Divergence of Bowman-Birk protease inhibitor family into seed-specific and environmentally responsive subfamilies in legume and soybean: Implication for legume seed composition improvement.

Authors: WANG, ZHIBO - Danforth Plant Science Center; JIANG, HE - Danforth Plant Science Center; LIU, KESHUN - Danforth Plant Science Center; LOHANI, NEETA - Danforth Plant Science Center; MISRA, SAURAV - Kansas State University; SHEN, WENHAO - Danforth Plant Science Center; GOMEZ-LUCIANO, LUIS - Danforth Plant Science Center; POKHREL, SURESH - Danforth Plant Science Center; COLLIER, RAY - Wisconsin Crop Innovation Center; KAEPPLER, SHAWN - Wisconsin Crop Innovation Center; An, Yong Qiang

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 8/27/2025
Publication Date: 8/27/2025
Citation: Wang, Z., Jiang, H., Liu, K., Lohani, N., Misra, S., Shen, W., Gomez-Luciano, L., Pokhrel, S., Collier, R., Kaeppler, S., An, Y. 2025. Divergence of Bowman-Birk protease inhibitor family into seed-specific and environmentally responsive subfamilies in legume and soybean: Implication for legume seed composition improvement.. bioRxiv. https://doi.org/10.1101/2025.08.26.658694.
DOI: https://doi.org/10.1101/2025.08.26.658694

Interpretive Summary: Soybeans (Glycine max) are one of two major crops grown in the US and are primarily used for animal feed. Bowman-Birk inhibitors (BBIs) are an ancient class of serine protease inhibitors that originated prior to the emergence of angiosperms. They have been preserved in legumes and cereals, likely providing an advantage in nature selection. However, accumulation of BBIs in seeds compromises nutritional and economic values for its use as feeds. It is critical to understand the molecular evolution, regulation and functions of BBIs for designing effective strategies to reduce BBI accumulation in seeds for increasing soybean nutritional value without affecting soybean agronomic performance. The study showed that BBI accumulation in soybeans is controlled by two sets of genes. One set of genes is specifically responsible for the accumulation of BBIs in seeds of soybean and legume crops while the other set are specifically associated with plant response to environmental stresses. BBIs are not required by the core plant growth and development. Using gene editing technologies, we removed almost all genes responsible for accumulation of BBIs in seeds and developed new soybeans with over 70% reduction of BBI activity in seeds and no significant changes in soybean growth. Our findings offer new insights into the molecular evolution, regulation, and functional specialization of BBIs in plants and demonstrate that the feasibility of genome editing to enhance the nutritional value of soybeans with minimal impact on agronomic performance, therefore lay a foundation for designing effective strategies to reduce BBI activities in soybeans and other species to meet modern agricultural needs.

Technical Abstract: Bowman-Birk inhibitors (BBIs) are an ancient class of serine protease inhibitors that originated prior to the emergence of angiosperms. Although BBIs have been lost in many divergent plant lineages, they have been retained in legumes (Fabaceae) and cereals (Poaceae). However, the molecular evolution and regulation of BBIs remain largely unclear. Our study reveals that BBIs in legumes and cereals are encoded by two distinct gene families, with further diversification in legumes into two subfamilies. One legume BBI subfamily is specifically expressed in seeds, while the other subfamily, along with BBIs in cereals, shows no significant expression in any of the examined tissues, including seed, root, leaf, and flower. Soybeans have a large BBI gene family. Comprehensive characterization of soybean BBI family indicate that its expansion is attributed to whole-genome, segmental, and tandem duplications. Protein sequence and structural analyses suggest that key functional domains including endoplasmic reticulum (ER) secretion signals, double-headed inhibitory loops, and binding sites for trypsin and chymotrypsin are largely conserved. Eight genes in the seed-specific subfamily exhibit expression during seed maturation, but not embryogenesis. Conversely, the five genes of the other non-expression subfamily are highly inducible by abiotic and biotic stimuli, each responding to a distinct subset of examined environmental signals. The specific expression of BBIs during seed maturation and in response to environmental stresses, two processes dispensable for plant growth and development, suggest that BBIs are not integral to core development. Their preservation in both legumes and cereals is likely driven by their role in enhancing plant fitness under natural selection, consistent with their established function in stress tolerance. Using a CRISPR/Cas9 approach, we generated soybean lines with knockout of seed-specific BBIs in soybean, resulting in a 69.4–73.7% reduction in trypsin inhibitor activity and a 76.4–79.4% reduction in chymotrypsin inhibitor activity. The edited soybeans showed no statistically significant differences in key agronomic traits, supporting the conclusion that BBI functions are non-essential at the standard growth condition. While seed specific BBIs may provide evolutionary advantages, the presence of BBIs in seeds reduce the nutritional value of soybeans as feed and food, highlighting a conflict between evolutionary benefits and anti-nutritional effects in modern agriculture. Our findings offer new insights into the molecular evolution, regulation, and functional specialization of BBIs in plants and demonstrate that the feasibility of genome editing to enhance the nutritional value of soybeans with minimal impact on agronomic performance, therefore lay a foundation for designing effective strategies to engineer BBI activities in soybean and other species to meet modern agricultural needs.