The key goal in developmental biology is to understand the context-specific role of a gene. And this can be difficult to achieve by conventional knockout mutants or by constitutive overexpression lines. We used the modular green gate cloning system to generate a resource for inducible cyto-specific expression in the three main meristems of the model plant Arabidopsis thaliana.
Using the same modular cloning strategy, this method can be applied to other plant species that are immutable to transformation. Visual demonstration of this procedure is valuable because analyzing the effect of trans-activation in the meristems of the stem and the short apex requires dissection before imaging, which can be quite challenging. To begin, sterilize the seeds as outlined in the text protocol.
Next, prepare half-strength Murashige and Skoog medium at pH 5.8 and add one percent sucrose and 0.9%agar. After autoclaving, add Dex dissolved in DMSO to the induction plates at a final concentration between 10 and 30 micromolar. Add an equal amount of DMSO to the control plates.
Put the seeds for root imaging on plates and stratify them for 48 hours in darkness and at four degrees Celsius. Put the plates in a vertical position in a plant incubator and grow them for five days. Five days after germination, use confocal laser scanning microscopy to image the seedlings.
To begin, prepare and grow the seeds as outlined in the text protocol. Six to seven days after germination, transfer each seedling to soil in a separate pot. If inducing the plants by watering, use a 25 micromolar DEX solution in water, prepared from a stock of 25 millimolar DEX dissolved in ethanol.
Water every two to three days until the desired time of induction. If inducing by dipping, prepare a one-liter beaker with 750 milliliters of water containing 0.02%Silwet L77 and DEX at 25 micromolar final concentration or an equivalent amount of DMSO for the DEX and mock treatment. Dip a single plant into the induction or mock solution for 30 seconds.
Repeat every two to three days, until the desired time of imaging. To begin, prepare and grow the seeds as outlined in the text protocol. Six to seven days after germination, transfer each seedling to soil in a separate pot.
When the stem is around one centimeter long, spray the influorescent SAM with between 10 and 50 micromolar DEX solution in water. For induction and imaging at later stages of development, induce SAMs of longer stems or side shoots. 24 to 48 hours after induction, dissect the SAMs and proceed to imaging.
First, transfer the seedlings from the plate to a 10 microgram per millileter solution of propidium iodide and counterstain them for five minutes. Place the roots into a microscope imaging chamber and image them using a confocal laser scanning microscope with a 63x water immersion objective. To visualize propidium iodide fluorescence, use an excitation wavelength of 488 nanometers and collect emission between 590 and 660 nanometers.
For mTurquoise2 fluorescence, use 458 nanometer excitation and collect emission between 460 and 615 nanometers using sequential scanning. First, fix a stem with a finger on the opposite side of the desired section. Using a razor blade, cut a segment of approximately three centimeters and perform several fine cuts.
Rinse the razor blade in a Petri dish containing tap water and collect the stem sections. Either stain the sections or directly mount them onto microscope slides. For staining, prepare one milliliter of a 250 microgram per milliliter solution of propidium iodide in a reaction tube.
Remove the tap water from the Petri dish with a pipette and replace it with the propidium iodide solution and stain for five minutes. Then, remove the propidium iodide solution and rinse with water. Using either fine forceps or a fine paintbrush, transfer the stained sections to microscope slides.
Use a confocal laser scanning microscope with a 25x dipping water immersion lens to image the samples. Use a 561 nanometer laser light to excite propidium iodide fluorescence and collect emission from 570 to 620 nanometers. Use a 405 nanometer laser light to excite the mTurquoise2 fluorophore effector encoded by the driver line constructs and collect emission from 425 to 475 nanometers.
First, use forceps to cut the stem two centimeters below the shoot tip. Hold the stem in one hand and use fine forceps to remove the flower buds and large primordia. To remove young primordia, fix the SAM in an upright position in a Petri dish containing three percent agarose.
Next, use a binocular and forceps to remove any young primordia close to the SAM. Transfer the dissected SAM to a tube containing a 250 microgram per milliliter solution of propidium iodide. Let the sample stain for five to 10 minutes while making sure the sample stays fully submerged during the staining procedure.
Place the stained SAM into a small Petri dish containing three percent agarose medium. Cover the SAM with double distilled water. Use a confocal laser scanning microscope equipped with a 25x dipping water immersion lens to image the dissected SAMs.
Use a 561 nanometer laser light to excite propidium iodide fluorescence and collect emissions from 570 to 620 nanometers. Use a 405 nanometer laser light to excite the mTurquoise2 fluorophore and collect emission from 425 to 475 nanometers. Record image stacks spanning 50 micrometers in the Z direction with a step size of 0.5 micrometers.
Induction with DEX leads to cell-type specific mTurquoise2 expression in the root endodermis the phloem precursors and cambium, and the stem cells in the shoot apical meristem. As a test case for transactivation, an effector line encoding the secondary cell wall master transcription factor VND7 fused to the V16 activation domain, is generated. After five days of single treatment with either 15 microliters of DEX or DMSO, stem sections are prepared for the visualization of the ectopic lignification in the start sheath.
The start sheath cells in induced samples show a strong signal for the propidium iodide channel and some cells show the typical reticulate thickening of the cell wall in xylem cells. After watching this video, you should have a clear understanding of how to induce gene expression in different tissues and how to prepare your samples for imaging. Following this procedure, the established driver lines can be used with a wide variety of effector constructs generated by other users according to their research interests.
This shortcut should help plant researchers to rapidly assess the effect of cell-type specific expression or knockdown of a gene of interest in a time reserved manner. This cell-type specific induction system requires the use of the corticosteroid dexamethasone. Direct contact should be avoided, so it is important to wear gloves and a face mask during treatment.
