Enrichment of White Clover Leaf Epidermal Cells for Omics-Based Analysis of the Proanthocyanidin Biosynthesis Pathway

Consumption by ruminants of high-protein forages, such as white clover, with insignificant levels of proanthocyanidins in herbage is associated with pasture bloat. Proanthocyanidins and anthocyanins are both products of the flavonoid pathway and share common metabolic intermediates and possibly biosynthetic enzymes. White clover leaves accumulate proanthocyanidins only in glandular trichomes, but produce anthocyanins in the epidermal cell layer. This makes white clover amenable to single cell-type studies of proanthocyanidin and anthocyanidin biosynthesis and possible interactions within the flavonoid pathway.

This study aims to provide insight into mechanisms regulating anthocyanin and proanthocyanidin biosynthesis in white clover leaves with a view to enabling metabolic engineering for bloat safety. Methods were developed for enrichment and purification of specific tissues in white clover leaves to enable analysis of the proanthocyanidin and anthocyanin pathway at the resolution of single cell types. We have applied LC-MS analysis and RNA-Seq to different tissues, namely trichomes, epidermis and mesophyll, as well as single cell types isolated using Fluorescence Activated Cell Sorting (FACS) and Laser Capture Microdissection (LCM).