** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** PLANTS CONTAIN SEVERAL HUNDRED ENZYMES (NAMED PROTEASES) WHICH ARE CAPABLE OF DEGRADING PROTEINS; YET, THE BIOLOGICAL FUNCTIONS OF THE VAST MAJORITY OF PROTEASES HAVENOT BEEN CLEARLY DEFINED. DURING LEAF SENESCENCE (VISIBLE AS A GRADUAL LOSS OF GREEN LEAF COLOR), LEAF PROTEINS ARE DEGRADED, ALLOWING THE REMOBILIZATION OF PROTEIN-DERIVED NITROGEN TO DEVELOPING SEEDS/GRAINS. IN ANNUAL CROPS INCLUDING BARLEY, THIS PROCESS CONTROLS SEED COMPOSITION AND QUALITY. AVAILABLE KNOWLEDGE SUGGESTS THAT TWO PROTEASE 'FAMILIES' ARE FUNCTIONALLY RELEVANT FOR NITROGEN REMOBILIZATION, BUT THE DETAILS ARE POORLY DEFINED. THIS LACK OF UNDERSTANDING HINDERS EFFORTS AIMED AT IMPROVING OVERALL CROP NITROGEN USE EFFICIENCY AND GRAIN/SEED PROTEIN CONCENTRATION (GPC). OUR PROJECT ADDRESSES THE OUTLINED PROBLEM USING TWO SPECIFIC OBJECTIVES, NAMELY1. IDENTIFICATION OF SENESCENCE-ASSOCIATED PROTEASES USING ADVANCED BIOCHEMICAL TECHNIQUES; AND2. FUNCTIONAL CHARACTERIZATION OF IDENTIFIEDPROTEASES IN ISOLATED BARLEY LEAF CELLS AND IN WHOLE BARLEY PLANTS IN WHICH CANDIDATE PROTEASES HAVE BEEN EXPERIMENTALLY KNOCKED OUT. THE USE OF ISOLATED CELLS WILL ALLOW FASTER EXPERIMENTAL PROGRESS THAN WORK WITH INTACT PLANTS, SERVING AS A SCREENING TOOL FOR THE MORE LIMITED NUMBER OF ENZYMES THAT CAN BE CHARACTERIZED AT THE WHOLE-PLANT LEVEL. BARLEY LINES WITH KNOCKOUTS IN ONE OR SEVERAL PROTEASES WILL BE TESTED FOR NITROGEN CONTENT OF SENESCING LEAVES AND FOR GRAIN PROTEIN CONCENTRATION (GPC).THOSE PROTEASES WHOSE KNOCKOUT RESULTS IN DIMINISHED NITROGEN REMOBILIZATION FROM LEAVES AND LOWER GPC WILL BECOME NOVEL TARGETS FOR REGULATING THIS ECONOMICALLY IMPORTANT TRAIT. LOWERING GPC MAY BE DIRECTLY APPLICABLE TO MALT BARLEY BREEDING, ESPECIALLY FOR VARIETIES USED IN DRYLAND FARMING. IN CONTRAST, HIGHER GPC MAY BE ACHIEVED BY INCREASING THE ACTIVITY OF IDENTIFIED PROTEASES, USING BREEDING OR MOLECULAR APPROACHES.