This work was funded by the German Science Foundation (DFG) and the Excellence Initiative of the German federal and state governments.

1. Crosslinking of plant material
<ol>
 <li>Harvest 1-2g of Arabidopsis leaves (6 to 10cm rosette diameter) in a 50-ml plastic tube and fill up to 40mL with crosslinking buffer. </li>
 <li>Make sure leaves stay immersed, for example by stuffing a tailored filter sponge right above buffer surface. Then place tubes in a desiccator.</li>
 <li>Vacuum infiltrate for 10min. To each tube add 2.5mL of 2M glycine to stop crosslinking, and invert tubes to warrant equal dispersion of glycine.</li>
 <li>Vacuum infiltrate for another 5min and discard fluid while harvesting leaves on a sieve.</li>
 <li>Wash leaves twice with 1L of water in a glass beaker, then dry leaves thoroughly with paper towels.</li>
 <li>Collect leaves in a fresh plastic tube and freeze in liquid nitrogen. Store leaves at -80&deg; C until chromatin isolation.</li>
</ol>
2. Isolation of chromatin
<ol>
 <li>Leaves were ground with mortar and pistil that were kept in liquid nitrogen until use.</li>
 <li>Transfer powder to a 50-mL plastic tube and suspend in 30mL extraction buffer #1.</li>
 <li>Incubate for 15min at 4&deg;C on an overhead shaker.</li>
 <li>Filtrate suspension through four layers of miracloth into a fresh 50-mL plastic tube.</li>
 <li>Spin for 20min at 2,800 x g at 4&deg;C.</li>
 <li>Remove supernatant and suspend pellet with a paint brush in 1mL extraction buffer #2. First add a small volume of buffer #2, then suspend pellet with a paint brush, and then add the rest of buffer.</li>
 <li>Transfer suspension to a 1.5mL microfuge tube and spin for 10min at 12,000 x g at 4&deg;C.</li>
 <li>Meanwhile, prepare 2mL-microfuge tubes containing 1.5mL extraction buffer #3.</li>
 <li>Remove supernatant from the spun tube (step 2.7) and suspend pellet in 300&mu;L of extraction buffer #3. First add a small volume of buffer #3, suspend pellet with a paint brush, and then add the remaining buffer.</li>
 <li>Carefully pipet the suspended pellet (step 2.9) on top of the 1.5 mL extraction buffer #3 prepared in step 2.8. Make sure the two phases won't mix. Spin for 1h at 16,000 x g at 4&deg;C.</li>
 <li>Remove supernatant and suspend chromatin pellet with a paint brush in 300&mu;L of sonication buffer.</li>
 <li>Sonicate chromatin with a sonotrode, or in an ultrasonic bath, to a DNA size of approximately 400bp. When sonicating, make sure chromatin will not be heated above approximately 30 &deg;C to protect heat-sensitive crosslinks. The duration of sonication needs to be determined experimentally, because it varies significantly between different sonication devices. For guidance, settings for two different devices are provided: </li>
</ol>
For sonication with a Bandelin brand sonotrode expose chromatin in 0.6mL microfuge tube five times to 20 bursts with settings 25% power, cycle = 50. While doing so, cool sample after every 20th bursts in an ice bath.
For sonication with a Diagenode Bioruptor ultrasonic bath, fill bath with water and ice and sonicate ten times for 30 seconds in 1.5mL microfuge tubes with setting 'high'. After every 30 seconds, cool samples for another 30 seconds.
<ol start="13"><li>Spin sonicated sample for 5min at 16,000g at 4&deg;C to precipitate undissolved material. The supernatant can directly be used for immunoprecipitation. Alternatively, samples can be shock frozen in liquid nitrogen and stored at -80&deg;C until further use.</li></ol>
3. Immunoprecipitation
<ol>
 <li>Prepare 2-mL microfuge tubes containing 40&mu;L protein-A agarose and 1.8mL of antibody-binding buffer. Add 200&mu;L of the chromatin preparation (step 2.13).</li>
 <li>Incubate tubes for 1h on an overhead shaker.</li>
 <li>Discard protein-A agarose beads and use supernatant for immunoprecipitation. </li>
 <li>Remove a 40&mu;L-aliquot to determine chromatin concentration ('input'). </li>
 <li>Add 30&mu;L protein-A agarose and the amount of modification-specific antibody that is indicated in the below table of specific reagents and equipment to 400&mu;L sonicated chromatin suspension. </li>
 <li>Incubate for 2.5h or overnight on an overhead shaker at 4&deg;C.</li>
 <li>Spin down beads for 2min at 440 x g.</li>
 <li>Wash bead pellet with 900&mu;L of each wash buffer (see the below list). For each buffer, incubate beads in the buffer for 10min on an overhead shaker at 4&deg;C, then spin for 2min at 440 x g, discard supernatant, and add the next buffer.
<ol>
<li>Low salt wash buffer</li>
<li>High salt wash buffer</li>
<li>LiCl wash buffer</li>
<li>TE wash buffer </li>
</ol></li>
 <li> After the final wash, completely remove any remaining buffer.</li>
</ol>
4. De-crosslinking of histones and DNA, and purification of precipitated DNA
<ol>
 <li>Add 100&mu;L of de-crosslinking buffer to the agarose pellet and the 40&mu;l 'input' sample from step 3.4, mix for 5min on a vortex mixer, spin samples, and incubate at 65&deg;C overnight. </li>
 <li>Purify DNA with a commercial DNA or PCR purification kit. </li>
 <li>Typically 5&mu;l of a 1:5 dilution of the final column eluate are sufficient to perform conventional locus-specific real-time quantitative RT-PCR (RT-qPCR). </li>
</ol>
5. Table of buffers
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Crosslinking buffer</td>
 </tr>
 <tr>
 <td>Sodium butyrate</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>400mM</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 8.0</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>&beta;-Mercaptoethanol</td>
 <td>5mM</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>3% (v/v) </td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Extraction buffer #1</td>
 </tr>
 <tr>
 <td>Sodium butyrate</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>400mM</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 8.0</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>&beta;-Mercaptoethanol</td>
 <td>5mM</td>
 </tr>
 <tr>
 <td>Complete Protease Inhibitor</td>
 <td>1x</td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Extraction buffer #2</td>
 </tr>
 <tr>
 <td>Sodium butyrate</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>250mM</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 8.0</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>&beta;-Mercaptoethanol</td>
 <td>5mM</td>
 </tr>
 <tr>
 <td>MgCl2</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>1% (w/v)</td>
 </tr>
 <tr>
 <td>Complete Protease Inhibitor</td>
 <td>1x</td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Extraction buffer #3</td>
 </tr>
 <tr>
 <td>Sodium butyrate</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>1.7M</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 8.0</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>&beta;-Mercaptoethanol</td>
 <td>5mM</td>
 </tr>
 <tr>
 <td>MgCl2</td>
 <td>2mM</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>0.15% (w/v)</td>
 </tr>
 <tr>
 <td>Complete Protease Inhibitor</td>
 <td>1x</td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Sonication buffer</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 8.0</td>
 <td>25mM</td>
 </tr>
 <tr>
 <td>EDTA-NaOH, pH 8.0</td>
 <td>5mM</td>
 </tr>
 <tr>
 <td>SDS</td>
 <td>0.5% (w/v)</td>
 </tr>
 <tr>
 <td>Complete Protease Inhibitor</td>
 <td>1x</td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Antibody binding buffer</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 8.0</td>
 <td>50mM</td>
 </tr>
 <tr>
 <td>EDTA-NaOH, pH 8.0</td>
 <td>1mM </td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>150mM</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>0.1% (w/v)</td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">Low salt wash buffer</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>150mM</td>
 </tr>
 <tr>
 <td>SDS</td>
 <td>0.1% (w/v)</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>1% (w/v)</td>
 </tr>
 <tr>
 <td>EDTA-NaOH, pH 8.0</td>
 <td>2mM</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 8.0</td>
 <td>20mM</td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">High salt wash buffer</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>500mM</td>
 </tr>
 <tr>
 <td>SDS</td>
 <td>0.1% (w/v)</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>1% (w/v)</td>
 </tr>
 <tr>
 <td>EDTA-NaOH, pH 8.0</td>
 <td>2mM</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 8.0</td>
 <td>20mM</td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td colspan="2">LiCl wash buffer</td>
 </tr>
 <tr>
 <td>Lithium chloride</td>
 <td>250mM</td>
 </tr>
 <tr>
 <td>Nonidet-P40</td>
 <td>1% (v/v)</td>
 </tr>
 <tr>
 <td>Sodium desoxycholate</td>
 <td>1% (w/v)</td>
 </tr>
 <tr>
 <td>EDTA-NaOH, pH 8.0</td>
 <td>1mM</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 8.0</td>
 <td>20mM</td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td>TE wash buffer </td>
 </tr>
 <tr>
 <td>EDTA-NaOH, pH 8.0</td>
 <td>10mM</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 8.0</td>
 <td>1mM</td>
 </tr>
 </tbody>
</table>
<table border="1">
 <tbody>
 <tr>
 <td>Decrosslinking buffer</td>
 </tr>
 <tr>
 <td>Tris-HCl, pH 6.8</td>
 <td>62.5mM</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>200mM</td>
 </tr>
 <tr>
 <td>SDS</td>
 <td>2% (w/v)</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>10mM</td>
 </tr>
 </tbody>
</table>
6. Representative results: 
Expression of the Arabidopsis thaliana PR2 defense gene encoding a &beta;-1,3-glucanase with antimicrobial activity12, was induced by benzothiadiazole (BTH), a synthetic analog of the plant hormone salicylic acid3. Figure 1 demonstrates that activation of PR2 expression is associated with an increase in the acetylation of lysine residues on histone H3 and H4 on the PR2 promoter. Since nucleosome density decreases during gene activation13, histone acetylation values were normalized for the signal obtained with an antibody to an invariant region of histone 3.
<img src="/files/ftp_upload/3096/3096fig1.jpg" alt="Figure 1"/> Figure 1 BTH induces histone acetylation on the PR2 promoter in Arabidopsis. Plants were treated with 100&mu;M BTH or a wettable powder control. After 72h, leaves were collected and chromatin was isolated. The signal obtained after precipitation with acetylation-specific antibodies (as indicated in the figure) was normalized to the signal obtained by precipitation with an antibody to an invariant domain of histone H3. Precipitated chromatin was quantified by RT-qPCR. Data are standardized for histone modification levels in the absence of BTH treatment.

<ol>
	<li>Eberharter, A., Becker, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation:+a+switch+between+repressive+and+permissive+chromatin.">Histone acetylation: a switch between repressive and permissive chromatin.</a> EMBO rep. 3, 224-229 (2002).</li><li>Ruthenburg, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+of+lysine+4+on+histone+H3:+intricacy+of+writing+and+reading+a+single+epigenetic.">Methylation of lysine 4 on histone H3: intricacy of writing and reading a single epigenetic.</a> 25, 15-30 (2007).</li><li>Jaskiewicz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modification+acts+as+a+memory+for+systemic+acquired+resistance+in+the+plant+stress+response.">Chromatin modification acts as a memory for systemic acquired resistance in the plant stress response.</a> EMBO rep. 12, 50-55 (2011).</li><li>Ng, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+histone+modifications+are+associated+with+transcriptional+poising+and+activation+of+the+phaseolin+promoter.">Ordered histone modifications are associated with transcriptional poising and activation of the phaseolin promoter.</a> Plant Cell. 18, 119-132 (2006).</li><li>Offermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illumination+is+necessary+and+sufficient+to+induce+histone+acetylation+independent+of+transcriptional+activity+at+the+C4-specific+phosphoenolpyruvate+carboxylase+promoter+in+maize.">Illumination is necessary and sufficient to induce histone acetylation independent of transcriptional activity at the C4-specific phosphoenolpyruvate carboxylase promoter in maize.</a> Plant Physiol. 141, 1078-1088 (2006).</li><li>Deng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+histone+acetyltransferase+AtHAC1+in+the+regulation+of+flowering+time+via+repression+of+FLOWERING+LOCUS+C+in+Arabidopsis.">Involvement of the histone acetyltransferase AtHAC1 in the regulation of flowering time via repression of FLOWERING LOCUS C in Arabidopsis.</a> Plant Physiol. 143, 1660-1668 (2007).</li><li>Benhamed, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+GCN5,+HD1,+and+TAF1/HAF2+interact+to+regulate+histone+acetylation+required+for+light-responsive+gene+expression.">Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression.</a> Plant Cell. 18, 2893-2903 (2006).</li><li>Bowler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+techniques+for+plant+cells.">Chromatin techniques for plant cells.</a> Plant Journal. 39, 776-789 (2004).</li><li>Orlando, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+chromosomal+proteins+in+vivo+by+formaldehyde+decrosslinked-chromatin+immunoprecipitation.">Mapping chromosomal proteins in vivo by formaldehyde decrosslinked-chromatin immunoprecipitation.</a> Trends Biochem. 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No conflicts of interest declared.

Most critical steps in the protocol are the crosslinking of DNA and histones, the type and amount of leaf material, grinding of the material, and the sonication of chromatin. For a more detailed discussion of these issues, see refs. 9 and 10.
Sonication and crosslinking: 
It is important to disrupt chromatin during sonication to fragments of about 400bp. Exhaustive sonication will destroy chromatin by disrupting DNA and histones, whereas insufficient sonication will leave long chromatin fragments. These affect the specificity of the method, because histone modifications distal from the tested locus cannot be discriminated from local modifications. Sonication efficiency is best tested by collecting 20&mu;L chromatin from samples before and after sonication, de-crosslinking DNA and histones, isolating the DNA and determining the length of the sheared DNA by agarose gel electrophoresis. RNAse should be added to the samples before electrophoresis, because the chromatin preparation still contains large amounts of RNA at this stage of purification that might interfere with the visualization of fragmented DNA. The samples are useful for chromatin immunoprecipitation if DNA from non-sheared chromatin is longer than 10kbp, whereas DNA from sheared chromatin forms a smear with maximum signal intensity around 400bp of DNA. 
We routinely use 3% (v/v) formaldehyde for chromatin crosslinking in intact leaves, but 1% (v/v) formaldehyde might be sufficient in most cases. In our hands, low formaldehyde concentrations allow for shorter sonication times and background signals in consequent PCR reactions. However, insufficient amounts of formaldehyde often result in low reproducibility of the PCR signal between independent experiments. This might be due to insufficient penetration of leaves with formaldehyde at low concentrations. Make sure to exchange your formaldehyde stock frequently as the compound tends to polymerize with time. Formaldehyde solutions are stable only for approximately one month, and this period is hardly prolonged by methanol stabilization. It is advised to test different formaldehyde concentrations when setting up experimental conditions. 
Amount of plant material and grinding: 
It is important to infiltrate low amounts of leaf material in a large volume of buffer to avoid leaves sticking to each other. Leaf sticking often results in insufficient infiltration of formaldehyde. Furthermore, too much plant material will block pores of the miracloth tissue. The grinding efficiency considerably depends on using a mortar and pestle with the perfect fit. We routinely grind with a liquid nitrogen-cooled pestle and mortar in the absence of additional liquid nitrogen three times for 50 seconds until the material is ground to a fine powder.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Protein-A-Agarose</td>
			<td>Roche</td>
			<td>11 134 515 001</td>
			<td>depends on the antibody class</td>
		</tr>
		<tr>
			<td>Protein-G-Agarose</td>
			<td>Roche</td>
			<td>11 243 233 001</td>
			<td>depends on the antibody class</td>
		</tr>
		<tr>
			<td>Anti-hyperacetyl-H4</td>
			<td>Millipore</td>
			<td>06-946</td>
			<td>we used 5&mu;L</td>
		</tr>
		<tr>
			<td>Anti-acetyl-H4K5</td>
			<td>Millipore</td>
			<td>07-327</td>
			<td>we used 5&mu;L</td>
		</tr>
		<tr>
			<td>Anti-acetyl-H4K8</td>
			<td>Millipore</td>
			<td>07-328</td>
			<td>we used 5&mu;L</td>
		</tr>
		<tr>
			<td>Anti-acetyl-H4K12</td>
			<td>Millipore</td>
			<td>07-595</td>
			<td>we used 5&mu;L</td>
		</tr>
		<tr>
			<td>Anti-acetyl-H4K16</td>
			<td>Millipore</td>
			<td>07-329</td>
			<td>we used 5&mu;L</td>
		</tr>
		<tr>
			<td>Anti-acetyl-H4K18</td>
			<td>Millipore</td>
			<td>07-354</td>
			<td>we used 1&mu;L</td>
		</tr>
		<tr>
			<td>Anti-acetyl-H3K9</td>
			<td>Millipore</td>
			<td>07-352</td>
			<td>we used 5&mu;L</td>
		</tr>
		<tr>
			<td>Anti-acetyl-H3K14</td>
			<td>Millipore</td>
			<td>07-353</td>
			<td>we used 1&mu;L</td>
		</tr>
		<tr>
			<td>Anti-trimethyl-H3K4</td>
			<td>Diagenode</td>
			<td>pAB-003-50</td>
			<td>we used 5&mu;L</td>
		</tr>
		<tr>
			<td>Anti-dimethyl-H3K4</td>
			<td>Millipore</td>
			<td>07-030</td>
			<td>we used 5&mu;L</td>
		</tr>
		<tr>
			<td>Anti-monomethyl-H3K4</td>
			<td>Abcam</td>
			<td>ab8895</td>
			<td>2,5 -10&mu;L</td>
		</tr>
		<tr>
			<td>Anti-H3</td>
			<td>Abcam</td>
			<td>ab1791</td>
			<td>we used 1&mu;L to check histone density</td>
		</tr>
		<tr>
			<td>Bioruptor</td>
			<td>Diagenode</td>
			<td>UCD-200 TO</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sonotrode MS72</td>
			<td>Bandelin </td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Miracloth</td>
			<td>Calbiochem</td>
			<td>475855</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Complete Protease Inhibitor</td>
			<td>Roche</td>
			<td>11836145001</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

detection of histone modifications in plant leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

Watch this Scientific Journal Video about Detection of Histone Modifications in Plant Leaves at JoVE.com

Detection of Histone Modifications in Plant Leaves

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

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JoVE Journal

Biology

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Video: Detection of Histone Modifications in Plant Leaves

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...