The authors thank Greyson Butler and Margaret Barrett for help in carrying out the experiments, Judy Stone for advice on grain homogenization, and Lynn Hannum for advice on fluorometry. This work was supported by the National Science Foundation (IOB 0443676), by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423, and by grants from the Colby College Division of Natural Sciences.

1. Preparation of Effector and Reporter Gene Constructs
<ol>
	<li>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.</li>
	<li>Build reporter constructs that include the promoter whose activity is to be measured, placed upstream of an open reading frame, such as GUS. For...

<ol>
	<li>Chen, K., An, Y. Q. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+responses+to+gibberellin+and+abscisic+acid+in+barley+aleurone.">Transcriptional responses to gibberellin and abscisic acid in barley aleurone.</a> J. Integ. Plant Biol. 48, 591-612 (2006).</li><li>Lanahan, M. B., Ho, T. H. D., Rogers, S. W., Rogers, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gibberellin+response+complex+in+cereal+alpha-amylase+gene+promoters.">A gibberellin response complex in cereal alpha-amylase gene promoters.</a> Plant Cell. 4, 203-211 (1992).</li><li>Shen, Q., Zhang, P., Ho, T. H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+nature+of+abscisic+acid+(ABA)+response+complexes;+composite+promoter+units+that+are+necessary+and+sufficient+for+ABA+induction+of+gene+expression.">Modular nature of abscisic acid (ABA) response complexes; composite promoter units that are necessary and sufficient for ABA induction of gene expression.</a> Plant Cell. 8, 1107-1119 (1996).</li><li>G&#243;mez-Cadenas, A., Zentella, R., Walker-Simmons, M. K., Ho, T. H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibberellin/abscisic+acid+antagonism+in+barley+aleurone+cells:+site+of+action+of+the+protein+kinase+PKABA1+in+relation+to+gibberellin+signaling+molecules.">Gibberellin/abscisic acid antagonism in barley aleurone cells: site of action of the protein kinase PKABA1 in relation to gibberellin signaling molecules.</a> Plant Cell. 13, 667-679 (2001).</li><li>Johnson, R. R., Shin, M., Shen, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wheat+PKABA1-interacting+factor+TaABF1+mediates+both+abscisic+acid-suppressed+and+abscisic+acid-induced+gene+expression+in+bombarded+aleurone+cells.">The wheat PKABA1-interacting factor TaABF1 mediates both abscisic acid-suppressed and abscisic acid-induced gene expression in bombarded aleurone cells.</a> Plant Mol. Biol. 68, 93-103 (2008).</li><li>Harris, L. J., Martinez, S. A., Keyser, B. R., Dyer, W. E., Johnson, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+TaABF1+during+abscisic+acid+and+gibberellin+signaling+in+aleurone+cells+of+cereal+grains.">Functional analysis of TaABF1 during abscisic acid and gibberellin signaling in aleurone cells of cereal grains.</a> Seed Science Res. 23, 89-98 (2013).</li><li>Shen, Q., Casaretto, J., Zhang, P., Ho, T. H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+definition+of+ABA-response+complexes:+the+promoter+units+necessary+and+sufficient+for+ABA+induction+of+gene+expression+in+barley+(Hordeum+vulgare).">Functional definition of ABA-response complexes: the promoter units necessary and sufficient for ABA induction of gene expression in barley (Hordeum vulgare).</a> Plant Mol. Biol. 54, 111-124 (2004).</li><li>Ritchie, S., Gilroy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abscisic+acid+signal+transduction+in+the+barley+aleurone+is+mediated+by+phospholipase+D+activity.">Abscisic acid signal transduction in the barley aleurone is mediated by phospholipase D activity.</a> Proc. Natl. Acad. Sci. USA. 95, 2697-2702 (1998).</li><li>Takemiya, A., Shimazaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidic+acid+inhibits+blue+light-induced+stomatal+opening+via+inhibition+of+protein+phosphatase+1.">Phosphatidic acid inhibits blue light-induced stomatal opening via inhibition of protein phosphatase 1.</a> Plant Physiol. 153, 1555-1562 (2010).</li><li>Zou, X., Seeman, J. R., Neuman, D., Shen, Q. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+WRKY+gene+from+creosote+bush+encodes+an+activator+of+the+abscisic+acid+signaling+pathway.">A WRKY gene from creosote bush encodes an activator of the abscisic acid signaling pathway.</a> J. Biol. Chem. 279, 55770-55779 (2004).</li><li>Ishibashi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+are+involved+in+gibberellin/abscisic+acid+signaling+in+barley+aleurone+cells.">Reactive oxygen species are involved in gibberellin/abscisic acid signaling in barley aleurone cells.</a> Plant Physiol. 158, 1705-1714 (2012).</li><li>Piskurewicz, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gibberellic+acid+signaling+repressor+RGL2+inhibits+Arabidopsis+seed+germination+by+stimulating+abscisic+acid+synthesis+and+ABI5+activity.">The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity.</a> Plant Cell. 20, 2729-2745 (2008).</li><li>Hong, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-mediated+regulation+of+a+rice+ABA+responsive+element+binding+factor.">Phosphorylation-mediated regulation of a rice ABA responsive element binding factor.</a> Phytochemistry. 72, 27-36 (2011).</li><li>Lopez-Molina, L., Mongrand, S., McLachlin, D. T., Chait, B. T., Chua, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABI5+acts+downstream+of+ABI3+to+execute+an+ABA-dependent+growth+arrest+during+germination.">ABI5 acts downstream of ABI3 to execute an ABA-dependent growth arrest during germination.</a> Plant J. 32, 317-328 (2002).</li><li>Zhou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOS2-LIKE+PROTEIN+KINASE5,+an+SNF1-RELATED+PROTEIN+KINASE3-Type+protein+kinase,+is+important+for+abscisic+acid+responses+in+Arabidopsis+through+phosphorylation+of+ABSCISIC+ACID-INSESENSITIVE5.">SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-Type protein kinase, is important for abscisic acid responses in Arabidopsis through phosphorylation of ABSCISIC ACID-INSESENSITIVE5.</a> Plant Physiol. 168, 659-676 (2015).</li><li>Zong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+regulation+of+ABA+signaling+and+biosynthesis+by+a+bZIP+transcription+factor+targets+drought-resistance-related+genes.">Feedback regulation of ABA signaling and biosynthesis by a bZIP transcription factor targets drought-resistance-related genes.</a> Plant Physiol. 171, 2810-2825 (2016).</li></ol>

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 cells2,3,4,5,<sup class="xre...

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&#160;(&#946;-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 TaABF15,6 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 protocols2,3,4,5,6 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.

<table>
	<tbody>
		<tr>
			<td>GeneElute HP plasmid Maxiprep kit</td>
			<td>Sigma</td>
			<td>NA0310-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-vis spectrophotometer</td>
			<td>Nanodrop</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Himalaya barley grains</td>
			<td>/</td>
			<td>/</td>
			<td>A variety of hulless barley (store in the dark at 4&#176; C)</td>
		</tr>
		<tr>
			<td>sodium succinate</td>
			<td>Sigma</td>
			<td>S2378</td>
			<td>Reagent for Imbibing Solution</td>
		</tr>
		<tr>
			<td>calcium chloride (dihydrate)</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td>Reagent for Imbibing Solution</td>
		</tr>
		<tr>
			<td>Imbibing Solution</td>
			<td>home made</td>
			<td>/</td>
			<td>20 mM sodium succinate, 20 mM calcium chloride, pH 5.0. Sterilize by autoclaving before use.</td>
		</tr>
		<tr>
			<td>chloramphenicol</td>
			<td>Sigma</td>
			<td>C0378</td>
			<td>Prepare a 10 mg/mL stock solution in 70% ethanol.</td>
		</tr>
		<tr>
			<td>vermiculite</td>
			<td>Fisher</td>
			<td>NC0430369</td>
			<td>Used for vermiculite plates.</td>
		</tr>
		<tr>
			<td>filter paper circles (90 mm)</td>
			<td>Whatman</td>
			<td>1001 090</td>
			<td>Used for vermiculite and for pre-bombardment grain preparation</td>
		</tr>
		<tr>
			<td>Vermiculite Plates</td>
			<td>home made</td>
			<td>/</td>
			<td>Add 50 mL of vermiculite to a glass petri dish. Place a 90 mm paper circle on top of the vermiculite. Autoclave.</td>
		</tr>
		<tr>
			<td>forceps (fine pointed)</td>
			<td>Fisher</td>
			<td>13-812-42</td>
			<td>Used for removing seed coat from barley grains.</td>
		</tr>
		<tr>
			<td>forceps (ultra fine point)</td>
			<td>Fisher</td>
			<td>12-000-122</td>
			<td>Used for removing seed coat from barley grains.</td>
		</tr>
		<tr>
			<td>gold microcarriers (1.6 &#956;m)</td>
			<td>BioRad</td>
			<td>1652264</td>
			<td></td>
		</tr>
		<tr>
			<td>macrocarriers</td>
			<td>BioRad</td>
			<td>1652335</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride (dihydrate)</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td>Prepare a 2.5 M stock solution and store 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>spermidine</td>
			<td>Sigma</td>
			<td>S0266</td>
			<td>Prepare a 100 mM stock solution and store 500 &#956;L aliquots at -20&#176; C (use within 2 months).</td>
		</tr>
		<tr>
			<td>rupture discs (1550 psi)</td>
			<td>BioRad</td>
			<td>1652331</td>
			<td></td>
		</tr>
		<tr>
			<td>stopping screens</td>
			<td>BioRad</td>
			<td>1652336</td>
			<td></td>
		</tr>
		<tr>
			<td>macrocarrier holders</td>
			<td>BioRad</td>
			<td>1652322</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolistic particle delivery system</td>
			<td>BioRad</td>
			<td>PDS-1000/He</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium phosphate monobasic monohydrate</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td>Reagent for 1M sodium phosphate pH 7.2</td>
		</tr>
		<tr>
			<td>sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S9763</td>
			<td>Reagent for 1M sodium phosphate pH 7.2</td>
		</tr>
		<tr>
			<td>1M sodium phosphate pH 7.2</td>
			<td>home made</td>
			<td>/</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>dithiothrietol (DTT)</td>
			<td>Sigma</td>
			<td>43819</td>
			<td>Dissolve in water to 1 M. Store at -20&#176; in 1 mL aliquots.</td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>Dissolve in water to 10 mg/mL. Store at -20&#176; C.</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Prepare a 50% solution in water.</td>
		</tr>
		<tr>
			<td>Grinding Buffer</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 10 mL of 1 M sodium phosphate pH 7.2, 500 &#956;L of 1 M DTT, 100 &#956;L of 10 mg/mL leupeptin, and 40 mL of 50% glycerol. Add water to 100 mL.</td>
		</tr>
		<tr>
			<td>stainlesss steel beads (5 mm)</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL tubes</td>
			<td>Eppendorf</td>
			<td>22363352</td>
			<td>This specific model of tube is recommended for use with the homogenizer.</td>
		</tr>
		<tr>
			<td>bead homogenizer (TissueLyser)</td>
			<td>Qiagen</td>
			<td>85210</td>
			<td></td>
		</tr>
		<tr>
			<td>12mm x 75 mm glass test tubes</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>luciferin</td>
			<td>Goldbio</td>
			<td>LUCK-100</td>
			<td>Prepare a 25 mM stock solution and store 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td>Prepare a 100 mM stock solution and store 250 &#956;L aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>Reagent for 1M Tris sulfate pH 7.7.</td>
		</tr>
		<tr>
			<td>sulfuric acid</td>
			<td>Sigma</td>
			<td>258105</td>
			<td>Reagent for 1M Tris sulfate pH 7.7.</td>
		</tr>
		<tr>
			<td>1M Tris sulfate pH 7.7</td>
			<td>home made</td>
			<td>/</td>
			<td>Dissolve 12.1 g Tris base in 100 mL of water. Adjust pH to 7.7 with sulfuric acid.</td>
		</tr>
		<tr>
			<td>magnesium chloride</td>
			<td>Sigma</td>
			<td>M9397</td>
			<td>Dissolve in water to 2 M.</td>
		</tr>
		<tr>
			<td>Luciferase Assay Buffer (LAB)</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 3 mL of 1 M Tris sulfate pH 7.7, 500 &#956;L of 2 M magnesium chloride, 1 mL of 1 M DTT, and 200 &#956;L of 0.5 M EDTA. Add water to 50 mL.</td>
		</tr>
		<tr>
			<td>Luciferase Assay Mixture</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 15 mL of LAB, 800 &#956;L of 25 mM luciferin, 200 &#956;L of 100 mM ATP, and 4 mL of water. This makes enough assay mixture (20 mL) for 100 luciferase assays.</td>
		</tr>
		<tr>
			<td>luminometer (Sirius)</td>
			<td>Berthold</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>4-methylumbelliferyl-&#946;-D-glucuronide (MUG)</td>
			<td>Goldbio</td>
			<td>MUG1</td>
			<td>Dissolve in DMSO to 100 mM.</td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Sigma</td>
			<td>S8032</td>
			<td>Prepare a 2% stock solution in water and store 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>96 well plates (standard)</td>
			<td>Fisher</td>
			<td>12565501</td>
			<td></td>
		</tr>
		<tr>
			<td>GUS assay buffer</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 2.5 mL of MUG, 5 mL of 1 M sodium phosphate pH 7.2, 400 &#956;L of 0.5 M EDTA, 1 mL of 1 M DTT, 100 &#956;L of 10 mg/ml leupeptin, 20 mL of methanol, and 1 mL of 2% sodium azide. Add water to 100 mL.</td>
		</tr>
		<tr>
			<td>TempPlate sealing film</td>
			<td>USA Scientific</td>
			<td>2921-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates (black)</td>
			<td>Costar</td>
			<td>3916</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium carbonate</td>
			<td>Sigma</td>
			<td>S7795</td>
			<td>Prepare a 200 mM solution in water.</td>
		</tr>
		<tr>
			<td>4-methylumbelliferone</td>
			<td>Sigma</td>
			<td>M1381</td>
			<td>Prepare a 100 &#956;M solution in water. Freeze 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>microplate fluouresence reader</td>
			<td>Bio-Tek</td>
			<td>FLX-800</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring gene expression in bombarded barley aleurone layers with increased throughput

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 (&#946;-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.

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 TaABF15,<sup class="xre...

Watch this Scientific Journal Video about Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput at JoVE.com

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

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.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

The overall goal of this procedure is to measure gene expression from reporter constructs after introducing them into barley aleurone cells by particle bombardment. This method can help answer key questions in plant hormone regulated gene expression such as how specific signaling molecules act to regulate abscisic acid into barley induced genes. The main advantage of this technique is that it provides higher throughput than previous methods, allowing a large number of gene constructs and incubation conditions to be tested.Demonstrating the protocol will be Grace, a student in my lab. First, place a few embryoless grains into the lid of a 90 millimeter plastic petri dish containing 20 milliliters of imbibing solution and 20 microliters of chloramphenicol. Using sterile forceps, gently remove the transparent seed coat from each grain.Peeling the seed coats from the barley grain is a critical step. If the seed coats are not fully removed, the gold microcarriers will be prevented from entering the aleurone cells. But if the peeling is done too roughly, the aleurone cells can be damaged.After peeling the seed coat from all of the grains, transfer them from the petri dish to a vermiculite plate. Then, place the peeled embryoless grains in an incubator at 24 degrees Celsius for 16-20 hours. Now, label a one point five milliliter tube from each treatment that will be included in the bombardment experiment.To each tube, add two point five micrograms of UBI luciferase internal control plasmid, two point five micrograms of a GUS reporter plasmid and the desired amount of an effector plasmid. Add ultra-pure water to each tube to bring the total volume to five microliters. For each treatment, add 50 microliters of microcarriers suspended in 50%glycerol into each one point five milliliter tube containing plasmid DNA.At the final step of the microcarrier coding procedure, pipette eight microliters of suspended microcarriers onto each of the five macrocarriers and allow them to air dry. Set up the particle bombardment apparatus by turning on the vacuum pump, opening the helium valve, and then turning on the particle delivery system. Place a 1550 psi rupture disk into the retaining cap and mount it on the particle delivery system.Place a macrocarrier containing the desired plasmid combination with a stopping screen into the microcarrier launch assembly. Insert the assembly into the top slot of the bombardment chamber. Next, arrange eight 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 milliliter petri dish.Place the dish with the grains into the bombardment chamber at a distance of six centimeters from the stopping screen. Close the door and press the vacuum switch to evacuate the bombardment chamber to 95 kilopascals. Quickly turn the vacuum switch to the hold position.Then hold down the fire switch to bombard the sample. After releasing the vacuum, transfer the bombarded grains to a labeled 60 millimeter petri dish containing four milliliters of imbibing solution with one milligram per milliliter of chloramphenicol. Repeat until all of the bombardments have been completed.Then, incubate the bombarded grains on a shaking platform at 100 rpm at 24 degrees Celsius for 24 hours. Using forceps, remove the eight bombarded grains from the 60 millimeter petri dish and blot them dry on a paper towel. Divide the grains into two labeled two milliliter tubes, each containing 800 microliters of grinding buffer and a five millimeter stainless steel bead.After repeating the previous step for all bombarded grains, place up to 48 two milliliter tubes into two racks of a bead homogenizer. Then mount the racks on the homogenizer and shake the samples at 30 hertz for three minutes. When finished, remove the racks from the homogenizer and place them on ice for five minutes to re-cool the samples.Now, mount the racks on the homogenizer in the opposite direction. Shake the samples again at 30 hertz for three minutes. After removing the racks from the homogenizer, place them on ice for five minutes to re-cool the samples.Following this, centrifuge the grain extracts at 16, 000 G at four degrees Celsius for 10 minutes. Immediately after centrifugation, decant the clear supernatants into a second set of microcentrifuge tubes. Briefly vortex each tube and store on ice.After transferring the supernatant recover the stainless steel beads from the pellets so they can be washed and reused. Now add 200 microliters of GUS assay buffer to each well of a 96-well plate. Place 50 microliters of each grain extract into one of the wells and mix with the pipette tip.Then, seal the top of the plate with sealing film. Place the 96-well plate in an incubator in the dark at 37 degrees Celsius for 20 hours. Following incubation, centrifuge the 96-well plate at 4, 000 G at four degrees Celsius for 10 minutes.When finished, place the plate on ice. Now, add 250 microliters of zero point two molar of sodium carbonate to each well of a black 96-well plate. Add six point two five microliters of each centrifuged GUS assay mixture into each well of the black plate and mix with the pipette tip.Include wells with six point two five microliters of 10-40 at 100 micromolar, four methylumbelliferone as standards. Place the black plate into a plate fluorometer. Read the fluorescence of methylumbelliferone setting the gain of the instrument, such that a well containing six point two five microliters of 40 micromolar, four methylumbelliferone and 250 microliters of zero point two molar sodium carbonate give a reading of 100 fluorescence units.In this experiment, an HVA1 GUS reporter construct was introduced into aleurone cells together with a UBI TaABF1 effector construct. While a TaABF1 HVA1 ratio of just zero point zero one was sufficient to cause a three-fold induction of the HVA1 reporter gene in the absence of exogenis absyssic acid or ABA. Higher levels of the TaABF1 construct resulted in progressively greater HVA1 expression in a dosed dependent manner.Both exogenis ABA and TaABF1 could strongly induce expression of the HVA1 GUS reporter construct. In either case, the presence of one butanol strongly inhibited the HVA1 induction, which suggests that phospholipase D could play an important role in the activation of HVA1 gene expression by ABA through TaABF1. ABA was shown to strongly inhibit gibberellin induced expression for the Amy32b GUS reporter construct.While neither hydrogen peroxide nor sodium nitroprusside caused any significant reduction. These results, therefore, do not support a role for nitric oxide and hydrogen peroxide in aleurone cell ABA responses. Once mastered, this technique can allow many different samples to be measured for gene expression in a relatively short time.For example, bombardment of 200 different grain samples can be done by two investigators in a couple of hours. Then, on the following day, a single investigator can prepare all of the protein extracts in two hours. The modified GUS assay carried out in 96-well plates also requires much less labor than other protocols that use in the future tube assays.

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JoVE Journal

Genetics

6.4K ビュー.  Colby College. The overall goal of this procedure is to measure gene expression from reporter constructs after introducing them into barley aleurone cells by particle bombardment. This method can help answer key questions in plant hormone regulated gene expression such as how specific signaling molecules act to regulate abscisic acid into barley induced genes. The main advantage of this technique is that it provides higher throughput than previous methods, allowing a large number of gene constructs and incu...