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.

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 microcentrifuge tube. Immerse the dissected seedlings in it.

Tightly attach a 10-milliliter syringe to the microcentrifuge 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 ERL1 trafficking routes in transgenic plants showed that 30 minutes of exposure to brefeldin A solution resulted in ERL1-YFP detection in large compartments known as BFA bodies. It indicated that brefeldin A could block ERL1 trafficking suggesting recycling or endocytosis of ERL1.

When transgenics were treated with wortmannin for 30 minutes, ring-like structures called Wm bodies were highlighted by ERL1-YFP. It showed that ERL1-YFP were transported to multivesicular bodies, indicating that ERL1 followed the root to vacuoles for degradation. 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 microcentrifuge tube. Immerse the dissected seedlings in it. Tightly attach a 10-milliliter syringe to the microcentrifuge 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 ERL1 trafficking routes in transgenic plants showed that 30 minutes of exposure to brefeldin A solution resulted in ERL1-YFP detection in large compartments known as BFA bodies. It indicated that brefeldin A could block ERL1 trafficking, suggesting recycling or endocytosis of ERL1. When transgenics were treated with wortmannin for 30 minutes, ring-like structures called Wm bodies were highlighted by ERL1-YFP.

It showed that ERL1-YFP were transported to multivesicular bodies, indicating that ERL1 followed the root to vacuoles for degradation. To begin the sample preparation, dissect the true leaf from the pharmacologically treated Arabidopsis thaliana transgenic sample using a sharp razor blade. Place the true leaf on a glass slide in a drop of water with the abaxial side up.

Slowly cover the true leaf with a cover slip without air bubbles. For imaging, set up the beam path by selecting a 514-nanometer laser for yellow fluorescent protein or YFPX citation. Obtain high-image quality by using high laser power.

Turn on PMT or HyD and define the upper and lower emission band thresholds based on the spectrum for the YFP fluorophore. Set a detection window of 530 to 570 nanometers to collect the YFP signal. For the sequential image of the second color, click on the Seq button and add a new channel.

In Seq 2, select a 555 nanometer laser for the red fluorescent protein or RFP excitation and set a detection window of 570 to 630 nanometers to collect the RFP signal. Select a 63x/1.2 W Corr lens on the system for imaging the subcellular membrane traffic activity within stomatal precursor cells. Start the format with an image size of 1024 by 1024 pixels.

Then, optimize it based on the zoom factor and the objective settings for a good resolution. Next, set up the speed of the scan head. Starting with a speed of 400 hertz, and optimize this based on the specific situation of the samples.

To zoom on a region of interest without changing the objective lens, enter a zoom factor of one and optimize this based on the specific needs of the experiment. The line average refers to the number of times each X-line will be scanned to obtain an average result. Start with a two X-line average for imaging the membrane trafficking events and optimize as needed.

Select the xyt scan mode in the acquisition mode to enable the time series utility. Next, select the waiting period between the time points under Time Interval. Define the number of frames to be collected by selecting Frames.

Use the Z-Stack scan mode with the maximum intensity projection to collect the three dimensional information regarding the membrane trafficking event within the entire cell. Then select the xyz scan mode in acquisition mode, and then the Z-Stack utility will become accessible. Select the Z-Wide option.

Under the scan mode, define the Z-Stack’s top and bottom images using the Begin and End buttons. Next, define the thickness of the Z-Step by clicking on the Z-Step Size. To process the image, click on the primary Process tab in confocal software.

Under the Open projects tab on the far left side, select the interested Z-Stack file. Then, in the middle tab named ProcessTools, select the Projection function. Choose Maximum in the dropdown panel of method and click on the Apply button.

A Z-Stack projection image will be generated under the Open projects tab. To generate the video from the Time Series images, click the File tab in Fiji software and Open function to open all the Time Series images one by one in the correct order. Alternatively, drag the images onto Image J to open the data in the correct order.

Then, find the Stack function in the Image tab and choose Images to Stack to generate a video. Once done, save the file in the avi format with the desired frame rate. In the true leaf of seven-day-old transgenic plants bearing ERL1 promoter ERL1-YFP, scattered cells were highlighted by the YFP signal where ERL1-YFP labeled the plasma membrane.

Multiple punctate signals were also observed, suggesting that ERL1 was also localized to the endosomes. RFP-Ara7 highlighted multiple punctate signals in almost all the cells, indicating the multivesicular bodies or MVBs in these cells. When RFP-Ara7 merged with the ERL1-YFP signal, most punctate overlapped, suggesting that some ERL1-YFP molecules were transported to the MVBs.

Time series of true leaves bearing ERL1-YFP and MVB marker protein, RFP-Ara7 showed that YFP labeled endosomes moved together with RFP-Ara7 within the stomatal lineage cells. It confirmed that ERL1-YFP was localized to the multivesicular bodies.