Forraje

Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa (Medicago sativa L.)
M. S. Srinivasa Reddy*†, Fang Chen*†, Gail Shadle*, Lisa Jackson*, Hugh Aljoe*‡, and Richard A. Dixon*§
*Plant Biology Division and ‡Agriculture Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401 Edited by Rodney B. Croteau, Washington StateUniversity, Pullman, WA, and approved September 27, 2005 (received for review July 8, 2005)

Improving the digestibility of forages provides a means to enhance animal performance and protect the environment against excessive animal waste. Increased lignin content during maturity, and corresponding changes in lignin composition, correlate with decreased digestibility of forages. These relationshipshave yet to be investigated in isogenic systems. By targeting three specific cytochrome P450 enzymes of the lignin pathway for antisense downregulation, we generated transgenic alfalfa lines with a range of differences in lignin content and composition. There was a strong negative relationship between lignin content and rumen digestibility, but no relationship between lignin composition anddigestibility, in these transgenic lines. Models for genetic manipulation of forage digestibility based on the changes in lignin composition that increase paper-pulping efficiency in trees are therefore invalid. Down-regulation of 4-coumarate 3-hydroxylase provided the largest improvements in digestibility yet seen in a forage crop.
forage digestibility lignin modification metabolic engineering

eeding andgrazing studies have shown that small changes in forage digestibility significantly impact animal performance (1), and improving digestibility is an important goal of forage breeding programs. Lignin, a polymer of hydroxylated and methoxylated phenylpropane units (monolignols) linked by means of oxidative coupling (2), exerts a negative influence on digestibility. Lignin is found in secondarilythickened plant cell walls and is critical for structural integrity of the wall and the strength of stems (3, 4). Angiosperm lignin contains two major monolignols, monomethoxylated guaiacyl (G) and dimethoxylated syringyl (S) units, polymerized through at least five different linkage types (5). Gymnosperm lignin is low in S units and highly condensed because the high G unit composition facilitatesadditional interunit bonding. Gymnosperm wood is therefore less amenable than angiosperm wood to the harsh chemical and physical treatments (pulping) necessary to remove lignin from cellulose during paper production (6), and pulping properties are improved by genetic modification of trees to increase the S G ratio (5). In many forage crops, lignin content and S G ratio increase with stem maturity(7, 8), and both parameters correlate negatively with forage digestibility in ruminant animals (8–12). The amount of G lignin also has been linked with reduced cell wall degradability in forages (13), although studies with synthetic lignins (14) have questioned effects of lignin composition on forage digestibility. Most of the above studies used materials with different lignin contents and orcompositions as a result of divergent selection (15), natural genetic variation (16), delignification (17), or maturity (18). Such studies are complicated by the many uncontrolled genetic and developmental variables potentially affecting digestibility (19). Alfalfa (Medicago sativa L.) is an autotetraploid, autogamous forage legume, and inbred lines are therefore not available. Generating isogenictransgenic lines in which lignin content or composition is modified by altering expression
www.pnas.org cgi doi 10.1073 pnas.0505749102

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of a single gene provides a strategy for better elucidating the lignin digestibility relationship in alfalfa. Lignin biosynthesis is now believed to proceed according to the pathway in Fig. 1 (20–24). Genes encoding all of the enzymes in Fig. 1 have been...