PROTEASES -- TARGETS FOR IMPROVING NITROGEN REMOBILIZATION EFFICIENCY FROM SENESCING CEREAL LEAVES

Plant genomes encode several hundred proteases; yet, the biological function of the vast majority of these enzymes has not been defined. Two phases of the plant life cycle are characterized by large-scale protein degradation: (1) During seed germination, storage proteins present in the cotyledons or the endosperm are hydrolyzed to make organic nitrogen available for seedling growth. (2) During leaf or whole-plant senescence, chloroplast proteins are degraded in preparation for nitrogen remobilization to sink organs such as developing seeds. In annual crops including barley, most seed protein nitrogen is derived from this remobilization process, which therefore controls seed composition and quality.Chloroplast protein degradation in senescing organs may be initiated by plastidial proteases. Molecular approaches have shown that several pathways exist whereby stromal proteins, thylakoid proteins, or both are transported to lytic vacuoles with high protease activity; some of those pathways depend on autophagy. Available data indicate that papain-like cysteine proteases (PLCPs) and serine proteases control bulk degradation of chloroplast proteins in senescing leaves. Work in Arabidopsis and in crops has identified several functionally important enzymes, but the picture is far from complete. This situation constitutes a gap in our understanding of a fundamental plant physiological process. Furthermore, as senescence and particularly senescence-associated nitrogen remobilization control crop yield and quality, this lack of understanding hinders efforts aimed at improving seed / grain protein content and crop nitrogen use efficiency.This project proposes to address the problem outlined above through two specific objectives, namely1. Identification of senescence-associated proteases at the protein and activity levels (activity-based profiling, zymography, mass spectrometry); and2. Functional characterization of upregulated proteases in planta and in vitro (transient expression in protoplasts; biochemical analysis of purified proteases; stable barley transformation).Long-term goals of this project are aimed at closing the knowledge gap identified above, and applying gained knowledge to the development of barley varieties with 1) enhanced nitrogen remobilization efficiency, and 2) grain protein content adapted to end use.