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Improving Saccharification Efficiency of Alfalfa Stems Through Modification of the Terminal Stages of Monolignol Biosynthesis

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Abstract

A series of transgenic lines of alfalfa (Medicago sativa) were generated in which either one of the two potentially terminal enzymes of the monolignol pathway, cinnamoyl CoA reductase (CCR) or cinnamyl alcohol dehydrogenase (CAD) was down-regulated by expression of antisense transgenes. Levels of CCR enzymatic activity were reduced to between 10% to 65% of the control level, and levels of CAD activity were similarly reduced to between 5% to 40% of the control. Biomass yields were reduced in the most strongly down-regulated lines for both transgenes, but many of the lines exhibited reduced lignin levels but normal biomass and flowering time. In vitro dry matter digestibility was increased for most transgenic lines compared to controls. Saccharification efficiency was determined by measuring the release of sugars from cell walls directly, or after sulfuric acid pre-treatment and subsequent digestion with a mixture of cellulase and cellobiase. Several CCR down-regulated lines had significantly enhanced saccharification efficiency with both pre-treated and untreated tissues, whereas CAD down-regulation had less impact on sugar release when compared to that from CCR lines with similar lignin contents. One CCR line with a 50–60% improvement in saccharification efficiency exhibited normal biomass production, indicating the potential for producing high yielding, improved feedstocks for bioethanol production through genetic modification of the monolignol pathway.

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Abbreviations

CAD:

cinnamyl alcohol dehydrogenase

CCR:

cinnamoyl coenzyme A reductase

G:

guaiacyl

HCT:

hydroxycinnamoyl transferase

S:

syringyl

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Acknowledgements

We thank Drs Ajith Anand and Stephen Temple for critical reading of the manuscript, and Dennis Walker for assistance with fiber quality analysis. This work was supported by the US Department of Energy (award number DE-FG02-06ER64303), Forage Genetics International and the Samuel Roberts Noble Foundation. This report was prepared as an account of work partly sponsored by the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of the authors expressed herein do not necessarily reflect those of the United States Government or any agency thereof.

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Authors and Affiliations

  1. Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK, 73401, USA

    Lisa A. Jackson, Gail L. Shadle, Rui Zhou, Jin Nakashima, Fang Chen & Richard A. Dixon

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  1. Lisa A. Jackson
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  2. Gail L. Shadle
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  3. Rui Zhou
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  4. Jin Nakashima
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  5. Fang Chen
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  6. Richard A. Dixon
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Correspondence to Richard A. Dixon.

Additional information

Lisa A. Jackson, Gail L. Shadle: These authors contributed equally to this work.

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Improving saccharification efficiency of alfalfa stems through modification of the terminal stages of monolignol biosynthesis. (DOC 410 KB)

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Jackson, L.A., Shadle, G.L., Zhou, R. et al. Improving Saccharification Efficiency of Alfalfa Stems Through Modification of the Terminal Stages of Monolignol Biosynthesis. Bioenerg. Res. 1, 180–192 (2008). https://doi.org/10.1007/s12155-008-9020-z

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