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

拟南芥幼苗中叶绿素生物合成的高分辨率成像

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.

作者聚焦：去硫醇化过程中叶绿素合成的无创高分辨率测量

拟南芥脱硫早期叶绿素生物合成动力学测定的非侵入性测定

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.

使用iReenCAM系统对 拟南芥 幼苗进行叶绿素荧光测量和分析

首先，打开iReenCAM系统，并确保在自动启动的PS服务器软件中正确配置系统。激活调度程序软件，然后单击“实验”，然后单击“新建实验”，以创建测量的实验计划。为实验提供描述性名称并填写详细信息。单击“添加操作”（Add Action） 计划单板测量并检查测量日期。单击“生成列表”（Generate List） 以定义测量轮次之间的时间间隔。选择每轮的开始和结束时间以及轮次之间的间隔。单击生成后，查看屏幕左侧生成的列表中光脉冲之间的时间范围和间隔。选择测量方案并将所有修改保存到数据库中以备将来参考。在测量之前，小心地取下覆盖含有四天大拟南芥幼苗的培养皿的铝箔。将板水平放置在测量室内。根据先前为测量设置的日期和时间，在腔室中自动感应光化光脉冲。检查第一轮是否在调度程序中按时执行。执行的回合应具有“OK”状态，而其他回合应具有“待处理”状态。用于叶绿素测量的幼苗荧光成像将作为每一轮的结果自动生成。

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 ...

科研

生物学

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

拟南芥 种子表面灭菌和生长室培养

Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

拟南芥幼苗叶绿素荧光数据提取与分析

使用iReenCAM系统对 拟南芥 幼苗进行叶绿素荧光测量和分析

使用iReenCAM系统对拟南芥幼苗进行叶绿素荧光测量和分析