Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.)

Authors

  • Fang Chen,

    1. Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401, USA, and
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    • These authors contributed equally to this work.

  • Marry S. Srinivasa Reddy,

    1. Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401, USA, and
    2. Forage Genetics International, N5292 South Gills Coulee Road, West Salem, WI 54669, USA
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    • These authors contributed equally to this work.

  • Stephen Temple,

    1. Forage Genetics International, N5292 South Gills Coulee Road, West Salem, WI 54669, USA
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  • Lisa Jackson,

    1. Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401, USA, and
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  • Gail Shadle,

    1. Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401, USA, and
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  • Richard A. Dixon

    Corresponding author
    1. Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401, USA, and
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*For correspondence (fax +1 580 224 6609; e-mail radixon@noble.org).

Summary

Genes encoding seven enzymes of the monolignol pathway were independently downregulated in alfalfa (Medicago sativa) using antisense and/or RNA interference. In each case, total flux into lignin was reduced, with the largest effects arising from the downregulation of earlier enzymes in the pathway. The downregulation of l-phenylalanine ammonia-lyase, 4-coumarate 3-hydroxylase, hydroxycinnamoyl CoA quinate/shikimate hydroxycinnamoyl transferase, ferulate 5-hydroxylase or caffeic acid 3-O-methyltransferase resulted in compositional changes in lignin and wall-bound hydroxycinnamic acids consistent with the current models of the monolignol pathway. However, downregulating caffeoyl CoA 3-O-methyltransferase neither reduced syringyl (S) lignin units nor wall-bound ferulate, inconsistent with a role for this enzyme in 3-O-methylation ofS monolignol precursors and hydroxycinnamic acids. Paradoxically, lignin composition differed in plants downregulated in either cinnamate 4-hydroxylase or phenylalanine ammonia-lyase. No changes in the levels of acylated flavonoids were observed in the various transgenic lines. The current model for monolignol and ferulate biosynthesis appears to be an over-simplification, at least in alfalfa, and additional enzymes may be needed for the 3-O-methylation reactions of S lignin and ferulate biosynthesis.

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