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Park S.-H.,University of Arizona | Ong R.G.,Michigan State University | Mei C.,The Institute for Sustainable and Renewable Resources | Sticklen M.,Michigan State University
Journal of Visualized Experiments | Year: 2014

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. © JoVE 2006-2014. All Rights Reserved. Source

Park S.-H.,Michigan State University | Mei C.,The Institute for Sustainable and Renewable Resources | Pauly M.,Michigan State University | Pauly M.,University of California at Berkeley | And 6 more authors.
Crop Science | Year: 2012

Conversion of lignocellulosic biomass into fermentable sugars for biofuels requires expensive pretreatment processes involving the breakdown of the cell wall structure and/ or removal of lignin to increase accessibility of enzymes to the crop structural carbohydrates. Lignin is synthesized from precursors through a complex biosynthesis pathway. One of the important enzymes in this pathway is cinnamoylcoenzyme A reductase (CCR), which catalyzes the transformation of feruloyl and p-coumaryl thioesters to their respective aldehydes. In an attempt to reduce lignin content and potentially accelerate deconstruction of maize (Zea mays L.) stover structural carbohydrates into fermentable sugars, expression of maize CCR (ZmCCR1; EC was downregulated via ribonucleic acid interference (RNAi). Thirty first generation independent ZmCCR1_RNAi transgenic lines were produced. Among 10 out of 30 randomly tested, six lines showed significantly reduced ZmCCR1 transcription. The second generation of these ZmCCR1 downregulated transgenic plants exhibited brown coloration of midribs, husk, and stems and 7.0 to 8.7% reduction in Klason lignin. Also, crystalline cellulose was slightly increased in the lignin downregulated maize stover and further increased conversion of the ammonia fiber expansion (AFEX)-pretreated maize stover into fermentable sugars. The third generation of CCR downregulated plants showed further reduced CCR transcription as compared to their second generation of transgenic (T1) plants. © Crop Science Society of America. Source

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