RNAi and CRISPR-Cas silencing E3-ring ubiquitin ligase AiP2 mitigates proteome rebalancing and enhances soybean seed protein content.

Abstract: The majority of plant-sourced protein in the world’s food supply is derived from soybean. Soybean is a key nutrition source for global animal feed and in its incorporation into plant-based foods, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content resulting in breeding or engineering for enhanced protein reduces oil mitigating any economic advantage. If one or more seed proteins are silenced that total seed protein content remains unchanged as other proteins increase in accumulation to compensate, a biological process termed proteome rebalancing. Together, the inverse relationship of protein to oil as well as proteome rebalancing has impeded efforts to increase the economic performance of soybean production. AiP2 is an E3-ring ubiquitin ligase that targets the seed-specific transcription factor, ABI3. Silencing both soybean AiP2 genes (AiP2a/AiP2b) by RNAi enhanced seed protein content by up to seven percentage-points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AiP2a or AiP2b by a CRISPR Cas 9-mediated mutation increased seed protein content, this effect was greater when both genes were inactivated. Transactivation assays in soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2s albumin, and oleosin genes, indicating that AiP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean which may also be applicable to other protein-source crops.