The video details the process of manually crossing homozygous transgenic Arabidopsis thaliana plants to produce F1 seeds. It guides on removing older flowers, dissecting a flower to leave the pistil, transferring pollen from another plant, and verifying successful genetic cross by checking for fluorescent protein signals in the seeds.

﻿preparation 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 ﻿Preparation of Dual-Colored F1 Transgenic Arabidopsis thaliana at JoVE.com

Preparation of Dual-Colored F1 Transgenic Arabidopsis thaliana  

﻿Preparation of Dual-Colored F1 Transgenic Arabidopsis thaliana

To begin, grow homozygous transgenic Arabidopsis thaliana plants bearing ERL1-YFP and a red fluorescent protein, or RFP-labeled marker protein RFP ARA7 side-by-side in a growth room until flowering. On the young inflorescence of plant A, remove all the older flowers and calyx, except for one recently opened flower. Carefully remove the younger flowers and floral merostems.In the recently opened flower, remove the sepals, petals, and stamen using a pair of sharp tweezers, leaving only the pistil on the inflorescence. From plant B, take mature stamen from an opened flower and deposit the pollen grains into the stigma of the dissected pistil of plant A.Label this manually crossed flower, indicating its parents and the date of the genetic cross. When the calyx turns yellow or brown, harvest the F1 seeds to check for a successful F1 plant having yellow fluorescent protein, or YFP, and RFP signals.

Research

JoVE Journal

Biology

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...