Small proteins, great promises: geographic bioprospecting of Bowman-Birk protease Inhibitors and domestication side-effects in African cowpea (Vigna unguiculata L.)

Societal Impact Statement: The legume crop cowpea is grown worldwide, but 90% of the world's total share is produced in Africa. It is a promising species due to its resilience properties, balance of macro and micronutrients and presence of health-promoting bioactive compounds. In African countries, cowpea has a crucial role in guaranteeing food security as a subsistence crop for families and commercial income for small farmers. The discovery of compounds with high nutraceutical value and bioactive properties supports socio-economic policies to improve health and nutrition, especially in low- and middle-income countries. In turn, this encourages biodiversity protection and crop enhancement programmes. Summary: Bowman–Birk protease inhibitors (BBIs) are a restricted group of small proteins in plants mainly involved in defence mechanisms against pests. BBIs are demonstrated to be active components capable of reducing the viabilities of different cancer cell lines. BBI bioactivity is directly linked to the inhibition capacity, but the variability and the efficiency against the physiological targets of different BBI isoforms remain still unexplored. We analysed the natural genetic diversity of two main genes encoding BBI trypsin-trypsin (BBI-TT) and trypsin-chymotrypsin (BBI-TC) in wild and domesticated cowpea samples mainly spread in Sub-Saharan Africa. We analysed DNA sequences and respective amino acidic isoforms/isoproteins to explore signs of natural selection and haplotype relationships. Moreover, we calculated the binding energy between BBIs and their biological targets to identify which are the most efficient inhibitors and their geographical locations. We found a high level of haplotype diversity for both genes, almost exclusively in wild accessions and detected positive and negative selection signals in the amino acid sequences. Furthermore, in the wild diversity pool, some BBI-TT and BBI-TC mature proteins were potentially better interactors with the physiological targets. The long interaction between plant-pathogen has selected new and useful isoforms in wild lineages that have allowed the chances of survival of the species to improve. On the other hand, the domestication process has produced an intense bottleneck leaving only poorly efficient BBI variants. In addition to providing information on the natural diversity and evolution of BBIs, our work discusses the potential applications in agriculture and human health.