The video demonstrates a pharmacological analysis of dual-colored F1 Arabidopsis thaliana through the treatment of transgenic seedlings with Brefeldin A and wortmannin. It examines ERL-1 trafficking routes, revealing that Brefeldin A blocks ERL-1 trafficking, while wortmannin promotes its transport to multivesicular bodies for degradation.

pharmacological analysis of dual-colored f1 transgenic arabidopsis thaliana

Spatiotemporal Investigation of ERL1 Dynamics via Confocal Imaging

Membrane traffic is a conserved cellular process that distributes membrane components (also known as cargoes), including proteins, lipids, and other biological products, between different organelles within a eukaryotic cell or across the plasma membrane to and from the extracellular space1. This process is facilitated by a collection of membranes and organelles named the endomembrane system, which consists of the nuclear membrane, the endoplasmic reticulum, the Golgi apparatus, the vacuole/lysosomes, the plasma membrane, and multiple endosomes1. The endomembrane system enables the modification, packaging, and transport of membrane components using dynamic vesicles that shuttle between these organelles. Membrane trafficking events are crucial to cell development, growth, and environmental adaptation and, thus, are under stringent and complex regulation2. Currently, multiple approaches in molecular biology, chemical biology, microscopy, and mass spectrometry have been developed and applied to the field of membrane trafficking and have greatly advanced the understanding of the spatiotemporal regulation of the endomembrane system3,4. Molecular biology is used for classical genetic manipulations of the putative players involved in membrane trafficking, such as altering the gene expression of the protein of interest or labeling the protein of interest with certain tags. Tools in chemical biology include the usage of molecules that specifically interfere with the traffic of certain routes4,5. Mass spectrometry is powerful for identifying the components in an organelle that has been mechanically isolated by biochemical approaches3,4. However, membrane traffic is a dynamic, diverse, and complex biological process1. To visualize the membrane trafficking process in live cells under various conditions, light microscopy is an essential tool. Continuous progress has been made in advanced microscope techniques to overcome the challenges in measuring the efficiency, kinetics, and diversity of the events4. Here, this study focuses on the widely adopted methodologies in chemical/pharmacological biology, molecular biology, and microscopy to study membrane trafficking events in a naturally simplified and experimentally accessible system, the stomatal developmental process.
Stomata are micropores on plant aerial surfaces that open and close to facilitate gas exchange between the inner cells and the environment6,7,8. Hence, stomata are essential for photosynthesis and transpiration, two events that are crucial for plant survival and growth. Stomatal development is dynamically adjusted by environmental cues to optimize the plant&#39;s adaptation to the surroundings9. Dating back to studies in 2002, the identification of the receptor protein Too Many Mouths (TMM) opened the door to a new era of investigating the molecular mechanisms of stomatal development in the model plant Arabidopsis thaliana10. After just a few decades, a classical signaling pathway has been identified. From upstream to downstream, this pathway includes a group of secretory peptide ligands in the epidermal patterning factors (EFP) family, several cell-surface leucine-rich-repeat (LRR) receptor kinases in the EREECTA (ER) family, the LRR receptor protein TMM, a MAPK cascade, and several bHLH transcription factors including SPEECHLESS (SPCH), MUTE, FAMA, and SCREAM (SCRM)11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26. Previous work indicates that one of the receptor kinases, ER-LIKE 1 (ERL1), demonstrates active subcellular behaviors upon EPF perception20. ERL2 also dynamically traffics between the plasma membrane and some intracellular organelles27. Blocking the membrane trafficking steps causes abnormal stomatal patterning, resulting in stomatal clusters on the leaf surface28. These results suggest that membrane traffic plays an essential role in stomatal development. This study describes a protocol to spatiotemporally investigate the ERL1 dynamics using protein-protein subcellular co-localization analysis combined with pharmacological treatment using some membrane trafficking inhibitors.

Author Spotlight: Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells  

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

This technique studies the membrane trafficking events in stomatal development, the micropores on plant aerial surface, to facilitate photosynthesis and the transpiration. Various techniques are used to study plant stomatal development. This includes molecular biology, cell biology or microscopy, chemical biology, mass spectrometry, biochemistry, and structural biology.Individual stomata form asynchronously. Each stoma is formed by three transcend cell fate transitions. So collecting enough stomata lineage cells at a particular cell fate is one of the big challenges.Membrane trafficking is essential for cell survival and growth. Stomata lineage cell is no exception. This protocol addresses the commonly used approach to study membrane trafficking events in stomata lineage cells.Plants face challenging environment in the face of climate change. Stomata are passages for plant gas exchange, and are highly flexible to environmental stress for adaptation. We are interested in studying how abiotic stress affects the stomata development by focusing on the membrane trafficking of certain proteins in stomata lineage cells.

Watch this Scientific Journal Video about Pharmacological Analysis of Dual-Colored F1 Transgenic Arabidopsis thaliana at JoVE.com

Pharmacological Analysis of Dual-Colored F1 Transgenic Arabidopsis thaliana   

Pharmacological Analysis of Dual-Colored F1 Transgenic Arabidopsis thaliana

To begin the pharmacological analysis of dual-colored F1 Arabidopsis thaliana, dissect and remove the cotyledons of seven-day old transgenic seedlings. Add 500 microliters of a 30-micromolar Brefeldin A, or BFA solution, in a 1.5-milliliter micro-centrifuge tube. Immerse the dissected seedlings in it.Tightly attach a 10-milliliter syringe to the micro-centrifuge tube and apply a vacuum for one minute. Remove the syringe before incubating the sample in the solution for 30 minutes before imaging. Next, immerse the dissected seven-day old seedling into 25-millimolar wortmannin.Apply the vacuum for one minute before incubating the immersed sample for 30 minutes. Examining ERL-1 trafficking routes in transgenic plants showed that 30 minutes of exposure to Brefeldin A solution resulted in ERL-1-YFP detection in large compartments known as BFA bodies. It indicated that Brefeldin A could block ERL-1 trafficking suggesting recycling or endocytosis of ERL-1.When transgenics were treated with wortmannin for 30 minutes, ring-like structures called WM bodies were highlighted by ERL-1-YFP. It showed that ERL-1-YFP were transported to multivesicular bodies indicating that ERL-1 followed the route to vacuoles for degradation.

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Research

JoVE Journal

Biology

44 vistas.  Rutgers University. Para comenzar el análisis farmacológico de Arabidopsis thaliana F1 de dos colores, diseccione y retire los cotiledones de plántulas transgénicas de siete días de edad. Agregue 500 microlitros de una solución de Brefeldina A o BFA de 30 micromolares en un tubo de microcentrífuga de 1,5 mililitros. Sumerja las plántulas disecadas en él.Conecte firmemente una jeringa de 10 mililitros al tubo de microcentrífuga y aplique una aspiradora durante un minuto. Retire la jeringa antes d...