In cereals, lysine is the most limiting essential amino acid, and attempts have been made over the decades to improve the nutritional quality of these grains by increasing lysine content, while maintaining favourable agronomic traits. The high proportion of lysine-poor prolamin storage proteins in cereals are associated with the sub-optimal nutritional quality of cereal grains. To this end, the single lys3 mutation caused by the mutation in the PBF transcription factor in barley has been shown to significantly increases lysine content but reduces grain size and the content of both starch and gluten. Yet, the low agronomic yield and germination defects have presented as barriers for the commercial uptake of these barley mutants.
Conventional breeding strategies were used to combine the malting barley cultivar Sloop with the high lysine/low gluten lines, Risø 56 and Risø 1508, or the Ethiopian genotype R118 with a decreased D-hordein content to generate the single null B-, C- and D-hordein lines, respectively. These single-null lines were intercrossed to produce double-null lines that further reduced the gluten content combined with elevated lysine content.
The mechanisms activated in the grains of these double-null mutant lines to compensate for the loss of these major gluten proteins and decreased starch content remain unknown. In the current study, data-independent acquisition (DIA) mass spectrometry analyses were performed in parallel with phenotypic characterisations. The aim was to study the large-scale quantitative changes in proteins within the hordein double null lines in comparison to their parent lines. Functional annotation and bioinformatic analyses were carried out to uncover the protein classes related to the increased lysine content and gluten reduction in the double null lines. Balanced changes in the induced and suppressed protein abundances confirms the dual regulatory mechanisms of nutrient accumulation and energy metabolism in the mutant lines, resulting in differences both in the hordein levels and composition as well as in the lysine content of the double nulls. This research serves as a framework for future proteomics-assisted crop development in order to study pleiotropic effects on safety and nutrition quality-related improvements.