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      RNAi downregulation of three key lignin genes in sugarcane improves glucose release without reduction in sugar production

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          Abstract

          Background

          Sugarcane is a subtropical crop that produces large amounts of biomass annually. It is a key agricultural crop in many countries for the production of sugar and other products. Residual bagasse following sucrose extraction is currently underutilized and it has potential as a carbohydrate source for the production of biofuels. As with all lignocellulosic crops, lignin acts as a barrier to accessing the polysaccharides, and as such, is the focus of transgenic efforts. In this study, we used RNAi to individually reduce the expression of three key genes in the lignin biosynthetic pathway in sugarcane. These genes, caffeoyl-CoA O-methyltransferase ( CCoAOMT), ferulate 5-hydroxylase ( F5H) and caffeic acid O-methyltransferase ( COMT), impact lignin content and/or composition.

          Results

          For each RNAi construct, we selected three events for further analysis based on qRT-PCR results. For the CCoAOMT lines, there were no lines with a reduction in lignin content and only one line showed improved glucose release. For F5H, no lines had reduced lignin, but one line had a significant increase in glucose release. For COMT, one line had reduced lignin content, and this line and another released higher levels of glucose during enzymatic hydrolysis. Two of the lines with improved glucose release (F5H-2 and COMT-2) also had reduced S:G ratios.

          Conclusions

          Along with improvements in bagasse quality for the production of lignocellulosic-based fuels, there was only one line with reduction in juice sucrose extraction, and three lines with significantly improved sucrose production, providing evidence that the alteration of sugarcane for improved lignocellulosic ethanol production can be achieved without negatively impacting sugar production and perhaps even enhancing it.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13068-016-0683-y) contains supplementary material, which is available to authorized users.

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          Most cited references48

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          The origin and evolution of lignin biosynthesis.

          Lignin, a phenolic polymer derived mainly from hydroxycinnamyl alcohols, is ubiquitously present in tracheophytes. The development of lignin biosynthesis has been considered to be one of the key factors that allowed land plants to flourish in terrestrial ecosystems. Lignin provides structural rigidity for tracheophytes to stand upright, and strengthens the cell wall of their water-conducting tracheary elements to withstand the negative pressure generated during transpiration. In this review, we discuss a number of aspects regarding the origin and evolution of lignin biosynthesis during land plant evolution, including the establishment of its monomer biosynthetic scaffold, potential precursors to the lignin polymer, as well as the emergence of the polymerization machinery and regulatory system. The accumulated knowledge on the topic, as summarized here, provides us with an evolutionary view on how this complex metabolic system emerged and developed.
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            The genetics of lignin biosynthesis: connecting genotype to phenotype.

            The processes underlying lignification, which for many years have been the near-exclusive purview of chemists and biochemists, have more recently been approached using both classical forward genetic screens and targeted reverse genetic approaches such as antisense suppression, RNAi, and characterization of insertional mutants. In this review, we provide an overview of the current understanding of lignin biosynthesis and structure, with emphasis on mutant and transgenic plants that have contributed to this knowledge. We also discuss ongoing work aimed at elucidating the relationship between lignin structure and function in vivo, as well as the phenotypic consequences arising from genetic manipulation of the lignin biosynthetic pathway.
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              Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants.

              A set of plasmids has been constructed utilizing the promoter, 5' untranslated exon, and first intron of the maize ubiquitin (Ubi-1) gene to drive expression of protein coding sequences of choice. Plasmids containing chimaeric genes for ubiquitin-luciferase (Ubi-Luc), ubiquitin-beta-glucuronidase (Ubi-GUS), and ubiquitin-phosphinothricin acetyl transferase (Ubi-bar) have been generated, as well as a construct containing chimaeric genes for both Ubi-GUS and Ubi-bar in a single plasmid. Another construct was generated to allow cloning of protein coding sequences of choice on Bam HI and Bam HI-compatible restriction fragments downstream of the Ubi-1 gene fragment. Because the Ubi-1 promoter has been shown to be highly active in monocots, these constructs may be useful for generating high-level gene expression of selectable markers to facilitate efficient transformation of monocots, to drive expression of reference reporter genes in studies of gene expression, and to provide expression of biotechnologically important protein products in transgenic plants.
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                Author and article information

                Contributors
                patrick.bewg@qut.edu.au
                crpoovai@syr.edu
                wlan2@wisc.edu
                +1 608 890 2429 , jralph@wisc.edu , http://www.biochem.wisc.edu/faculty/ralph/
                315-443-0453 , hcoleman@syr.edu
                Journal
                Biotechnol Biofuels
                Biotechnol Biofuels
                Biotechnology for Biofuels
                BioMed Central (London )
                1754-6834
                20 December 2016
                20 December 2016
                2016
                : 9
                : 270
                Affiliations
                [1 ]Queensland University of Technology, Brisbane, QLD 4000 Australia
                [2 ]Department of Biology, Syracuse University, Syracuse, NY 13244 USA
                [3 ]Department of Biological Systems Engineering, University of Wisconsin, Madison, WI USA
                [4 ]US Department of Energy, Great Lakes Bioenergy Research Center (GLBRC), Wisconsin Energy Institute, University of Wisconsin, Madison, WI 53726 USA
                [5 ]Department of Biochemistry, University of Wisconsin, Madison, WI 53726 USA
                Author information
                http://orcid.org/0000-0001-7157-5176
                http://orcid.org/0000-0002-0677-3085
                http://orcid.org/0000-0002-6093-4521
                http://orcid.org/0000-0002-4923-601X
                Article
                683
                10.1186/s13068-016-0683-y
                5168864
                28031745
                7c880c16-86e6-4ae7-9ae3-dfa93d1e79f6
                © The Author(s) 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 29 September 2016
                : 6 December 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100000923, Australian Research Council;
                Funded by: FundRef http://dx.doi.org/10.13039/501100001055, Sugar Research and Development Corporation;
                Funded by: FundRef http://dx.doi.org/10.13039/501100000038, Natural Sciences and Engineering Research Council of Canada;
                Funded by: FundRef http://dx.doi.org/10.13039/100006132, Office of Science;
                Categories
                Research
                Custom metadata
                © The Author(s) 2016

                Biotechnology
                lignin biosynthesis,ferulate 5-hydroxylase,caffeic acid o-methyltransferase,caffeoyl-coa o-methyltransferase,sugarcane,rnai

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