Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa):: effects on lignin composition in specific cell types

Independent antisense down-regulation of 10 individual enzymes in the monolignol pathway has generated a series of otherwise isogenic alfalfa (Medicago sativa) lines with varying lignin content and composition. These plants show various visible growth phenotypes, and possess significant differences in vascular cell size and number. To better understand the phenotypic consequences of lignin modification, the distributions of lignin content and composition in stems of the various alfalfa lines at the cellular level were studied by confocal microscopy after staining for specific lignin components, and by chemical analysis of laser capture dissected tissue types. Although all antisense transgenes were driven by the same promoter with specificity for vascular, fiber and parenchyma tissues, the impact of down-regulating a specific transgene varied in the different tissue types. For example, reducing expression of ferulate 5-hydroxylase reduced accumulation of syringyl lignin in fiber and parenchyma cells, but not in vascular elements. The results support a model for cell type-specific regulation of lignin content and composition at the level of the monolignol pathway, and illustrate the use of laser capture microdissection as a new approach to spatially resolved lignin compositional analysis.