Investigating the Effects of Cruciferin Double Knockouts on Amino Acid Profiles, Dry Seed Proteome, and Oxidative Stress Levels.

As plant seeds mature, they accumulate large quantities of seed storage proteins, which are a vital source of carbon, nitrogen, and sulfur necessary for establishing the seedling, especially during the transition from the heterotrophic to the photoautotrophic stage. However, seed storage proteins in many crop seeds are deficient in essential amino acids, which cannot be synthesized by humans and monogastric animals and must be obtained from the diet. Lysine and tryptophan are the most deficient amino acids in cereal seeds, while methionine is the most deficient amino acid in legumes. In the last few decades, extensive research has been done to improve the nutritional quality of seed crops. However, much of this effort was hindered due to the conserved natural phenomenon of proteomic rebalancing that 'resets' the seed's protein-bound amino acid composition despite major alterations to the proteomic sink. Neither the underlying regulatory mechanism nor the natural function of proteomic rebalancing is well understood. To address this gap, we used the model organism to investigate the impact of cruciferin (CRU) seed storage protein double knockouts on key biological processes. Amino acid analysis showed that the protein-bound amino acid composition and levels did not change in the mutants despite major alterations in the proteome, especially in the double mutant lacking both CRUA and CRUC ( ). This mutant also has the highest free amino acid changes and experienced the most oxidative stress damage compared to other mutants based on analysis of protein carbonylation and glutathione levels. The mutant that lacks CRUA and CRUB ( ), on the other hand, was the least affected in all the traits examined. These results suggest that CRUs are not functionally redundant, and that each CRU is not replaceable by another in . The results also show that seed protein-bound amino acid composition is fully rebalanced in the double CRU mutants despite major proteome alteration.