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

Imaging ad alta risoluzione della biosintesi della clorofilla nella piantina di 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.

Autore in primo piano: Misurazione non invasiva ad alta risoluzione della sintesi della clorofilla durante la de-eziolazione

Test non invasivo per la determinazione della cinetica della biosintesi della clorofilla durante le prime fasi della deeziolazione dell'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.

Misurazione e analisi della fluorescenza della clorofilla nelle piantine di Arabidopsis utilizzando il sistema iReenCAM

Per iniziare, accendi il sistema iReenCAM e assicurati che il sistema sia configurato correttamente nel software del server PS avviato automaticamente. Attivare il software di pianificazione, quindi fare clic su Esperimenti, seguito da Nuovo esperimento, per creare il piano sperimentale per la misurazione. Specificare un nome descrittivo per l'esperimento e inserire i dettagli.Fare clic su Aggiungi azione per pianificare la misurazione di una piastra e controllare le date per la misurazione. Fare clic su Genera elenco per definire gli intervalli di tempo tra i cicli di misurazione. Scegli l'ora di inizio e di fine per ogni round e gli intervalli tra i round.Dopo aver fatto clic su Genera, esaminare l'intervallo di tempo e gli intervalli tra gli impulsi luminosi dall'elenco generato sul lato sinistro dello schermo. Scegliere il protocollo di misura e salvare tutte le modifiche nel database per riferimento futuro. Poco prima della misurazione, rimuovere con cautela il foglio di alluminio che copre la piastra di Petri contenente le piantine di arabidopsis di quattro giorni.Posizionare la piastra orizzontalmente all'interno della camera di misurazione. Sulla base della data e dell'ora precedentemente impostate per la misurazione, gli impulsi luminosi attinici vengono indotti automaticamente nella camera. Controlla se il primo round viene eseguito a tempo debito nello scheduler.Il round eseguito dovrebbe avere lo stato OK"e gli altri hanno In sospeso. L'imaging fluorescente delle piantine per le misurazioni della clorofilla verrà generato automaticamente come risultato di ogni ciclo eseguito.

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 ...

Ricerca

Biologia

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

Thaliana di Arabidopsis Sterilizzazione e coltivazione della superficie del seme in camera di crescita

Data Extraction and Analysis of Chlorophyll Fluorescence in Arabidopsis Seedlings

Estrazione e analisi dei dati della fluorescenza della clorofilla nelle piantine di Arabidopsis