Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Podsumowanie

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

Streszczenie

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (β-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

Wprowadzenie

The barley aleurone layer is a well-established model system for the study of hormone-regulated gene expression in plants¹. In particular, a number of genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The GA and ABA signaling pathways are intertwined, as the expression of some GA-activated genes is inhibited by ABA, and vice-versa¹.

A valuable strategy for understanding the role of particular actors in GA/ABA signaling has been the introduction of effector gene constructs via particle bombardment, followed by transient expression of reporter constructs that allow the resulting effect on downstream gene expression to be determined. The use of reporter genes such as GUS (β-glucuronidase) or Luciferase allows the sensitive and quantitative measurement of gene expression specifically within the cells that have received the effector construct. For example, the introduction of an effector construct encoding the transcription factor TaABF1^5,6 established that ABA-induced genes such as HVA1 are induced by TaABF1, while GA-induced genes such as Amy32b are repressed. Particle bombardment as an experimental strategy has been used by multiple laboratories to investigate diverse aspects of GA/ABA signaling. Such work has led to the identification of promoter elements important for the activation of both GA-induced² and ABA-induced genes³, and to the discovery of protein kinases⁴ and transcription factors⁵ that regulate the expression of these genes.

The existing protocols^2,3,4,5,6 for particle bombardment and subsequent measurement of transient gene expression are quite labor intensive, as each set of bombarded barely grains is homogenized by hand in a mortar and pestle and the enzyme assays are carried out individually. This manuscript reports an improved protocol that partially automates and streamlines the homogenization step and the GUS assays to allow significantly more throughput, permitting a larger number of treatments to be tested in the same experiment, and/or the inclusion of more replicates for each treatment to obtain more statistically robust results. Representative results are shown for the expression of HVA1 and Amy32b reporter constructs, regulated by the transcription factor TaABF1 as well as by GA, ABA, and other regulatory molecules.

Protokół

1. Preparation of Effector and Reporter Gene Constructs

1. Build effector constructs that employ a constitutive promoter (e.g. maize ubiquitin, UBI) to drive expression from a desired open reading frame. For example, construct a plasmid containing UBI::TaABF1 to drive strong constitutive expression of TaABF1⁵.
2. Build reporter constructs that include the promoter whose activity is to be measured, placed upstream of an open reading frame, such as GUS. For example, construct a plasmid containing HVA1::GUS to measure expression from the HVA1 promoter^2,5.
3. Build an internal control construct (e.g. UBI::luciferase).
4. Using a silica binding column protocol (see Table of Materials), prepare high-quality plasmid for each of the required gene constructs. Using ultraviolet spectroscopy (see Table of Materials), carefully measure the concentration of each plasmid preparation.

2. Preparation of Barley Grains for Bombardment

1. Set up a spreadsheet (see Figure 1 for an example) indicating the plasmids that will be bombarded, the appropriate hormone treatments, and other relevant information for each treatment that will be part of the experiment.
2. Using a cutting board and a sterile razor blade, cut off the embryos from grains of Himalaya barley (40 for each treatment included in the experiment). Exclude grains that are discolored or substantially smaller than average.
3. Place all the embryoless grains into a 50 mL tube. Add 40 mL of water and rock for 10 minutes.
4. Pour out the water carefully (to avoid losing grains) and add 40 mL of 10% bleach. Rock for 20 minutes.
5. Pour out the bleach and add 40 mL of sterile water. Rock for 5 minutes. Repeat this sterile water wash four more times.
6. Add 40 mL of Imbibing Solution (20 mM sodium succinate, 20 mM calcium chloride, pH 5.0) and 40 μL of chloramphenicol stock solution (10 mg/mL).
7. Transfer most of the Imbibing Solution (with chloramphenicol) from the tube into a Vermiculite Plate (see Table of Materials). Then transfer the grains onto the surface of the wet filter paper in the Vermiculite Plate. Spread out the grains evenly. Pour in enough imbibing solution to keep the grains partially (but not completely) submerged. Place the lid on top of the Vermiculite Plate.
8. Incubate the embryoless grains at 24 °C for about 48 h with fluorescent lighting. Add additional Imbibing Solution as necessary.
9. Open up a 90 mm plastic petri dish and pour 20 mL of Imbibing Solution into the dish itself and 20 mL into the inverted lid. Add 20 μL of chloramphenicol (10 mg/mL) to each. Sterilize two pairs of fine point forceps by dipping them in alcohol.
10. Place a few of the embryoless grains into the lid of the petri dish. Using the forceps, gently remove the (transparent) seed coat from each grain. While removing the seed coat from the smooth side of the grain, do not damage the underlying (aleurone) cells. Transfer the peeled grain to the petri dish containing Imbibing Solution.
11. After peeling the seed coat from all of the grains, return them to the Vermiculite Plate. Incubate the peeled embryoless grains at 24 °C for 16 - 20 hours.
12. Just before using the grains for bombardment, remove surface moisture by shaking them between filter papers in a petri dish.

3. Preparation of Plasmid DNA for Bombardment

1. Label a 1.5 mL tube for each treatment that will be included in the bombardment experiment. To each tube add 2.5 µg of UBI::luciferase internal control plasmid, 2.5 µg of a GUS reporter plasmid, and the desired amount (typically 0.025 µg to 2.5 µg) of an effector plasmid. Add water to bring the total volume to 5 µL.
2. Prepare 1.6 µm gold microcarriers (see Table of Materials) following published protocols⁶.
3. For each treatment, add 50 µL of suspended microcarriers into the 1.5 mL tube containing plasmid DNA. Coat the microcarriers with plasmid DNA according to manufacturer's protocols⁶.
4. At the final step, when the plasmid-coated microcarriers are suspended in 48 µL 100% ethanol, pipet 8 μL of suspended microcarriers onto each of five macrocarriers and allow them to air dry.

4. Particle Bombardment of Embryoless Barley Grains

1. Set up the particle bombardment apparatus (see Table of Materials)⁶.
2. Place a 1550 psi rupture disk into the retaining cap and mount it on the instrument.
3. Place a macrocarrier containing the desired plasmid combination (together with a stopping screen) into the microcarrier launch assembly. Insert the assembly into the top slot of the bombardment chamber. Set the distance between the rupture disc retaining cap and the macrocarrier cover lid at the standard distance (6 mm), and place the stopping screen support at the (standard) middle position, allowing a macrocarrier travel distance of 11 mm (Figure 2A).
4. Arrange 8 embryoless grains in a tight radial pattern (with the thinner ends pointing inward) on a filter paper circle that is taped down flat to the lid of a 90 mm petri dish (see Figure 2B).
5. Place the dish with the grains into the bombardment chamber, at a distance of 6 cm from the stopping screen.
6. Evacuate the bombardment chamber to 95 kPa and then bombard the sample.
7. After releasing the vacuum, transfer the bombarded grains to a labeled 60 mm petri dish containing 4 mL of Imbibing Solution containing 1 mg/ml of chloramphenicol.
8. Repeat until all of the bombardments have been completed. Incubate (on a shaking platform at 100 rpm) at 24 °C for 24 hours.

5. Extraction of Soluble Protein from the Bombarded Grains

1. Using forceps, remove the 8 bombarded grains from a 60 mm petri dish and blot them dry on a paper towel. Divide the grains into two labeled 2.0 mL tubes (4 grains per tube) each containing 800 μL Grinding Buffer (100 mM Na phosphate pH 7.2, 5 mM DTT, 10 µg/mL leupeptin, 20% glycerol) and a 5 mm stainless steel bead. Repeat this process for all of bombarded grains.
2. Place (up to 48) 2.0 mL tubes into the two racks of the bead homogenizer (see Table of Materials). Mount the racks on the homogenizer.
3. Shake the samples at 30 Hz for 3 minutes.
4. Remove the homogenizer racks from the homogenizer and place them on ice (5 min) to re-cool the samples.
5. Mount the racks back on the homogenizer - in the opposite orientation. Shake the samples again at 30 Hz for 3 minutes.
6. Cool the racks on ice, then repeat Steps 5.3 to 5.5. This will add up to a total of 12 minutes of shaking.
7. Centrifuge the grain extracts at 16,000 x g at 4 °C for 10 minutes.
8. Immediately after the centrifugation, decant the clear supernatants into a second set of microcentrifuge tubes. Give each tube a quick vortex. Store the tubes on ice.
9. After transferring the supernatant, recover the steel beads from the pellets so they can be washed and reused.

6. Measurement of Luciferase Activity from Grain Extracts

1. For each tube of grain extract to be assayed, prepare a 12 mm x 75 mm glass test tube containing 200 μL of Luciferase Assay Mixture (45 mM Tris sulfate pH 7.7, 15 mM MgCl₂, 20 mM DTT, 1.5 mM EDTA, 1 mM luciferin, 1 mM ATP).
2. Add 100 μL of the first grain extract to the first assay tube. Vortex quickly to mix well. Immediately place the tube into the tube holder of the luminometer and close the door. When the measurement is finished, remove the tube from the luminometer – leaving the door open for placement of the next tube.
3. Repeat this process until all the samples have been measured.

7. Measurement of GUS Activity from Grain Extracts

1. Add 200 μL of GUS Assay Buffer (50 mM Na phosphate pH 7.2, 2.5 mM 4-methylumbelliferyl--D-glucuronide, 10 mM DTT, 2 mM EDTA, 10 µg/mL leupeptin, 20% methanol, 0.02% sodium azide) to each well of a 96 well plate.
2. Place 50 μL of each grain extract into the corresponding well. Mix with the tip after adding. Seal the top with sealing film.
3. Incubate the 96 well plate at 37 °C in the dark for 20 hours.
4. Centrifuge the 96 well plate at 4,000 x g at 4 °C for 10 minutes and place it on ice.
5. Add 250 μL of 0.2 M Na₂CO₃ to each well of a black 96 well plate.
6. Add 6.25 μL of each centrifuged GUS assay mixture into the corresponding well (containing 0.2 M Na₂CO₃) of the black 96 well plate. Mix with the tip after adding. Include wells with 6.25 μL of 10 μM, 40 μM, and 100 μM 4-methylumbelliferone as standards.
7. Place the black plate into a plate fluorometer and read the fluorescence of methylumbelliferone. Take the readings at an excitation wavelength of 360 nm, and an emission wavelength of 460 nm. Set the gain of the instrument such that a well containing 6.25 μL of 40 μM 4-methylumbelliferone and 250 μL of 0.2 M Na₂CO₃ gives a reading of 1000 fluorescence units.

8. Data Analysis

1. Enter the values for the luciferase and GUS assays into a spreadsheet. Exclude from consideration any sample with a luciferase reading of lower than 20,000 relative luminescence units s^-1, as this indicates that the gene constructs were not successfully delivered to the aleurone cells.
2. For each successfully bombarded grain extract (representing a group of four embryoless grains), calculate the normalized GUS expression from the raw GUS and luciferase data as follows: normalized GUS = [(GUS expression - GUS blank) x (2,000,000)] / (luciferase expression – luciferase blank).
   NOTE: This normalizes the amount of GUS activity to what would have been observed in a bombardment that gave a luciferase reading of 2,000,000.
3. Report the relative level of expression of a particular reporter construct in the presence of effector constructs or hormone treatments being tested by comparing to the level of expression in the absence of effectors or hormones.

Wyniki

The technique described here can be used to introduce any test gene construct into aleurone cells of barley grains. The level of expression from the test gene may then be conveniently measured (Figure 2). The higher throughput protocol described here greatly increases the efficiency of the seed grinding and enzyme assay steps. This method has been used to assess the ability of the transcription factor TaABF1^5,

Dyskusje

The introduction of effector gene constructs via particle bombardment, followed by transient expression of reporter constructs is a valuable strategy for dissecting the role of particular actors in GA/ABA signaling and in the resulting hormone-regulated gene expression.
However, existing protocols for carrying out such experiments in barley aleurone cells^2,3,4,5,

Materiały

Name	Company	Catalog Number	Comments
GeneElute HP plasmid Maxiprep kit	Sigma	NA0310-1KT
UV-vis spectrophotometer	Nanodrop	ND-1000
Himalaya barley grains	/	/	A variety of hulless barley (store in the dark at 4° C)
sodium succinate	Sigma	S2378	Reagent for Imbibing Solution
calcium chloride (dihydrate)	Fisher	C79-500	Reagent for Imbibing Solution
Imbibing Solution	home made	/	20 mM sodium succinate, 20 mM calcium chloride, pH 5.0. Sterilize by autoclaving before use.
chloramphenicol	Sigma	C0378	Prepare a 10 mg/mL stock solution in 70% ethanol.
vermiculite	Fisher	NC0430369	Used for vermiculite plates.
filter paper circles (90 mm)	Whatman	1001 090	Used for vermiculite and for pre-bombardment grain preparation
Vermiculite Plates	home made	/	Add 50 mL of vermiculite to a glass petri dish. Place a 90 mm paper circle on top of the vermiculite. Autoclave.
forceps (fine pointed)	Fisher	13-812-42	Used for removing seed coat from barley grains.
forceps (ultra fine point)	Fisher	12-000-122	Used for removing seed coat from barley grains.
gold microcarriers (1.6 μm)	BioRad	1652264
macrocarriers	BioRad	1652335
calcium chloride (dihydrate)	Fisher	C79-500	Prepare a 2.5 M stock solution and store 1 mL aliquots at -20° C.
spermidine	Sigma	S0266	Prepare a 100 mM stock solution and store 500 μL aliquots at -20° C (use within 2 months).
rupture discs (1550 psi)	BioRad	1652331
stopping screens	BioRad	1652336
macrocarrier holders	BioRad	1652322
Biolistic particle delivery system	BioRad	PDS-1000/He
sodium phosphate monobasic monohydrate	Sigma	S9638	Reagent for 1M sodium phosphate pH 7.2
sodium phosphate dibasic	Sigma	S9763	Reagent for 1M sodium phosphate pH 7.2
1M sodium phosphate pH 7.2	home made	/	Combine 6.9 g of sodium phosphate monobasic monohydrate with 7.1 g of sodium phosphate dibasic. Add water to 100 mL. Add NaOH to get pH 7.2.
dithiothrietol (DTT)	Sigma	43819	Dissolve in water to 1 M. Store at -20° in 1 mL aliquots.
leupeptin	Sigma	L2884	Dissolve in water to 10 mg/mL. Store at -20° C.
glycerol	Sigma	G5516	Prepare a 50% solution in water.
Grinding Buffer	home made	/	Combine 10 mL of 1 M sodium phosphate pH 7.2, 500 μL of 1 M DTT, 100 μL of 10 mg/mL leupeptin, and 40 mL of 50% glycerol. Add water to 100 mL.
stainlesss steel beads (5 mm)	Qiagen	69989
2.0 mL tubes	Eppendorf	22363352	This specific model of tube is recommended for use with the homogenizer.
bead homogenizer (TissueLyser)	Qiagen	85210
12mm x 75 mm glass test tubes	Fisher
luciferin	Goldbio	LUCK-100	Prepare a 25 mM stock solution and store 1 mL aliquots at -20° C.
ATP	Sigma	A7699	Prepare a 100 mM stock solution and store 250 μL aliquots at -20° C.
Tris base	Sigma	T1503	Reagent for 1M Tris sulfate pH 7.7.
sulfuric acid	Sigma	258105	Reagent for 1M Tris sulfate pH 7.7.
1M Tris sulfate pH 7.7	home made	/	Dissolve 12.1 g Tris base in 100 mL of water. Adjust pH to 7.7 with sulfuric acid.
magnesium chloride	Sigma	M9397	Dissolve in water to 2 M.
Luciferase Assay Buffer (LAB)	home made	/	Combine 3 mL of 1 M Tris sulfate pH 7.7, 500 μL of 2 M magnesium chloride, 1 mL of 1 M DTT, and 200 μL of 0.5 M EDTA. Add water to 50 mL.
Luciferase Assay Mixture	home made	/	Combine 15 mL of LAB, 800 μL of 25 mM luciferin, 200 μL of 100 mM ATP, and 4 mL of water. This makes enough assay mixture (20 mL) for 100 luciferase assays.
luminometer (Sirius)	Berthold	/
4-methylumbelliferyl-β-D-glucuronide (MUG)	Goldbio	MUG1	Dissolve in DMSO to 100 mM.
sodium azide	Sigma	S8032	Prepare a 2% stock solution in water and store 1 mL aliquots at -20° C.
96 well plates (standard)	Fisher	12565501
GUS assay buffer	home made	/	Combine 2.5 mL of MUG, 5 mL of 1 M sodium phosphate pH 7.2, 400 μL of 0.5 M EDTA, 1 mL of 1 M DTT, 100 μL of 10 mg/ml leupeptin, 20 mL of methanol, and 1 mL of 2% sodium azide. Add water to 100 mL.
TempPlate sealing film	USA Scientific	2921-1000
96 well plates (black)	Costar	3916
sodium carbonate	Sigma	S7795	Prepare a 200 mM solution in water.
4-methylumbelliferone	Sigma	M1381	Prepare a 100 μM solution in water. Freeze 1 mL aliquots at -20° C.
microplate fluouresence reader	Bio-Tek	FLX-800