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

Summary

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.

Abstract

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.

Introduction

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 wal....

Protocol

1. Preparation of dsRNAi Constructs Used for the Down-regulation of ZmCCR1

1. 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 described in Figure 1.
2. Amplify the large fragment by polymerase chain reaction (PCR) from the ZmCCR1 cDNA template usin....

Results

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.......

Discussion

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<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
N6OSM (Osmotic medium)			Made in-house
N6E (Callus induction)			Made in-house
N6S media (Selection media)			Made in-house
Regeneration medium			Made in-house
Rooting medium			Made in-house
10% Neutral buffered formalin (1 L)			Made in-house
Bio-Rad PSD-1000/He Particle Delivery device	Hercules, CA, United States
Zeiss PASCAL confocal laser scanning microscope	Carl Zeiss, Jena, Germany		For brightfield microscopy, the images were recorded using a Zeiss (Jena, Germany) PASCAL confocal laser scanning microscope with a 488 nm excitation mirror, a 560 nm emission filter, and a 505-530 nm emission filter. Image analysis was performed using Laser scanning microscope PASCAL LSM version 3.0 SP3 software.
Excelsior ES Tissue Processor	Thermo Scientific, Pittsburgh, PA, United States
HistoCentre III Embedding Station	Thermo Scientific, Pittsburgh, PA, United States
Microtome Model Reichert 2030	Reichert, Depew, NY, United States
Emscope Sputter Coater model SC 500	Ashford, Kent, England
JEOL JSM-6400V Scanning Electron Microscope	JEOL Ltd., Tokyo, Japan
Fitzpatrick JT-6 Homoloid mill	Continental Process Systems, Inc., Westmont, IL
MA35 Moisture Analyzer	Sartorius
Critical point dryer, Balzers CPD	Leica Microsysstems Inc, Buffalo Grove, IL, United States
Screw-top high pressure tubes	Ace Glass, Vineland, NJ	#8648-27
Screw-top high pressure tube plugs	Ace Glass, Vineland, NJ	#5845-47