Evidence for Ancient Origins of Bowman-Birk Inhibitors from <i>Selaginella moellendorffii</i>

Abstract

Bowman-Birk Inhibitors (BBIs) are a well-known family of plant protease inhibitors first described 70 years ago. BBIs are known only in the legume (Fabaceae) and cereal (Poaceae) families, but peptides that mimic their trypsin-inhibitory loops exist in sunflowers (<i>Helianthus annuus</i>) and frogs. The disparate biosynthetic origins and distant phylogenetic distribution implies these loops evolved independently, but their structural similarity suggests a common ancestor. Targeted bioinformatic searches for the BBI inhibitory loop discovered highly divergent BBI-like sequences in the seedless, vascular spikemoss <i>Selaginella moellendorffii</i> Using de novo transcriptomics, we confirmed expression of five transcripts in <i>S. moellendorffii</i> whose encoded proteins share homology with BBI inhibitory loops. The most highly expressed, <i>BBI3</i>, encodes a protein that inhibits trypsin. We needed to mutate two lysine residues to abolish trypsin inhibition, suggesting BBI3's mechanism of double-headed inhibition is shared with BBIs from angiosperms. As <i>Selaginella</i> belongs to the lycopod plant lineage, which diverged ∼200 to 230 million years before the common ancestor of angiosperms, its BBI-like proteins imply there was a common ancestor for legume and cereal BBIs. Indeed, we discovered <i>BBI</i> sequences in six angiosperm families outside the Fabaceae and Poaceae. These findings provide the evolutionary missing links between the well-known legume and cereal <i>BBI</i> gene families.