학

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

애기장대(Arabidopsis thaliana) Seed Surface Sterilization and Cultivation in Growth Chamber

Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

애기장대 묘목의 엽록소 형광 데이터 추출 및 분석