Ectopic expression of a deletion variant of cystathionine gamma-synthase 2 (GmCGS2) in soybean increases expression of genes encoding sulfur amino acid-rich proteins

A deletion variant of GmCGS2 was ectopically expressed in soybean with a diverse range of expression levels and patterns. A combination of a soybean ubiquitin promoter and an in-frame deletion in GmCGS2, from K66 to S143, resulted in increased seed Met and Cys contents without incurring a yield penalty in a ten-location field trial. Transgenic events of this construct had increases of 41 to 114%, 10 to 12%, and 4.7 to 7.3% in free Met, total Cys, and total Met, respectively. RNAseq analysis of developing seed revealed increased transcript for three Cys-rich Bowman-Birk protease inhibitor (BBI) genes, and for two Met-rich 2S albumin genes, with little effect apparent on genes of primary sulfur metabolism. Trypsin-agarose chromatography, protease inhibition assays, and mass spectrometry analysis revealed increased BBI and 2S albumin proteins. The additional BBI protein was determined to be 1.5 to 1.8% of the protein in transgenic seed, sufficient to account for the increased Cys content. The observation that overexpressing an enzyme involved in Met biosynthesis that used Cys as a substrate still increased total Cys more than total Met suggested a lack of native Met-rich soybean proteins capable of incorporating the additional free Met. RNAseq analysis of developing seed was done for transgenic events e15 and e19 of the GmUBQ promoter construct compared to nulls.