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

Imagem de alta resolução da biossíntese de clorofila em mudas 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 em destaque: medição não invasiva de alta resolução da síntese de clorofila durante o desestiolamento

Ensaio Não Invasivo para Determinação da Cinética de Biossíntese de Clorofila durante os Estágios Iniciais da Desetiolação 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.

Medição e análise de fluorescência da clorofila em mudas de Arabidopsis utilizando o sistema iReenCAM

Para começar, ligue o sistema iReenCAM e verifique se o sistema está configurado corretamente no software do servidor PS iniciado automaticamente. Ative o software do agendador e clique em Experimentos, seguido de Novo Experimento, para criar o plano experimental para a medição. Forneça um nome descritivo para o experimento e preencha os detalhes.Clique em Adicionar ação para agendar a medição de uma placa e verificar as datas para a medição. Clique em Gerar Lista para definir os intervalos de tempo entre as rodadas de medição. Escolha o horário de início e término de cada rodada e os intervalos entre as rodadas.Depois de clicar em gerar, revise o período de tempo e os intervalos entre os impulsos de luz da lista gerada no lado esquerdo da tela. Escolha o protocolo de medição e salve todas as modificações no banco de dados para referência futura. Imediatamente antes da medição, remova cuidadosamente a folha de alumínio que cobre a placa de Petri contendo as mudas de arabidopsis com quatro dias de idade.Coloque a placa horizontalmente dentro da câmara de medição. Com base na data e hora previamente definidas para a medição, pulsos de luz actínica são induzidos automaticamente na câmara. Verifique se a primeira rodada é realizada em tempo hábil no agendador.A rodada realizada deve ter o status OK" e outras têm Pendente. Imagens fluorescentes de plântulas para medidas de clorofila serão geradas automaticamente como resultado de cada rodada 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 ...

Biologia

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

Arabidopsis thaliana | Esterilização e Cultivo de Superfícies de Sementes em Câmara de Crescimento

Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

Extração de dados e análise da fluorescência da clorofila em mudas de Arabidopsis