arabidopsis thaliana seed surface sterilization and cultivation in growth chamber

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.

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

Per iniziare, aggiungere le quantità necessarie di semi di arabidopsis thaliana Columbia zero in una micro provetta da centrifuga da due millilitri. Per la sterilizzazione superficiale dei semi, aggiungere etanolo al 70% nella provetta per due minuti e agitare delicatamente la provetta. Dopo aver rimosso l'etanolo, lavare i semi con acqua sterile per cinque minuti.Rimuovere l'acqua dal tubo e lasciare che i semi sedimentino sul fondo del tubo per gravità prima di rimuovere l'acqua rimanente. Per creare una sospensione acquosa di semi, aggiungere un volume uguale di acqua sterile alla provetta contenente i semi. Utilizzando un puntale di pipetta largo, distribuire uniformemente circa 30-40 semi in fila in ciascuna area sul piatto medio seguendo una griglia di semina.Lasciare asciugare l'acqua nei semi per 30 minuti. Sigillare la piastra con nastro adesivo microporoso e avvolgerla in un foglio di alluminio. Stratificare i semi per tre giorni a quattro gradi Celsius al buio per superare la dormienza dei semi e favorire una germinazione uniforme.Successivamente, srotola la pellicola dal piatto e trasferisci il piatto con i semi stratificati alla luce bianca per un'ora per indurre la germinazione. Avvolgere il piatto con un foglio di alluminio dopo il trattamento leggero per proteggere i semi dalla luce. Posizionate la piastra in posizione verticale in una camera di crescita e coltivatela per quattro giorni al buio a 21 gradi Celsius.

arabidopsis-thaliana-seed-surface-sterilization-cultivation-growth

Research

JoVE Journal

Biology

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

82 Views.  Masaryk University. Per iniziare, aggiungere le quantità necessarie di semi di arabidopsis thaliana Columbia zero in una micro provetta da centrifuga da due millilitri. Per la sterilizzazione superficiale dei semi, aggiungere etanolo al 70% nella provetta per due minuti e agitare delicatamente la provetta. Dopo aver rimosso l'etanolo, lavare i semi con acqua sterile per cinque minuti.Rimuovere l'acqua dal tubo e lasciare che i semi sedimentino sul fondo del tubo per gravità prima di rimuovere l'acqua ri...

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

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 dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...