This work was supported by the BMBF Forschungsinitiative &#8220;BioEnergie 2021 &#8211; Forschung f&#252;r die Nutzung von Biomasse&#8221; and the Cluster of Excellence &#8220;Tailor-made Fuels from Biomass&#8221;, which is funded through the Excellence Initiative by the German federal and state governments to promote science and research at German universities.

1. Growth of Wild Type N. tabacum&#160;Plants
<ol>
	<li>To grow N. tabacum L. cv. Petit Havana SR1 plants on soil, place tobacco seeds on appropriate pots filled with normal potting soil for germination. After 2 weeks, transfer seedlings to individual pots. Incubate plants in a greenhouse with constant 22 &#176;C (dark period) or 25 &#176;C (light period) and 70% relative air humidity. Provide illumination in a 16/8&#160;hr light/dark cycle (e.g. Philips IP65 400 W lamps; 180 &#956;mol&#183;sec-1&#183;m-2; &#955;= 400-700 nm) and water daily.</li>
</ol>
2. Transient Expression of TrCel5A and TrCel5A-mCherry Proteins
<ol>
	<li>Under sterile conditions, pick single colonies of Agarobacterium tumefaciens (GV3101::pMP90RK GmR, KmR, RifR) containing either pTRAkc-ER-TrCel5A or pTRAkc-ER -TrCel5A-mCherry and transfer to an Erlenmeyer flask containing 10 ml&#160;yeast extract broth (YEB, prepared as per manufacturers specifications) medium and kanamycin (50 mg/ml), rifampicin (50 mg/ml), and carbenicillin (100 mg/ml) for selection. Incubate for 36-40 hr at 26 &#176;C and 180 rpm. Note: The design of appropriate constructs for protein expression is outside the scope of this paper, but for guidance, the methods of Maclean et al.13 and Munro and Pellham14 can be followed for construct design.</li>
	<li>Take 200 &#181;l&#160;of each culture to inoculate 20 ml&#160;of YEB with the antibiotic concentrations mentioned above and incubate under the same conditions as before.</li>
	<li>After 36-40 hr, preinduce the cultures by adding 2 &#181;l&#160;acetosyringon (200 &#181;M final concentration), 100 &#181;l&#160;40% (w/v) glucose solution and 200 &#181;l&#160;of 1 M MES buffer pH 5.6. Incubate overnight.</li>
	<li>Prepare 100 ml&#160;of a 2x infiltration buffer (100 g/L sucrose, 3.6 g/L glucose, 8.6 g/L Murashige and Skoog basal salts, adjust pH to 5.6 using potassium hydroxide).</li>
	<li>Measure OD600 of the cultures with 1:5 dilutions until it reaches an OD600 of 5-6. Calculate culture volume needed for 30 ml&#160;infiltration medium at OD600. Add 15 ml&#160;of 2x infiltration buffer, then add deionized H2O to make the buffer up to 30 ml.</li>
	<li>Take plants from greenhouse and use a pipette tip to nick the abaxial side of the 3rd to 5th leaf from the top to ease infiltration.</li>
	<li>Use a pipette tip to nick the abaxial side of the leaves to ease infiltration.</li>
	<li>Fill a syringe with infiltration medium and put it (without needle) on one of the previously made nicks. Support syringe with a finger on the adaxial leaf side. Press the medium carefully into the tissue of interveinal leaf zones. Note: After infiltration, cultivate plants in a greenhouse under the same conditions as described above. Also, maintain an equal proportion of untreated, non-transient expression plants (NTEPs) as controls, under the same conditions as infiltrated plants.</li>
</ol>
3. Assessment of TrCel5A-mCherry Expression in Plant Leaves
<ol>
	<li>After 4-6 days, take plants infiltrated with A. tumefaciens containing pTRAkc-ER-TrCel5A-mCherry from the greenhouse and place them in a dark room for fluorescence analysis.</li>
	<li>To visualize the fluorescence in leaf sections expressing TrCel5A-mCherry, light plants using a portable light source emitting green light at 515&#160;nm with a red filter (620-750&#160;nm). Note: In depth characterization of protein expression in the plant leaves can be achieved by thin-sectioning leaves with a vibratome and examination under light and fluorescence microscopy. For this follow the steps below:
	<ol>
		<li>Cut small sections of approximately 5 x 5 mm size from an infiltrated leaf and embed them in 4% agarose dissolved in 50 mM Phosphate buffer (pH 5.7).</li>
		<li>Cut section of 80-90 &#181;m size from embedded leaves using a vibratome. Place them on a microscope slide, cover with buffer or a cover slip and examine them using a microscope at 10X&#160;magnification with a fluorescence detector, using the same wavelength and filter stated above.</li>
	</ol>
	</li>
</ol>
4. TrCel5A Extraction from Tobacco Leaves
<ol>
	<li>Cut pieces from tobacco leaves to an approximate weight of 1 g from plants&#160;infected with A. tumefaciens containing pTRAkc-ER-TrCel5A and place in a mortar precooled with liquid nitrogen. Add an appropriate amount of liquid nitrogen to the samples. Grind leaves to powder using a precooled pestle.</li>
	<li>Add 2 ml&#160;of phosphate buffered saline (PBS) containing 1 mM phenylmethylsulfonyl fluoride to the ground leaf matter and mix until the majority of leaf matter is in suspension. Centrifuge extract at 15,000 x g for 20 min at 4 &#176;C and take protein-containing supernatant.</li>
	<li>Centrifuge the extract at 15,000 x g for 20 min at 4&#160;&#176;C and take protein-containing supernatant being careful not to disrupt the pellet. Discard the pellet.</li>
	<li>Partially purify TrCel5A by incubating the protein-containing supernatant at 55 &#176;C for 10 min. Allow to rest at room temperature for a further 10 min, then centrifuge at 15,000 x&#160;g for 10 min at RT. Repeat partial purification process for non-infiltrated plants to obtain control samples. Use these samples for all following experiments. Note: Protein solutions can be stored at 4 &#176;C for up to 1 week or at -20 &#176;C for up to 3 months.</li>
	<li>Repeat the partial purification process for non-infiltrated plants to obtain control samples. Use these samples for all following experiments. Note: Protein solutions can be stored at 4&#160;&#176;C for up to 1 week or at -20&#160;&#176;C for up to 3 months.</li>
</ol>
5. SDS-PAGE, Western Blotting, and Azo-CMC Zymography of TrCel5A-mCherry
<ol>
	<li>Purchase or prepare 12% (w/v) SDS-PAGE gels as per established methods15.</li>
	<li>Dissolve CMC&#160;in water to obtain a 1.5% (w/v) solution. To make CMC-SDS-PAGE gels, substitute 1 ml&#160;of 1.5% CMC for 1 ml&#160;of water in a 10 ml&#160;SDS-PAGE gel mix solution, resulting in a 0.1% (w/v) CMC + 12% (w/v) SDS-PAGE gel mix. Pour gel normally. Note: Make certain that the CMC in the original solution is completely dissolved by a combination of stirring and heating to 40 &#176;C (repeat directly prior to preparing the gel solution).</li>
	<li>Denature samples by boiling in the presence of SDS, as per established methods12. As a positive control, use a 1:500 dilution of a commercially available cellulase mixture from T. reesei, such as T. reesei ATCC 26921. As a negative control use a similarly prepared protein solution from non-infiltrated leaves (NTEPs).</li>
	<li>Carry out the electrophoresis as per normal protocols15, e.g.&#160;use 200 V for ~50 min with the electrophoresis halted once the dye front reached the bottom of the gel.</li>
	<li>Stain one SDS-PAGE gel using 0.1% (w/v) Coomassie brilliant blue and destain using a methanol/glacial acetic acid mixture. Note: Rapid staining and destaining can be performed by gently heating the staining and destaining solutions, i.e. by using a microwave for ~15 sec to warm (but not boil) first the staining and then the destaining solutions, changing the destaining solution after 10 min, or when cooled, and repeat with new destaining solution.</li>
	<li>Perform Western blotting of one SDS-PAGE gel using established protocols16,17. Use the following antibodies: a polyclonal rabbit antibody against a&#160;His-tag (primary) and a polyclonal goat anti-rabbit antibody Fc region which has been conjugated to an alkaline phosphatase (secondary). Note: secondary antibodies should be selected to enable visualization using nitroblue tetrazolium chloride/5-bromo-4-chloro-3&#8242;-indolyphosphate p-toluidine salt (NBT/BCIP), as described18.</li>
	<li>Perform in-gel protein refolding on the 0.15% (w/v) CMC SDS-PAGE gel by incubating the gel in 1.5% (w/v) &#946;-cyclodextrin, 20 mM phosphate-citrate buffer, pH 6.5, at room temperature with gentle shaking for 15 min19.</li>
	<li>Discard the &#946;-cyclodextrin solution and briefly wash the gel with H2O. Incubate at RT in 50mM sodium acetate buffer (pH 4.8) for a further 15 min. Perform CMC zymography using the refolded 0.15% CMC SDS-PAGE gel by incubating the gel in 30 mM sodium acetate buffer pH 4.8 for 20 min at 50 &#176;C.</li>
	<li>Perform CMC zymography using the refolded 0.15% (w/v) CMC SDS-PAGE gel by incubating the gel in 30 mM sodium acetate buffer&#160;(pH 4.8) for 20 min at 50&#160;&#176;C.</li>
	<li>Stain the gel for 10&#160;min using a 0.1% w/v Congo red solution and destain for 30 min or until clear bands are observed using 1 M NaCl. Wash the gel with 0.3% (v/v) acetic acid for clearer imaging.</li>
</ol>
6. 4-MUC Activity Assay of TrCel5A
<ol>
	<li>Prepare a stock solution of 10 mM 4-MUC in 50% (v/v) methanol, and store it in the dark at RT</li>
	<li>Immediately prior to the assay, prepare a 4-MUC 2x working solution, composed of 1 mM 4-MUC and 60 mM sodium acetate, pH 4.8.</li>
	<li>Perform this test on samples containing 100 &#956;l of leaf protein (with or without TrCel5A inducibly expressed), 100 &#956;l a commercially available cellulase mixture from T. reesei 1:1,000, and pure H2O, respectively. Run each sample in triplicate.</li>
	<li>Pipette 100 &#956;l of the 4-MUC working solution into separate wells of a 96-well plate, then add 100 &#956;l of each sample.</li>
	<li>Determine 0 min timepoint of 4-MU fluorescence through fluorescent spectroscopy, using an excitation wavelength of 360 nm and an emission wavelength of 465 nm.</li>
	<li>Incubate plates for 20 min at 50 &#176;C, covered with adhesive lids to prevent evaporation. Stop the reaction by freezing (or by combining&#160;100 &#956;l 0.15 M glycine, pH 10.0, and 100 &#956;l of sample in a separate plate).</li>
	<li>Determine the endpoint of 4-MU fluorescence through fluorescent spectroscopy, using an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Subtract 0 min from endpoint (20 min) timepoint to obtain the change in fluorescence values.</li>
</ol>
7. Azo-carboxymethyl Cellulose Activity Assay of TrCel5A
<ol>
	<li>Prepare a stock of 1.5% (w/v) Azo-CMC which is stored at RT, and a precipitation solution containing 18 mM zinc acetate, 0.5 M sodium acetate, pH 5 and 80% ethanol (v/v), store at room temperature.</li>
	<li>Immediately prior to the assay, prepare an Azo-CMC 2x working solution, composed of 1% (w/v) Azo-CMC (in H2O) and 60 mM sodium acetate, pH 4.8.</li>
	<li>Combine 50 &#956;l of sample and 50 &#956;l of the working solution in 1.5 ml&#160;tubes.</li>
	<li>Test samples containing 50 &#956;l of leaf protein (with or without TrCel5A transiently expressed); 100 &#956;l of a commercially available cellulase mixture from T. reesei diluted 1:1,000; and pure H2O. Run samples and standards in triplicate.</li>
	<li>Incubate samples for 20 min at 50 &#176;C. Stop the reaction by adding 250 &#956;l of precipitation solution. Vortex each sample vigorously for 10 sec to ensure solutions were completely combined.</li>
	<li>Incubate samples at RT for 10 min and then vortex again. Centrifuge samples at 1,000 x g for 10 min. Remove 200 &#956;l&#160;of each sample supernatant to a 96-well plate, taking care not to disrupt the pellet.</li>
	<li>Assay using absorbance spectroscopy at a wavelength of 590 nm.</li>
</ol>
8. PAHBAH Assay of TrCel5A
<ol>
	<li>Use a hole-punch to cut a circle (~5.5 mm diameter) of filter paper. Add 100 &#956;l&#160;60 mM NaAc, pH 4.8, and 100 &#956;l&#160;of samples, either containing leaf protein (with or without TrCel5A inducibly expressed) or a commercial T. reesei cellulase mixture, diluted 1:1,000, and incubate with filter paper circles for 20 hr at 50 &#176;C with 300 rpm shaking. For each sample incubated with filter paper, also incubate a duplicate solution without filter paper as individual sample-controls.</li>
	<li>Prepare a 330 mM p-hydroxy benzoic acid hydrazide (PAHBAH) solution A by dissolving PAHBAH in H2O with the addition of 5 ml&#160;concentrated HCl for a total of 100 ml&#160;solution, and store at RT. Note: Best results are achieved by adding the HCl to a smaller volume of PAHBAH slurry, i.e. 30 ml, which is stirred under temperature to help dissolution before making the volume to 100 ml&#160;with H2O.</li>
	<li>Prepare PAHBAH solution B, containing 50 mM sodium citrate, 10 mM calcium chloride, and 500 mM NaOH and&#160;store&#160;at RT.</li>
	<li>Immediately prior to the assay, prepare a 1.5x working solution of 1 ml&#160;PAHBAH reagent to 9 ml&#160;buffer solution, store on ice until use (to be used within 4 hr).</li>
	<li>Prepare samples in thermostable 0.2 or 0.5 ml&#160;tubes. Combine 5 &#956;l of each solution incubated with filter paper (step 8.1) 45 &#956;l of H2O and 100 &#956;l of PAHBAH working solution. Sample controls (samples incubated without filter paper) should be run under the same concentrations (5 &#956;l sample, 45 &#956;l H2O, 100 &#956;l PAHBAH working solution). Also test 5 standards (5 &#956;l) containing between 0 and 0.2 g/L of glucose, and pure H2O. Run samples and standards in triplicate.</li>
	<li>Incubate samples for exactly 10 min at 100 &#176;C, then cool at RT for exactly 10 min. Pipette solutions into a 96 well plate and assay spectroscopically at a wavelength of 410 nm. Subtract individual sample-controls (incubated without filter paper) from samples to obtain final values.</li>
</ol>

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The authors have nothing to disclose.

Recombinant expression of cellulases is a field of great interest, due to the desire for more efficient biofuel production systems1. Plants offer many possibilities for successful cellulase production, with features such as economic harvesting techniques and autohydrolysis methods being very desirable properties for large-scale biofuel production. Further, the use of different localizations for producing proteins allow, if required, posttranslational modifications20. Altering plants to enable consti...

Degradation of lignocellulosic biomass and its conversion into liquid fuel has been envisioned as a method to reduce reliance on fossil fuels. One significant hurdle in establishing economically feasible biomass processing systems is in the enzymatic degradation of cellulose and hemicellulose1. Plant expression systems show great potential for the production of enzymes on an industrial scale. Agricultural systems to harvest plants economically and in volume are already established, as they have been for thousands of years. The production of cellulases in plants is desirable due to factors like the ease of autohydrolysis2, and the potential for maximizing the use of lower enzyme levels by increasing the digestibility of plant cell walls1. Finally, plant systems allow the targeting of recombinant proteins to specific areas of plant cells and are able to posttranslationally modify enzymes where required3.

Nicotiana tabacum (tobacco) is very commonly used as a model organism for heterologous protein expression studies in plants, due to its rapid growth and biomass accumulation features4. Transient expression of recombinant proteins is a technique which enables protein production in short time periods5, while maintaining flexibility as to the localization and thus posttranslational modifications of the selected proteins, i.e. cellulases. This enables the production of the cellulase(s) for basic analysis, while also laying the groundwork for further expression strategies in plants. Using such a transient expression strategy, an endoglucanase from glycosyl hydrolase family 5, Cel5A, derived from the fungal host Trichoderma reesei (Hypocrea jecorina)6 is produced (hereafter referred to as TrCel5A). TrCel5A is a 42 kDa protein which is natively glycosylated and is highly active in hydroylzing cellulose chains7.

The transient expression technique described here is based on a relatively commonly used system, infiltrating the plant leaves with Agrobacteria carrying the gene of interest in an appropriate expression vector. To allow for rapid analysis of successful in planta expression, TrCel5A can also be expressed as a fusion protein with mCherry, a monomeric fluorescent protein originally derived from Discosoma sp. protein DsRed8, with an additional six Histidine residues (His-tag) fused to the C-terminus (TrCel5A-mCherry). Expression of the heterologous fusion protein, TrCel5A-mCherry, can thus be monitored within the growing plant by using green light to examine mCherry dispersal. If desired, thin sections of the plant material can be examined under green light microscopically to establish the specific localization of the protein. For this work, signal and transit peptides were incorporated in the construct, to localize the heterologous protein export to the endoplasmic reticulum9.

To analyze the activity of plant expressed cellulases, including TrCel5A, a number of cellulase activity tests can be run. After the extraction of the total soluble plant proteins, TrCel5A can be partially purified using a thermal incubation technique. Protein size is established using SDS-PAGE followed by Western blotting. Zymography can be used to analyze activity against substrates, e.g. cellulose which has been carboxy-methylated (making carboxymethyl cellulose: CMC) for solubility10. Cellulase and glucosidase activity can be monitored using the fluorophore 4-methylumbelliferyl (4-MU) associated with &#946;-D-cellobioside (combined: 4-MUC). Another method to assess endoglucanase activity involves the spectrometric analysis of CMC&#160;which has been associated with an azo-dye (Remazolbrilliant Blue R)11. In addition, protein activity of both endoglucanases and a range of cellulases can be monitored by the use of a sugar analysis test, such as the p-hydroxy benzoic acid hydrazide (PAHBAH) assay12. These techniques can be used to elucidate and quantify the activity of the expressed cellulase of interest.

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:308px;">4-methylumbelliferyl &#946;-D-cellobioside</td>
			<td style="width:127px;">SIGMA-ALDRICH</td>
			<td style="width:156px;">M6018</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>ROTH</td>
			<td>3738</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetosyringon (concentrated)</td>
			<td>ROTH</td>
			<td>6003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - kanamycin</td>
			<td>ROTH</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - rifampicin</td>
			<td>ROTH</td>
			<td>4163</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - carbenicillin</td>
			<td>ROTH</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibody - rabbit anti His(6) pAb</td>
			<td>ROCKLAND</td>
			<td>600-401-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibody - goat anti rabbit AP, FC pAb</td>
			<td>DIANOVA</td>
			<td>111-055-008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Azo-carboxymethyl cellulose</td>
			<td>MEGAZYME</td>
			<td>S-ACMC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-cyclodextrin</td>
			<td>SIGMA-ALDRICH</td>
			<td>C4805</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carboxymethyl cellulose</td>
			<td>ROTH</td>
			<td>3333.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellulase from Trichoderma reesei ATCC 26921</td>
			<td>SIGMA-ALDRICH</td>
			<td>C2730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Congo red</td>
			<td>SIGMA-ALDRICH</td>
			<td>75768</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomasie brilliant blue G 250</td>
			<td>ROTH</td>
			<td>9598.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D(+)-glucose</td>
			<td>ROTH</td>
			<td>8337</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>ROTH</td>
			<td>K928</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter paper - Rotilabo blotting paper</td>
			<td>ROTH</td>
			<td>CL67</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NBT/BCIP</td>
			<td>SIGMA-ALDRICH</td>
			<td>72091</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES buffer</td>
			<td>ROTH</td>
			<td>4256</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>ROTH</td>
			<td>8388</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium citrate</td>
			<td>ROTH</td>
			<td>3580</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecylsulfate</td>
			<td>SERVA</td>
			<td>20765</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>ROTH</td>
			<td>6771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Soil, Einheitserde classic&#160;</td>
			<td>PATZER GMBH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrometer - Infinite M200</td>
			<td>TECAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>SIGMA-ALDRICH</td>
			<td>S-5390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Hydroxy benzhydrazide&#160;</td>
			<td>SIGMA-ALDRICH</td>
			<td>H9882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>ROTH</td>
			<td>1058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV light source - BLAK RAY</td>
			<td>UVP</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vibratome - VT1000S</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

expression of recombinant cellulase cel5a from trichoderma reesei in tobacco plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

TrCel5A and TrCel5A-mCherry were expressed successfully in tobacco plants using the transient expression system (Figures 1A and 1B). Examination of the expression pattern of the TrCel5A-mCherry fusion protein revealed healthy leaves (Figure 1C), which showed widespread protein expression under green light (Figure 1D).
Extraction of the total soluble proteins was performed on tobacco plants which had leaves that contained the t...

Watch this Scientific Journal Video about Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants at JoVE.com

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

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12.7K Views.  RWTH Aachen University. Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Video: Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p&#8804;0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing seeds of Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum). We applied magnets under the culture dish. During the 4 days of application, we observed that the stem and root length increased. The group subjected to magnetic field treatment (n = 10) showed a 1.4 times faster rate of growth compared with the control group (n = 11) in a total of 8 days (p &#60;0.0005). This rate is 20% higher than that reported in previous studies. The tubulin complex lines did not have connecting points, but connecting points occur upon the application of magnets. This shows complete difference from the control, which means abnormal arrangements. However, the exact cause remains unclear. These results of growth enhancement of applying magnets suggest that it is possible to enhance the growth rate, increase productivity, or control the speed of germination of plants by applying static magnetic fields. Also, magnetic fields can cause physiological changes in plant cells and can induce growth. Therefore, stimulation with a magnetic field can have possible effects that are similar to those of chemical fertilizers, which means that the use of fertilizers can be avoided.

The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...