arabidopsis thaliana seed surface sterilization and cultivation in growth chamber

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.

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

Para começar, adicione as quantidades necessárias de sementes de arabidopsis thaliana Columbia zero em um tubo de microcentrífuga de dois mililitros. Para esterilização superficial das sementes, adicionar etanol a 70% ao tubo por dois minutos e agitar suavemente o tubo. Após a retirada do etanol, lave as sementes com água estéril por cinco minutos.Retire a água do tubo e deixe as sementes sedimentarem para o fundo do tubo por gravidade antes de remover a água restante. Para criar uma suspensão de água de sementes, adicione um volume igual de água estéril ao tubo que contém as sementes. Usando uma ponta de pipeta larga, distribua uniformemente aproximadamente 30 a 40 sementes seguidas em cada área na placa média seguindo uma grade de semeadura.Deixe a água secar nas sementes por 30 minutos. Sele a placa com fita adesiva de micropore e envolva-a em papel alumínio. Estratificar as sementes por três dias a quatro graus Celsius no escuro para superar a dormência e promover germinação uniforme.Em seguida, desembrulhe o papel alumínio da placa e transfira a placa com sementes estratificadas para a luz branca por uma hora para induzir a germinação. Embrulhe a placa com papel alumínio após o tratamento leve para proteger as sementes da luz. Coloque a placa em posição vertical em uma câmara de crescimento e cultive-a por quatro dias no escuro a 21 graus Celsius.

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

Research

JoVE Journal

Biology

Arabidopsis thaliana Seed Surface Sterilization and Cultivation in Growth Chamber

79 visualizações.  Masaryk University. Para começar, adicione as quantidades necessárias de sementes de arabidopsis thaliana Columbia zero em um tubo de microcentrífuga de dois mililitros. Para esterilização superficial das sementes, adicionar etanol a 70% ao tubo por dois minutos e agitar suavemente o tubo. Após a retirada do etanol, lave as sementes com água estéril por cinco minutos.Retire a água do tubo e deixe as sementes sedimentarem para o fundo do tubo por gravidade antes de remover a água restante. Para c...

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

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 ...