Minisymposium 3: Secondary metabolism

Abs # 13004: Understanding monolignol biosynthesis in Medicago sativa by a reverse genetics approach

Presenter:	Reddy, M.S. Srinivasa , msreddy@noble.org
Authors	Reddy, M.S. Srinivasa  (A)   Chen, Fang  (A)   Shadle, Gail  (A)   Jackson, Lisa  (A)   Dixon, Richard A (A)
Affiliations:	(A): The Samuel Roberts Noble Foundation

Lignin is a major structural component of secondarily thickened plant cell walls. It is a complex polymer of hydroxylated and methoxylated phenylpropane units. Dicotyledonous angiosperm lignins contain two major monomer species, termed guaiacyl and syringyl units. There is a considerable interest in genetic manipulation to alter the quantity and/or quality of the lignin polymer, due to its negative effects on paper pulping and forage digestibility. We are interested in lignin biosynthesis in alfalfa (Medicago sativa), the world’s most important forage legume, a species in which lignification negatively affects forage digestibility. Our approach is to use antisense gene downregulation, coupled with lignin compositional analysis and metabolite and transcript profiling, to decipher the regulatory architecture of the monolignol pathway. So far we have obtained transgenic alfalfa lines down-regulated for “4-coumarate 3-hydroxylase” (C3H), “cinnamate 4-hydroxylase” (C4H), “ferulate 5-hydroxylase” (F5H), phenylalanine ammonia-lyase (PAL) and hydroxycinnamoyl transferase (HCT). These transgenic lines show striking differences in lignin content and composition. For example, the H lignin content was increased by ten- fold in some of the C3H down-regulated lines compared to the controls. Greater variation was seen in the H/total lignin ratio which varied from 0.025 in controls to as high as 0.55 in one of the C3H down-regulated lines. Some of the C3H down-regulated lines were phenotypically shorter than the controls. One of the C4H down-regulated lines showed a five-fold decrease in total lignin and more than three-fold decrease in S/G lignin ratio. Similarly, there was a decreased S/G ratio in the F5H down-regulated lines. In addition, HCT and PAL down-regulated lines have been subjected to molecular analysis. Characterization of all these transgenic lines and their comparison with previously characterized COMT and CCoAOMT down-regulated transgenic alfalfa will be presented. Transcript profiling of the different developmental stages of the alfalfa stem and comparison of the transcript profiles between selected transgenic lines is in progress. Metabolic profiling of these transgenic lines is also being performed; wall-bound p-hydroxybenzaldehyde is increased and vanillin decreased in the C3H down-regulated lines, whereas soluble caffeic acid 3-O-glucoside accumulates in CCoAOMT down-regulated lines. Our overall approach represents the first detailed study of independent down-regulation of every enzyme of monolignol biosynthesis in a single species, and also addresses the issue of substantial equivalence for a forage crop engineered for improved digestibility.