Name: isolation of viable multicellular glands from tissue of the carnivorous plant, nepenthes
Uploaded: 2013-12-22T20:30:00
Description: Watch this Scientific Journal Video about Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes at JoVE.com

We thank the greenhouse team at the MPI-CE at the Botanical Garden of the Friedrich Schiller University, Jena, for cultivating plants and Wilhelm Boland and the Max Planck Society for support.

1. Plant Material Preparation
<ol>
	<li>All Nepenthes species were grown either in the greenhouses of the MPI for Chemical Ecology or the Botanical Garden in Jena, Germany.</li>
	<li>For the preparation of secreting tissue harvest a pitcher and remove the upper one third including lid, peristome, and slippery zone. The pitcher can be used either freshly or frozen in liquid nitrogen and stored at -80 &#176;C.</li>
</ol>
2. Microdissection of Pitcher Tissue
<ol>
	<li>The microsampling p...

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+for+nutrient+transport+in+the+pitchers+of+the+carnivorous+plant.">Pathways for nutrient transport in the pitchers of the carnivorous plant.</a> Nepenthes alata Ann. Bot. 84, 459-466 (1999).</li><li>Brandt, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microgenomics:+gene+expression+analysis+at+the+tissue-specific+and+single-cell+levels.">Microgenomics: gene expression analysis at the tissue-specific and single-cell levels.</a> J. Exp. Bot. 56, 495-505 (2005).</li><li>H&#246;lscher, D., Schneider, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+laser-assisted+microdissection+for+tissue+and+cell-specific+analysis+of+RNA,+proteins,+and+metabolites.">Application of laser-assisted microdissection for tissue and cell-specific analysis of RNA, proteins, and metabolites.</a> Prog. Bot. 69, 141-167 (2008).</li><li>Rottloff, S., M&#252;ller, U., Kilper, R., Mith&#246;fer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropreparation+of+single+secretory+glands+from+the+carnivorous+plant+Nepenthes.">Micropreparation of single secretory glands from the carnivorous plant Nepenthes.</a> Anal. Biochem. 394, 135-137 (2009).</li><li>Liu, W. H., Saint, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+quantitative+method+of+real+time+reverse+transcription+polymerase+chain+reaction+assay+based+on+simulation+of+polymerase+chain+reaction+kinetics.">A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics.</a> Anal. Biochem. 302, 52-59 (2002).</li><li>Werker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichome+diversity+and+development.">Trichome diversity and development.</a> Adv. Bot. Res. Inc. Adv. Plant Pathol. 31, 1-35 (2000).</li><li>Fahn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+secretory+cells.">Structure and function of secretory cells.</a> Adv. Bot. Res. Inc. Adv. Plant Pathol. 31, 37-75 (2000).</li><li>Juniper, B. E., Robins, R. J., Joel, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The carnivorous plants. , (1989).</li><li>Gershenzon, J., McCaskill, D., Rajaonarivony, J., Mihaliak, C., Karp, F., Croteau, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+secretory-cells+from+plant+glandular+trichomes+and+their+use+in+biosynthetic-studies+of+monoterpenes+and+other+gland+products.">Isolation of secretory-cells from plant glandular trichomes and their use in biosynthetic-studies of monoterpenes and other gland products.</a> Anal. Biochem. 200, 130-138 (1992).</li><li>Rontein, D., Onillon, S., 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=CYP725A4+from+yew+catalyzes+complex+structural+rearrangement+of+taxa-4(5),11(12)-diene+into+the+cyclic+ether+5(12)-oxa-3(11)-cyclotaxane.">CYP725A4 from yew catalyzes complex structural rearrangement of taxa-4(5),11(12)-diene into the cyclic ether 5(12)-oxa-3(11)-cyclotaxane.</a> J. of Biol. Chem. 283 (5), 6067-6075 (2008).</li><li>Aharoni, A., Jongsma, M. A., Bouwmeester, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+science?+Engineering+of+terpenoid+biosynthesis+in+plants.">Volatile science? Engineering of terpenoid biosynthesis in plants.</a> Trends Plant Sci. 10, 594-602 (2005).</li><li>Mith&#246;fer, A., Boland, W., Maffei, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+ecology+of+plant-insect+interactions.">Chemical ecology of plant-insect interactions.</a> Ann. Plant Rev. 34, 261-291 (2009).</li><li>Kennedy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+pests,+parasitoids,+and+predators:+Tritrophic+interactions+involving+the+genus+Lycopersicon.">Tomato pests, parasitoids, and predators: Tritrophic interactions involving the genus Lycopersicon.</a> Ann. Rev. Entomol. 48, 51-72 (2003).</li><li>Mith&#246;fer, A., Boland, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+defense+against+herbivores:+Chemical+aspects.">Plant defense against herbivores: Chemical aspects.</a> Ann. Rev. Plant Biol. 63, 431-450 (2012).</li><li>Rottloff, S., Stieber, R., Maischak, H., Turini, F. G., Heubl, G., Mith&#246;fer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+a+class+III+acid+endochitinase+from+the+traps+of+the+carnivorous+pitcher+plant+genus,+Nepenthes.">Functional characterization of a class III acid endochitinase from the traps of the carnivorous pitcher plant genus, Nepenthes.</a> J. Exp. Bot. 62, 4649-4647 (2011).</li></ol>

For a long time, studies of plant secretory structures, in particular trichomes have been performed essentially on anatomy and were purely descriptive. There is a large body of publications of this type as summarized in reviews by9-11. Our biochemical and molecular knowledge of plant secretory structures is still limited in spite of some early remarkable work e.g. on mint glandular trichomes showing that glandular trichomes are the site of biosynthesis of monoterpenoids which are secreted into the sub...

Plants produce a wide huge variety of compounds with different industrial and pharmaceutical applications, ranging from small molecular weight molecules to polymers. Such compounds are frequently produced and secreted by highly specialized structures, many of which are localized at the surface of the plants such as glandular trichomes or nectaries, or internally such as idioblasts or latex and resin ducts, respectively. However, relatively little is known about the biology of these specialized secretory structures, although all types of those glands display many features that are indicative of active metabolism. In particular, glands of carnivorous plants such as Nepenthes can be seen as model systems in plant cell biology1.
Carnivorous plants are the object of several studies since 1875 when Charles Darwin&#39;s &#39;Insectivorous Plants&#39; was published2. However, only in the last few years research on the molecular level has been done and still our knowledge is limited. For example, the protein composition of the digestion fluid in the pitcher plants of the genus Nepenthes is still not completely known. Only recently several hydrolytic proteins have been identified and a few of the corresponding genes as well3. In the pitfall-traps of Nepenthes (Figure 1), multicellular glands are localized at the inner bottom of the pitchers. These glands do both production and secretion of the digestive enzymes into the pitcher fluid and absorption of dissolved nutrients from the pitcher fluid4. Hence, these bifunctional glands represent a unique microtissue with a central position and function in the carnivory of Nepenthes that is worth to be studied in detail.
<img alt="Figure 1" src="/files/ftp_upload/50993/50993fig1.jpg" /> 
Figure 1. Nepenthes alata pitcher. The lower part of the pitcher contains the digestive fluid and glands on the inside.
<a href="http://www.jove.com/files/ftp_upload/50993/50993fig1highres.jpg" target="_blank">Click here to view larger image</a>.
In plant sciences, many experimental approaches rely on &#39;bulk material&#39; because no defined tissue is investigated but entire organs. As a consequence, their homogenization and analysis can only provide results that are diluted and proportionately with respect to individual tissues and cells5. To solve this problem, micropreparation techniques, for example the so-called laser capture microdissection (LCM), were developed and successfully used to harvest specific plant tissues and analyze their individual contents6. However, LCM is often limited if unstable and/or fast degrading cell components are targeted such as RNA. In such a case, the need of previous tissue fixation that is time consuming and often accompanied by degradation of the biomolecules, is of disadvantage. Moreover, due to the high water content of many plant tissues and the strength of cell walls, LCM often failed to prepare straight from fresh tissue7. Any approach to investigate cellular components that are present in secretory glands from genes, mRNA, and proteins to secondary compounds and their biosynthesis needs alternative techniques for the particular preparation. Thus, specific techniques to isolate single cells or even multicellular glands that remain intact after preparation are necessary.
Here a method is presented, employing a mechanized microsampling platform that features a direct microscopic visualization. This technique is both rapid and efficient in the preparation of individual glands from the pitchers of Nepenthes species. These multicellular glands are bifunctional, i.e. they are involved in secretion as well as in uptake processes, they are sessile, and not exposed cell layers are impregnated to form an endodermis. Entire glands can be isolated without neighboring tissue and deposited in a well-defined manner into a PCR tube. This technique is applied to study specific genes as well as gene expression in the glands&#39; tissues.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Name of Material/ Equipment</td>
		</tr>
		<tr>
			<td>RNAqueous-Micro Kit</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>AM1931</td>
			<td>Isolation of total RNA, 50 samples</td>
		</tr>
		<tr>
			<td>Dynabeads mRNA DIRECT Micro Kit</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>61011</td>
			<td>5 ml Dynabeads Oligo (dT)25</td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript CellsDirect cDNA Synthesis System</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>18080-200</td>
			<td>25 reactions</td>
		</tr>
		<tr>
			<td>RNaseOUT</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>10777-019</td>
			<td>Rnase inhibitor, 5,000 units</td>
		</tr>
		<tr>
			<td>Random Hexamers (0.4 &#956;g/&#956;l)</td>
			<td>QIAGEN GmbH, Germany</td>
			<td>79236</td>
			<td>100 &#956;l</td>
		</tr>
		<tr>
			<td>10 mM dNTP Mix, molecular biology grade</td>
			<td>Fermentas GmbH, Germany</td>
			<td>R0192</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase, (recombinant)</td>
			<td>Fermentas GmbH, Germany</td>
			<td>EP0402</td>
			<td>Supplied with 10x Taq buffer (with KCl or (NH4)2SO4) and 25 mM MgCl2</td>
		</tr>
		<tr>
			<td>TURBO DNase</td>
			<td>Life Technologies GmbH, Germany</td>
			<td>AM2238</td>
			<td>1,000 units</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Carl Roth GmbH Co. Germany</td>
			<td>T846.3</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler Low Range DNA ladder, ready-to-use</td>
			<td>Fermentas GmbH, Germany</td>
			<td>SM1193</td>
			<td>50 &#956;g</td>
		</tr>
		<tr>
			<td>Brilliant II SYBR Green QPCR Master Mix</td>
			<td>Agilent Technologies, Inc., USA</td>
			<td>600828</td>
			<td>Single kit</td>
		</tr>
		<tr>
			<td>RNaseZAP</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>R2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Carl Roth GmbH &#38; Co., Germany</td>
			<td>CK08.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Carl Roth GmbH &#38; Co., Germany</td>
			<td>2801.1 / 2855.1</td>
			<td></td>
		</tr>
		<tr>
			<td>GE NanoVue spectrophotometer</td>
			<td>GE Healthcare Europe GmbH, Germany</td>
			<td>28-9569-62</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastercycler gradient</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>5331 000.010</td>
			<td></td>
		</tr>
		<tr>
			<td>Mx3000P Real-Time PCR System</td>
			<td>Stratagene, USA</td>
			<td>401403</td>
			<td></td>
		</tr>
		<tr>
			<td>Agagel Standard Horizontal Gel Electrophoresis Chamber</td>
			<td>Biometra</td>
			<td>Discontinued</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>aura optik gmbh</td>
			<td>aureka</td>
			<td>Equipped with a microforceps</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss Microscopy, Jena, Germany</td>
			<td>SteREO Lumar.V12</td>
			<td>Equipped with an AxioCam</td>
		</tr>
		<tr>
			<td>Lumar Filter set 01</td>
			<td>Carl Zeiss, Microscopy, Jena, Germany</td>
			<td>485001-0000-000</td>
			<td>UV filter</td>
		</tr>
		<tr>
			<td>Lumar Filter set 09</td>
			<td>Carl Zeiss, Microscopy, Jena, Germany</td>
			<td>485009-0000-000</td>
			<td>non-UV filter</td>
		</tr>
	</tbody>
</table>

isolation of viable multicellular glands from tissue of the carnivorous plant, nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

For the preparation of single glands from Nepenthes pitcher tissue, a mechanized microdissection technique was developed and applied. The micropreparation platform needs to be equipped with microforceps, attached to the micromanipulator, which in turn are operated by a control panel. Individual glands, visible under the microscope, could be targeted with the forceps that grasped the gland tissue and removed it mechanically (Figure 4A).
The glandular tissue was transfe...

Watch this Scientific Journal Video about Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes at JoVE.com

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

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