** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** LONG-LIVED PERENNIAL PLANTS LIKE TREES ARE IMPORTANT IN PROVIDING FOOD, FIBER AND ECOSYSTEM SERVICES. NITROGEN IS AN ESSENTIAL NUTRIENT THAT IMPACTS PLANT GROWTH AND PRODUCTIVITY. ANNUAL PLANTS MUST ACQUIRE AND ASSIMILATE NITROGEN FROM SOIL TO SUPPORT GROWTH AND DEVELOPMENT. IN CONTRAST, NITROGEN ACQUISITION AND USE IN PERENNIAL PLANTS AND TREES IS MORE COMPLEX THAN ANNUAL PLANTS SINCE A MAJOR PORTION OF NITROGEN DEMAND IS MET BY INTERNAL SEASONAL CYCLING THAT REUSES ASSIMILATED AND REDUCED NITROGEN. SEASONAL NITROGEN CYCLING IS AN ANNUAL RECURSIVE PROCESS THAT INCREASINGLY CONTRIBUTES TO THE NITROGEN BUDGET AS TREES AGE. CONSEQUENTLY, SEASONAL NITROGEN CYCLING AFFORDS A COMPETITIVE ADVANTAGE IN NITROGEN LIMITED ENVIRONMENTS AND IS IMPORTANT TO THE ECOLOGY OF NITROGEN USE IN TREES AND IS A MAJOR FACTOR CONTRIBUTING TO NITROGEN USE EFFICIENCY.THE CONTRIBUTION OF STORED NITROGEN TO FOREST TREE NUTRIENT BUDGETS VARIES AMONG SPECIES. AND FOR POPLARS (POPULUS) SPRING GROWTH RELIES ALMOST EXCLUSIVELY ON NITROGEN FROM STORAGE RESERVES. BESIDES BEING IMPORTANT TO FOREST TREES, SEASONAL NITROGEN CYCLING AND NITROGEN RESERVE MOBILIZATION HAS LONG BEEN RECOGNIZED AS BEING IMPORTANT IN FRUIT TREES. IN THIS PROJECT, STATE-OF-THE-ART CRISPR GENE EDITING METHODS WILL BE USED IN COMBINATION WITH MOLECULAR BIOLOGY RESEARCH APPROACHES TO UNDERSTAND THE MOLECULAR AND GENETIC BASIS OF HOW SEASONAL NITROGEN CYCLING IS CONTROLLED AND REGULATED IN POPLAR. THIS BUILDS UPON THE DISCOVERY THAT THE PLANT HORMONE ETHYLENE REPRESSES THE EXPRESSION OF THE POPLAR BARK STORAGE PROTEIN GENES, WHICH PLAY A CENTRAL ROLE IN SEASON NITROGEN CYCLING. THIS WILL BE ACCOMPLISHED THROUGH (1) CRISPR GENE EDITING OF GENES THAT APPEAR TO REGULATE THIS PROCESS, (2) BIOCHEMICAL APPROACHES TO TEST IF THE PROTEINS ENCODED BY THESE GENE INTERACT, (3) MOLECULAR BIOLOGY APPROACHES TO DETERMINE IF THESE PROTEIN FACTORS BIND TO DNA REGULATORY SEQUENCES IN THE BARK STORAGE PROTEIN GENES, AND (4) DISCOVER IF OTHER REGULATORY FACTORS REGULATE BARK STORAGE PROTEIN GENE EXPRESSION AND SEASONAL NITROGEN CYCLING USING CRISPR-BASED SCREENS.THIS RESEARCH WILL ADVANCE UNDERSTANDING OF SEASONAL NITROGEN CYCLING AND DIRECTLY ADDRESSES THE RESEARCH PRIORITIES OF THE PHYSIOLOGY OF AGRICULTURAL PLANTS PROGRAM (A1152). SPECIFICALLY, THIS RESEARCH FALLS WITHIN THE RESEARCH PRIORITY OF NUTRIENT UPTAKE, ASSIMILATION, ACCUMULATION, AND/OR UTILIZATION, PARTICULARLY INCREASE EFFICIENCY IN USING NITROGEN, OR PHOSPHORUS OR OTHER SUPPLEMENTAL NUTRIENTS. IN ADDITION, THIS PROJECT WILL ALSO ADVANCE KNOWLEDGE CONCERNING REGULATORY FACTORS INVOLVED IN THE ESTABLISHMENT AND REGULATION OF N SOURCE-SINK RELATIONSHIPS.