The Tapetal Major Facilitator NPF2.8 is Required for Accumulation of Flavonol Glycosides on the Pollen Surface in Arabidopsis thaliana.

The exine of angiosperm pollen grains is usually covered by a complex mix of metabolites including pollen-specific hydroxycinnamic acid amides (HCAAs) and flavonoid glycosides. Whereas the biosynthetic pathways resulting in the formation of HCAAs and flavonol glycosides have been characterized, it is unclear, how these compounds are transported to the pollen surface. In this report we provide several lines of evidence that AtNPF2.8, a member of the nitrate/peptide NTR/PTR family of transporters is required for accumulation and transport of pollen-specific flavonol 3-O-sophorosides, characterized by a glycosidic β-1,2-linkage, to the pollen surface of Arabidopsis. Ectopic, transient expression of this flavonol sophoroside transporter, termed AtFST1, fused to green fluorescent protein (GFP) demonstrated localization of AtFST1 at the plasmalemma in epidermal leaf cells of Nicotiana benthamiana whereas the tapetum-specific AtFST1-expression was confirmed by promAtFST1:GFP-reporter lines. In vitro characterization of AtFST1-activity was achieved by microbial uptake assays based on 14C-labeled flavonol glycosides. Finally, rescue of an fst1-line by complementation with a genomic fragment of the AtFST1 gene restored flavonol glycoside accumulation of pollen grains to wild-type levels corroborating the requirement of AtFST1 for transport of flavonol-3-O-sophorosides from the tapetum to the pollen surface.