The author thanks Robert Holdcraft for technical assistance. This study was funded in part by a USDA CSREES Special Grant (2009-34155-19957) and hatch funds (NJ08192).

1. Local induction of volatiles: herbivore damage
<ol>
 <li>Two branches of blueberry plants are bagged with a spun polyester sleeve.</li>
 <li>Six gypsy moth caterpillars (2nd-3rd instars) are placed inside the bags and allowed to feed on plants for 2 days prior to volatile collection. Control plants do not receive any caterpillars. </li>
 <li>Volatile emissions are collected as described below (protocol &#35;7) on day 3 (Figure 1). Polyester sleeves remain on plants to prevent insects from escaping. </li>
</ol>
2. Local induction of volatiles: mechanical damage
<ol>
 <li>Mechanical damage to plants is inflicted by punching holes to mimic the amount of leaf area removed by gypsy moth. </li>
 <li>Five leaves per plant are injured with two 7-mm holes located at the base and upper portion of leaves at the end of days 1 and 2. The control treatment does not receive mechanical damage. </li>
 <li>Volatile emissions are collected as described below on day 3.</li>
</ol>
3. Local induction of volatiles: MeJA
<ol>
 <li>Blueberry plants are treated with 10 mL of either a 1 or 1.5 mM solution of MeJA in a 0.1&#37; Tween-20 solution. </li>
 <li>MeJA is applied using a 2-oz spray bottle. Control plants are sprayed with 10 mL of a 0.1&#37; Tween-20 solution. </li>
 <li>Plants are treated at 16:00 hr, and kept in the greenhouse for 15 hr inside a 17-cm diameter and 35-cm high Plexiglas chamber prior to volatile collections.</li>
 <li>Volatile emissions are collected as described below.</li>
</ol>
4. Systemic induction of volatiles: internal signaling
<ol>
 <li>A lower branch of a blueberry plant is bagged with a spun polyester sleeve and damaged by placing six (2nd-3rd instars) gypsy moth caterpillars inside the bag.</li>
 <li>The damaged branch remains outside the volatile collection chamber, while the branches above the damaged branch are placed inside the chamber (Figure 2).</li>
 <li>Caterpillars are allowed to feed on the bottom branch for 2 days. </li>
 <li>Starting on day 3, volatiles are collected from the undamaged portion of damaged plants.</li>
 <li>Volatiles are collected for 7 consecutive days. Control plants are treated in a similar manner but did not receive herbivore damage.</li>
</ol>
5. Vascular connectivity
<ol>
 <li>This study determined the degree of vascular connectivity among different branches within a blueberry plant.</li>
 <li>The end portion of a lower branch from a plant is cut to support a floral water pick containing 6 mL solution of a Rhodamine-B (Sigma-Aldrich) dye (0.25&#37; w/v), as described in Orians et al.11</li>
 <li>Movement of the dye through the plant is monitored daily for 7 days. </li>
 <li>After 7 days, the amount of red staining is visually assessed from different positions of the plant.</li>
</ol>
6. Exposure to HIPVs: external signaling
<ol>
 <li>Branches within a plant are either exposed to HIPVs from an adjacent branch or received no exposure to HIPVs. </li>
 <li>To expose branches to HIPVs, one lower branch is bagged with a polyester sleeve and six (2nd-3rd instars) gypsy moth caterpillars are placed inside bags (Figure 3). </li>
 <li>The plant is caged inside a Plexiglas chamber similar to those described above. Insects are allowed to feed on plants for 2 days, such that adjacent branches are exposed to volatiles emitted from the induced branch. </li>
 <li>After exposure (day 3), exposed branches are placed inside the volatile collection chamber, leaving the insect-injured branch outside (as seen in Figure 2). </li>
 <li>Volatiles from HIPV-exposed branches are collected as described below, and the amount of leaf area consumed from the lower branch are measured using Scion Image Software.</li>
</ol>
7. Exposure to HIPVs: priming
<ol>
 <li>To determine whether plants are "primed" after HIPV exposure, plants are treated as in protocol &#35;5.</li>
 <li>Half of the plants are exposed to HIPVs from an induced adjacent branch; while the other half is exposed to undamaged branches.</li>
 <li>On day 3, four early 2nd-instar gypsy moth caterpillars are placed on each plant and volatiles are collected as described below (Figure 4).</li>
 <li>After volatile collections, leaves initially damaged by gypsy moth caterpillars, as well as those that are damaged on day 3, are excised and amounts of leaf area consumed are measured.</li>
</ol>
8. Collection of volatiles
<ol>
 <li>HIPV emissions are sampled in the greenhouse using a push-pull system. The above ground portion of potted plants (stem, branches, and leaves), including caterpillars in the herbivore damage treatment, are placed inside a 4 L volatile collection chamber. A 2-piece guillotine supports the base of plants. Purified air enters the top of each chamber at a rate of 2 L/min. </li>
 <li>Volatiles are collected in traps filled with 30 mg of Alltech Super-Q adsorbent by pulling air from the chambers at a rate of 1 L/min. </li>
 <li>The volatile collection apparatus consists of four chambers, allowing for simultaneous collections of volatiles from four different plants (Figure 1).</li>
 <li>Collections are conducted during daytime from 09:00 to 17:00 h.</li>
 <li>After collection, all leaves from plants are harvested, oven dried at 60 &deg;C, and weighed, and chambers are rinsed with tap water and 70&#37; ethanol. </li>
</ol>
9. Analysis of volatiles
<ol>
 <li>The collected volatiles from Super-Q traps are eluted with dichloromethane (150 &mu;l) and 400 ng of n-octane is added as internal standard. </li>
 <li>Separation and quantification of compounds is done on a Hewlett Packard 6890 Series Gas Chromatograph (GC) (Figure 5), equipped with a flame ionization detector (FID) and an Agilent HP-1 column (10 m x 0.53 mm x 2.65 &mu;m), and using He as carrier gas (constant flow = 5 ml/min, velocity = 39 cm/sec). The temperature program started at 40 &deg;C kept for 1 min, increased at 14 &deg;C/min to 180 &deg;C (2 min), then at 40 &deg;C/min to 200 &deg;C, and kept at 200 &deg;C for 2 min.</li>
 <li>Tentative identification of compounds is carried out on a Varian 3400 GC coupled to a Finnigan MAT 8230 mass spectrometer (MS), equipped with a Supelco MDN-5S column (30 m x 0.32 mm x 0.25 &mu;m), and with He as the carrier gas. The MS is operated in electron ionization (EI) and total ion chromatogram (TIC) mode at 250 &deg;C (source temperature). The program started at 35 &deg;C (1 min), increased at 4 &deg;C/min to 170 &deg;C, then at 15 &deg;C/min to 280 &deg;C. </li>
 <li>Compounds are tentatively identified by comparison of spectral data with those from NIST library and by GC retention index, and by comparing their retention times with those of commercially available compounds.</li>
</ol>
10. Representative Results:
Twenty-two volatiles were identified from blueberry leaves (Figure 6). Figure 7 shows a representative chromatograph of undamaged blueberry leaves and leaves damaged by feeding by gypsy moth. Mechanical damage and feeding by gypsy moth caterpillars increased volatiles emissions locally from blueberry leaves compared to controls (Figure 8). Compared to caterpillar feeding, the MeJA treatment induced 11 out of the 17 compounds induced by gypsy moth (Figure 9). There was, however, no evidence of systemic induction of volatiles from undamaged leaves of gypsy moth-damaged plants seven days after initial feeding damage (i.e., lack of internal signaling) (Figure 10). In addition, after one week, very slow movement of the red dye was observed among branches of blueberry plants (Figure 11). There was high vascular connectivity among leaves within a single branch. However, there was intermediate-to-low connectivity between two branches vertically aligned within a shoot, and low connectivity between two branches on opposite sides of a shoot.
There was no difference between amounts of volatiles emitted from branches exposed to HIPVs versus those not exposed to HIPVs (Figure 12). However, HIPVs act as external defensive signals in blueberries. Gypsy moth caterpillars fed on leaves previously exposed to HIPVs consumed 71&#37; less leaf material than did those fed on unexposed control leaves (Figure 13). Furthermore, amounts of volatiles emitted per amount of leaf area consumed in HIPV-exposed branches were 4-fold higher compared to unexposed branches (Figure 14), indicating that leaves from HIPV-exposed branches were more responsive to herbivory (i.e., they were primed).
<img src="/files/ftp_upload/3440/3440fig1.jpg" alt="Figure 1"/> Figure 1. A push-pull system is used to collect volatiles from blueberry plants. Plants are placed inside glass chambers and clean air is passed over them. A filter containing an adsorbent material was attached to the side of each chamber to trap volatiles emitted from the plant. A vacuum is used to pull air from the inside of the chamber through the filter.
<img src="/files/ftp_upload/3440/3440fig2.jpg" alt="Figure 2"/> Figure 2. To study the systemic volatile response from blueberry plants, lower blueberry branches were either damaged by gypsy moth caterpillars (right chamber) or left undamaged (left chamber). After 2 days (on day 3), volatiles from undamaged upper branches from damaged and undamaged plants were collected. 
<img src="/files/ftp_upload/3440/3440fig3.jpg" alt="Figure 3"/> Figure 3. To test whether leaves from undamaged branches respond to HIPVs from damaged branches, I bagged one of the branches in each of blueberry plants. I then placed gypsy moth caterpillars inside the bags of half of the plants. The bags I used allow movement of volatiles from inside to the outside of bags. Plants were placed inside Plexiglas chambers to expose undamaged branches to HIPVs. Volatiles were then collected from exposed branches.
<img src="/files/ftp_upload/3440/3440fig4.jpg" alt="Figure 4"/> Figure 4. To test whether leaves from undamaged branches are "primed" after exposure to HIPVs, experiments were repeated as described in Figure 3 but gypsy moth caterpillars were placed on HIPV-exposed and unexposed branches inside each volatile collection chamber.
<img src="/files/ftp_upload/3440/3440fig5.jpg" alt="Figure 5"/> Figure 5. After volatile collections, samples are injected onto a gas chromatograph (GC) to identify and quantify the volatiles emitted from blueberry plants.
<img src="/files/ftp_upload/3440/3440fig6.jpg" alt="Figure 6"/> Figure 6. At least 22 compounds are emitted from blueberry leaves.
<img src="/files/ftp_upload/3440/3440fig7.jpg" alt="Figure 7"/> Figure 7. Typical chromatographs from undamaged blueberry leaves and leaves damaged by gypsy moth caterpillars. Volatiles are emitted at very low amounts from undamaged blueberry leaves. However, when leaves are damaged by gypsy moth caterpillars, emission of volatiles increased dramatically.
<img src="/files/ftp_upload/3440/3440fig8.jpg" alt="Figure 8"/> Figure 8. The graph shows the amounts of each of 22 volatiles emitted from undamaged leaves, mechanically-damaged leaves, and leaves damaged by gypsy moth caterpillars. Artificial damage to mimic the amount of leaf area removed by caterpillars increased volatile emission from blueberry leaves but the response was different from the volatile response of leaves to gypsy moth feeding.
<img src="/files/ftp_upload/3440/3440fig9.jpg" alt="Figure 9"/> Figure 9. Tested whether the JA pathway regulates volatile emission in blueberry leaves. Plants were sprayed with different amounts of MeJA. I found that increasing concentrations of exogenously-applied MeJA increased emissions of volatiles from blueberry leaves.
<img src="/files/ftp_upload/3440/3440fig10.jpg" alt="Figure 10"/> Figure 10. The graph shows the total amounts of volatiles emitted from control blueberry branches and from undamaged branches from gypsy moth damaged plants (systemic response). Volatiles were collected for a total of 7 consecutive days. I found no systemic induction of volatiles even 7 days after initial damage to lower branches of plants. 
<img src="/files/ftp_upload/3440/3440fig11.jpg" alt="Figure 11"/> Figure 11. I used rhodamine-B (red dye) to determine the degree of vascular connectivity among branches within blueberry plants. I found that approx. 80&#37;, 20&#37;, 5&#37; and 0&#37; of leaves from branches containing the dye, branches directly above the branch containing the dye, branches across the branch containing dye, and branches located in a different shoot within a blueberry plant, respectively, were fully stained with the dye.
<img src="/files/ftp_upload/3440/3440fig12.jpg" alt="Figure 12"/> Figure 12. The graph shows the amount of volatiles emitted from branches exposed to HIPVs and unexposed branches. I found that exposure to HIPVs did not affect volatile emissions in neighboring undamaged blueberry branches.
<img src="/files/ftp_upload/3440/3440fig13.jpg" alt="Figure 13"/> Figure 13. The graph shows the amount of feeding by gypsy moth caterpillars on blueberry branches exposed to HIPVs and unexposed branches. Caterpillars on HIPV-exposed branches consumed less amount of foliage compared with those on unexposed branches.
<img src="/files/ftp_upload/3440/3440fig14.jpg" alt="Figure 14"/> Figure 14. When I calculated the emission rate per area consumed, I found that HIPV-exposed branches had increased emission rates of volatiles compared with unexposed branches, indicating that exposure to HIPVs primed leaves in blueberry branches for an increased volatile response.

<ol>
	<li>Dicke, M., Van Loon, J. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multitrophic+effects+of+herbivore-induced+plant+volatiles+in+an+evolutionary+context.">Multitrophic effects of herbivore-induced plant volatiles in an evolutionary context.</a> Entomol. Exp. Appl. 97, 237-237 (2000).</li><li>Mumm, R., Dicke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+natural+plant+products+and+the+attraction+of+bodyguards+involved+in+indirect+plant+defense.">Variation in natural plant products and the attraction of bodyguards involved in indirect plant defense.</a> Can. J. Zool. 88, 628-628 (2010).</li><li>Hopke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivore-induced+volatiles:+the+emission+of+acyclic+homoterpenes+from+leaves+of+Phaseolus+lunatus+and+Zea+mays+can+be+triggered+by+a+&#946;-glucosidase+and+jasmonic+acid.">Herbivore-induced volatiles: the emission of acyclic homoterpenes from leaves of Phaseolus lunatus and Zea mays can be triggered by a &#946;-glucosidase and jasmonic acid.</a> FEBS Lett. 352, 146-146 (1994).</li><li>Rodriguez-Saona, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+electrophysiological+responses+of+the+emerald+ash+borer,+Agrilus+planipennis,+to+induced+volatiles+of+Manchurian+ash,+Fraxinus+mandshurica.">Behavioral and electrophysiological responses of the emerald ash borer, Agrilus planipennis, to induced volatiles of Manchurian ash, Fraxinus mandshurica.</a> Chemoecology. 16, 75-75 (2006).</li><li>Rodriguez-Saona, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivore+induced+volatiles+in+the+perennial+shrub,+Vaccinium+corymbosum,+and+their+role+in+inter-branch+signaling.">Herbivore induced volatiles in the perennial shrub, Vaccinium corymbosum, and their role in inter-branch signaling.</a> J. Chem. Ecol. 35, 163-163 (2009).</li><li>Karban, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage-induced+resistance+in+sagebrush:+volatiles+are+key+to+intra-+and+interplant+communication.">Damage-induced resistance in sagebrush: volatiles are key to intra- and interplant communication.</a> Ecology. 87, 922-922 (2006).</li><li>Frost, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within-plant+signalling+by+volatiles+overcomes+vascular+constraints+on+systemic+signalling+and+primes+responses+against+herbivores.">Within-plant signalling by volatiles overcomes vascular constraints on systemic signalling and primes responses against herbivores.</a> Ecol. Lett. 10, 490-490 (2007).</li><li>Heil, M., Bueno Silva, 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=Within-plant+signaling+by+volatiles+leads+to+induction+and+priming+of+an+indirect+plant+defense+in+nature.">Within-plant signaling by volatiles leads to induction and priming of an indirect plant defense in nature.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 5467-5467 (2007).</li><li>Turlings, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploitation+of+herbivore-induced+plant+odors+by+host-seeking+parasitic+wasps.">Exploitation of herbivore-induced plant odors by host-seeking parasitic wasps.</a> Science. 250, 1251-1251 (1990).</li><li>Makovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatiles+involved+in+the+nonhost+rejection+of+Fraxinus+pennsylvanica+by+Lymantria+dispar+larvae.">Volatiles involved in the nonhost rejection of Fraxinus pennsylvanica by Lymantria dispar larvae.</a> J. Agric. Food Chem. 44, 929-929 (1996).</li></ol>

I have nothing to disclose

The push-pull volatile collection apparatus described here represents a standard method for headspace collections of plant volatiles. This apparatus was used to determine the volatile response of blueberry leaves to herbivory by gypsy moth caterpillars and also allowed me to provide new evidence for the role of HIPVs in intra-plant signaling.
The results presented here show that caterpillar feeding, exogenously-applied MeJA , and mechanical wounding increased volatile emissions at the site of...

<table border="1">
<tr>
<td>Name of the reagent</td>
<td>Company</td>
<td>Catalogue number</td>
<td>Comments </td>
</tr>
<tr>
<td>Volatile collection chambers</td>
<td>Analytical Research Systems, Inc.</td>
<td>VCC-G6X12DT-1P</td>
<td>Gainesville, FL</td>
</tr>
<tr>
<td>Air compressor, 20 gal, oil free, 2 hp</td>
<td>Westward</td>
<td>3JR71 </td>
<td>Sold by Grainger, Inc.</td>
</tr>
<tr>
<td>Air delivery system</td>
<td>Analytical Research Systems, Inc.</td>
<td>VCS-ADS-4AFM4C</td>
<td>Gainesville, FL</td>
</tr>
<tr>
<td>Air collection system</td>
<td>Analytical Research Systems, Inc.</td>
<td>VCS-MVCS-4CX1P</td>
<td>Gainesville, FL</td>
</tr>
<tr>
<td>Vacuum pump 100-150V, &frac14; hp</td>
<td>Gast Manufacturing, Inc.</td>
<td><a href="http://www.grainger.com/Grainger/GAST-Vacuum-Pump-4F740">4F740</a></td>
<td>Sold by Grainger, Inc.</td>
</tr>
<tr>
<td>Methyl jasmonate</td>
<td>Sigma-Aldrich</td>
<td>J2500</td>
<td>St. Louis, MO</td>
</tr>
<tr>
<td>Tween-20 </td>
<td>Sigma-Aldrich</td>
<td>93773</td>
<td>St. Louis, MO</td>
</tr>
<tr>
<td>Rhodamine-B</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>St. Louis, MO</td>
</tr>
<tr>
<td>Plastic spray bottles, 2 oz</td>
<td>Setco Inc.</td>
<td>&nbsp;</td>
<td>Cranbury, NJ</td>
</tr>
<tr>
<td>Spun polyester sleeves</td>
<td>Rockingham Opportunities Corp.</td>
<td>&nbsp;</td>
<td>Reidsville, NC</td>
</tr>
<tr>
<td>Super-Q volatile collection traps</td>
<td>Analytical Research Systems, Inc.</td>
<td>VCT-1/4X3-SPQ</td>
<td>Gainesville, FL</td>
</tr>
<tr>
<td>Scion Image Software</td>
<td>Scion Corporation</td>
<td>&nbsp;</td>
<td>Frederick, MD</td>
</tr>
<tr>
<td>Dichloromethane</td>
<td>Sigma-Aldrich</td>
<td>270997</td>
<td>&nbsp;St. Louis, MO</td>
</tr>
<tr>
<td>Gas chromatograph HP 6890</td>
<td>Hewlett Packard</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Gas chromatograph Varian 3400 </td>
<td>Varian</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>n-octane</td>
<td>Sigma-Aldrich</td>
<td>296988</td>
<td>St. Louis, MO</td>
</tr>
<tr>
<td>Mass spectrometer MAT 8230 </td>
<td>Finnigan </td>
<td>&nbsp;</td>
<td>San Jose, CA</td>
</tr>
<tr>
<td>HP-1 GC column</td>
<td>Agilent Technologies</td>
<td>&nbsp;</td>
<td>Palo Alto, 
 CA</td>
</tr>
<tr>
<td>MDN-5S GC column</td>
<td>Supelco, Inc.</td>
<td>&nbsp;</td>
<td>Bellefonte, PA</td>
</tr>
</table>

herbivore-induced blueberry volatiles and intra-plant signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

Watch this Scientific Journal Video about Herbivore-induced Blueberry Volatiles and Intra-plant Signaling at JoVE.com

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

herbivore-induced-blueberry-volatiles-and-intra-plant-signaling

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16.3K Views.  Rutgers University. A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

Video: Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...