Name: lignin down-regulation of zea mays via dsrnai and klason lignin analysis
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Description: Watch this Scientific Journal Video about Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis at JoVE.com

The microscopic imaging was conducted via the services of the Michigan State University Center for Advanced Microscopy. Maize callus was purchased from the Maize Transformation Center of Iowa State University. The authors would like to thank Jeffrey R. Weatherhead of the MSU Plant Research Laboratory for his technical assistance on the carbohydrate analysis. This research was generously funded by the Corn Marketing Program of Michigan (CMPM) and the Consortium for Plant Biotechnology Research (CPBR).

1. Preparation of dsRNAi Constructs Used for the Down-regulation of ZmCCR1
<ol>
	<li>Design gene specific primers including necessary restriction enzyme sites for making a dsRNAi construct to knock-out the ZmCCR1 gene. Two primer sets were designed to amplify two fragment segments of ZmCCR1 cDNA: a 523 bp fragment from nucleotide 748 to 1271, and a 285 bp fragment from nucleotide 986 to 1271. The ZmCCR1 cDNA was provided from the Arizona Genome Institute (AGI). More details are descr...

<ol>
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H., 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=Downregulation+of+Maize+Cinnamoyl-Coenzyme+A+Reductase+via+RNA+Interference+Technology+Causes+Brown+Midrib+and+Improves+Ammonia+Fiber+Expansion-Pretreated+Conversion+into+Fermentable+Sugars+for+Biofuels.">Downregulation of Maize Cinnamoyl-Coenzyme A Reductase via RNA Interference Technology Causes Brown Midrib and Improves Ammonia Fiber Expansion-Pretreated Conversion into Fermentable Sugars for Biofuels.</a> Crop Sci. 52, 2687-2701 (2012).</li><li>Altpeter, F., 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=Particle+bombardment+and+the+genetic+enhancement+of+crops:+myths+and+realities.">Particle bombardment and the genetic enhancement of crops: myths and realities.</a> Mol Breeding. 15, 305-327 (2005).</li><li>Pichon, M., Courbou, I., Beckert, M., Boudet, A. M., Grima-Pettenati, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+two+maize+cDNAs+encoding+cinnamoyl-CoA+reductase+(CCR)+and+differential+expression+of+the+corresponding+genes.">Cloning and characterization of two maize cDNAs encoding cinnamoyl-CoA reductase (CCR) and differential expression of the corresponding genes.</a> Plant molecular biology. 38, 671-676 (1998).</li><li>Mansoor, S., Amin, I., Hussain, M., Zafar, Y., Briddon, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+novel+traits+in+plants+through+RNA+interference.">Engineering novel traits in plants through RNA interference.</a> Trends in plant science. 11, 559-565 (2006).</li><li>Hatfield, R. D., Jung, H. -. J. G., Ralph, J., Buxton, D. R., Weimer, P. 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Review and description of methods.</a> Journal of agricultural and food chemistry. 58, 9043-9053 (2010).</li><li>Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+structural+carbohydrates+and+lignin+in+biomass.">Determination of structural carbohydrates and lignin in biomass.</a> Laboratory Analytic Procedure. , (2008).</li><li>Bate, N. 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The accessibility of microbial cellulases to plant cell wall polysaccharides is largely dependent on the degree to which they are associated with phenolic polymers23. The conversion rate from lignocellulosic biomass to fermentable sugar is negatively correlated with lignin content deposited in plant secondadry cell walls. This correlation is ascribed to the physical properties of lignin such as hydrophobicity24, chemical heterogeneity, and the absence of regular hydrolysable intermonomeric linkages<...

The production of biofuels from lignocellulosic biomass is highly desirable due to its present abundance in the U.S.1, and in the case of the sustainable harvest of agricultural and forestry residues, the ability to not compete directly for cropland used for food and animal feed production. However, unlike maize grain, which is the main source of biofuel currently generated in the U.S., lignocellulosic materials are significantly more complex and difficult to break down. In addition to the long-chain carbohydrates, cellulose and hemicellulose, which are the main sources of sugars during fermentation of lignocellulosic materials, many types of plant cell walls also contain lignin, a phenylpropanoid polymer that provides strength, defense against pathogen attack, and hydrophobicity to cell walls. While necessary for plant growth and survival, lignin also presents a significant barrier to the successful enzymatic conversion of the cellulose and hemicellulose to soluble sugars. Materials with high lignin contents are generally less desirable materials for both the biofuel (through biological conversion pathways) and the pulp and paper industries due to the negative impacts on processing characteristics and product quality. Hence, genetic manipulation of plant materials for lignin reduction at a level that does not interfere with crop structural strength and defense systems might be important for the reduction of production costs for both the lignocellulosic biofuel and the pulp and paper industries.
In maize (Zea mays), lignin is covalently cross-linked to hemicellulose in the primary cell wall via ferulate and diferulate bridges2. The lignin-hemicellulose complex binds to cellulose microfibrils through hydrogen bonds, forming a complex matrix that confers integrity and strength to the secondary cell wall. The mechanical strength of plant cell walls is largely determined by the type of lignin subunits3-5. In previous studies, altering the proportions of lignin subunits has shown no clear trend on enzymatic digestibility6-11. However, reductions in lignin content generally show an improvement in conversions12,13 and may be a key to increasing the digestibility of plant material by hydrolytic enzymes including endocellulases, cellobiohydrolases, and &#946;-glucosidases14.
Genetic engineering to regulate the expression level of transcripts has been extensively practiced to improve crop traits. Advanced techniques, including anti-sense15 and co-suppression16 technologies, enable effective down-regulation of target genes. Complete gene knock-out has also been achieved using gene constructs encoding intron-spliced RNA with a hairpin structure17. Furthermore, a double stranded RNA interference (dsRNAi) technique, i.e. a powerful and effective gene expression mediator that works by either targeting transcript degradation or translation repression, provides a potent means to induce a wide range of suppression effects on the target mRNA18. Gene silencing techniques show several limitations. These techniques do not precisely regulate the level of transcription and it could cause unexpected silencing effects on other homologous genes.
In this method, we employed particle bombardment to carry the dsRNAi constructs into the maize genome. To date, a vast array of plant species have been successfully transformed using particle bombardment, Agrobacterium mediated transformation, electroporation, and microinjection methods. In maize genetic transformation, the particle bombardment method is advantageous over all the other methods because it is the most efficient. Particle bombardment is not dependent on bacteria, so the method is free of biological constraints such as the size of the gene, species of gene origin, or the plant genotype. The physical transgene delivery system enables high molecular weight DNA and multiple genes to be introduced into plant genomes and in certain cases into chloroplasts at high transformation efficiency19. The lignin reduction in the vascular system of the leaf mid-rib can be visualized via scanning electron microscopy (SEM) which is beneficial for examining the topography and composition of samples.
In maize plants, two of the cinnamoyl-CoA reductase (ZmCCR1: X98083 and ZmCCR2: Y15069) genes were found in the maize genome20. Cinnamoyl-CoA reductase catalyzes the conversion of the hydroxycinnamoyl-CoA esters into cinnamyl aldehydes. We chose the ZmCCR1 gene to down-regulate this enzyme because the gene is expressed in all lignifying tissues. The 523 nucleotides at the 3&#8217; terminus of the ZmCCR1 gene were chosen for a dsRNAi construct because the sequences appeared to be more diverse as compared to those of ZmCCR2. Thus, the dsRNAi construct would precisely bind only to ZmCCR1, avoiding off-target silencing21. A ZmCCR1_RNAi construct was engineered into the cytoplasmic expression system ImpactVector1.1-tag (IV 1.1) containing the green tissue specific promoter, ribulose-1, 5-bisphosphate carboxylase oxygenase (RuBisCO).
To study the effects of the dsRNAi construct on transgenic plants, the lignin content was quantified. The Klason (acid-insoluble) lignin measurement is known to be more accurate compared to the acid detergent lignin quantification methods which solubilize some of the lignin22. Therefore, the Klason lignin was measured in transgenic maize stalks. This procedure consists of a two-step acid hydrolysis that converts polymeric carbohydrates into soluble monosaccharides23. The hydrolyzed biomass was then fractionated into acid soluble and insoluble materials and the acid insoluble lignin was measured according to previous studies23,24. Ideally, lignin analysis should include extractions with water and ethanol prior to the hydrolysis step, in order to remove soluble materials that can interfere with the results, and a post-hydrolysis combustion of the lignin residue to account for any ash present in the residue. Without these steps, the lignin content of the sample could be artificially inflated. The full method is presented here, however for our experiments we were unable to perform both of these steps due to the small volume of material available for testing
Two other cell wall components, cellulose and hemicellulose were also analyzed in the lignin down-regulated transgenic maize lines. It has been reported that transgenic plants that have been down-regulated in either their phenylalanine ammonia-lyase (PAL)25, 4-coumarate:CoA ligase (4CL)26, or cinnamyl alcohol dehydrogenase (CAD)27 show an increase in other cell wall structural components. As a first step in our studies, crystalline cellulose was measured using the Updegraff method28. This method was originally devised for determination of cellulose in a large number of cellulolytic bacteria and fungi. Briefly, the milled maize stocks were treated with Updegraff reagent (acetic acid: nitric acid: water) to remove hemicellulose, lignin, and xylosans. The crystalline cellulose was completely hydrolyzed into glucose via Saeman hydrolysis by adding H2SO4. The crystalline cellulose was then assayed using the colorimetric anthrone method29. To verify if the hemicellulose contents were changed, the monosaccharide extracts from milled stalks were hydrolyzed using trifluoroacetic acid, derivatized using the alditol acetate method and then analyzed by gas chromatography (GC)30. The detailed procedures for crystalline cellulose content and matrix polysaccharides composition analyses are described in Foster et al. (2010)31.
Here, we describe the procedures used for lignin down-regulation in maize via a RNAi technology, particle bombardment transformation, and lignin analysis for accelerated deconstruction of maize lignocellulosic biomass into fermentable sugars for biofuels.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Media</td>
			<td>Chemical compositions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N6OSM</td>
			<td>4 g/l N6 salts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(Osmotic medium)</td>
			<td>1 ml/l N6 vitamin stock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 mg/l 2,4-D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/l myo-inositol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>0.69 g/L proline</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>30 g/l sucrose</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/L casein hydrolysate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>36.4 g/l sorbitol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>36.4 g/l mannitol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.5g/l gelrite, pH 5.8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Add filter sterilized silver nitrate (25uM) after autoclaving</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N6E</td>
			<td>4 g/l N6 salts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(Callus induction)</td>
			<td>1 ml/l (1000X) N6 vitamin stock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 mg/l 2,4-D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/l myo-inositol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.76 g/l proline</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>30 g/l sucrose</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/l casein hydrolysate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.5g/l gelrite, pH 5.8.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Add filter sterilized silver nitrate (25uM) after autoclaving</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N6S media</td>
			<td>4 g/l N6 salts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(Selection media)</td>
			<td>1 ml/l N6 vitamin stock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2 mg/l 2,4-D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/l myo-inositol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>0.69 g/L proline</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>30 g/L sucrose</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/L casein hydrolysate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>36.4 g/l sorbitol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>36.4 g/l mannitol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>2.5g/l gelrite, pH 5.8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Add filter sterilized silver nitrate (25uM) after autoclaving</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Regeneration medium</td>
			<td>4.3 g/L MS salts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 ml/L (1000X) MS vitamin stock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/L myo-inositol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>60 g/L sucrose</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>3 g/L gelrite, pH 5.8 (100x25 mm petri-plates)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Add filter sterilized bialaphos (3 mg/L) added after autoclaving.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rooting medium</td>
			<td>4.3 g/L MS salts</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>1 ml/L MS vitamin stock</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>100 mg/L myo-inositol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>30 g/L sucrose</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>3g/L gelrite, pH 5.8 (100x25 mm petri-plates).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Specific materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Screw-top high pressure tubes</td>
			<td>Pressure tube (#8648-27); Ace Glass, Vineland, NJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Plug (#5845-47); Ace Glass, Vineland, NJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral buffered formalin&#160;</td>
			<td>100ml of formalin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(1 liter)</td>
			<td>900ml of ddH2O</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>4.0 g of Sodium dihydrogen phosphate, monohydrate&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>(NaH2PO4.H2O)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad PSD-1000/He Particle Delivery device (Hercules, CA, United States)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss PASCAL confocal laser scanning microscope (Carl Zeiss, Jena, Germany)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excelsior ES Tissue Processor (Thermo Scientific, Pittsburgh, PA, United States).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HistoCentre III Embedding Station (Thermo Scientific, Pittsburgh, PA, United States)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Microtome Model Reichert 2030 (Reichert, Depew, NY, United States)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Emscope Sputter Coater model SC 500 (Ashford, Kent, England)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>JEOL JSM-6400V Scanning Electron Microscope (JEOL Ltd., Tokyo, Japan)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fitzpatrick JT-6 Homoloid mill; Continental Process Systems, Inc., Westmont, IL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MA35 Moisture Analyzer; Sartorius</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Critical point dryer, Balzers CPD (Leica Microsysstems Inc, Buffalo Grove, IL, United States)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

lignin down-regulation of zea mays via dsrnai and klason lignin analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

We have demonstrated a reduction in the lignin content in maize plants via RNAi. The particle bombardment transformation method yielded around 30% trnasformation efficiency. The gene silencing of ZmCCR1 was consistently observed in T0-T2 generations. The lignin reduced transgenics grew similarly to wildtype maize plants except for displaying brown coloration in the leaf mid-rib, husk, and stem. The histological assay has shown that the mutant lines exhibit a significant reduction in the cell wall thickness of th...

Watch this Scientific Journal Video about Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis at JoVE.com

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

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