TREES ARE LONG-LIVED PERENNIAL PLANTS PROVIDING FOOD, FIBER AND ECOSYSTEM SERVICES. NITROGEN (N) IS AN ESSENTIAL NUTRIENT THAT INFLUENCES PLANT GROWTH AND PRODUCTIVITY AND SIMILAR TO ANNUAL PLANTS TREES MUST FIRST ACQUIRE N FROM THE SOIL. SINCE TREES ARE PERENNIAL PLANTS A SIGNIFICANT PORTION OF THE N USED TO SUPPORT GROWTH AND DEVELOPMENT IS ALSO MET BY SEASONAL RECYCLING AND REUSE. THIS IS A KEY FACTOR OR TRAIT THAT CONTRIBUTES TO THE LONGEVITY AND PRODUCTIVITY OF TREES. TREE SEASONAL N CYCLING INVOLVES THE REMOBILIZATION OF N FROM SENESCING LEAVES DURING AUTUMN. THIS REMOBILIZED N IS STORED IN PERENNIAL TISSUES, LIKE BARK, OVER THE WINTER. THE FOLLOWING SPRING WHEN NEW GROWTH COMMENCES STORED N IS REMOBILIZED FROM STORAGE TISSUES TO EXPANDING BUDS AND SHOOTS. SEASONAL N CYCLING AND N RESERVE MOBILIZATION ACCOUNTS FOR A SIGNIFICANT AMOUNT OF THE N FOUND IN NEW GROWTH ONCE GROWTH COMMENCES IN THE SPRING OF BOTH FRUIT AND FOREST TREE CROPS. SINCE THIS IS A RECURSIVE PROCESS THAT OCCURS ANNUALLY, SEASONAL N CYCLING INCREASINGLY CONTRIBUTES TO THE OVERALL N BUDGET OF TREES OVER TIME.THE VAST MAJORITY OF WHAT IS KNOWN ABOUT SEASONAL N CYCLING OF TREES COMES FROM RESEARCH IN THE FOREST TREE SPECIES POPULUS (POPLARS). IN POPLAR SEASONAL N STORAGE RELIES UPON BARK ACCUMULATION OF SPECIFIC PROTEINS TERMED BARK STORAGE PROTEINS (BSPS). BASED ON PAST AND CURRENT RESEARCH IN POPLAR, WE HYPOTHESIZE THAT SEASONAL N STORAGE AND BSP ACCUMULATION INVOLVES 3 KEY STEPS. FIRST, N REMOBILIZED FROM SENESCING LEAVES INCREASES THE FLUX OF THE AMINO ACID GLUTAMINE TO BARK WHICH IN TURN ALTERS THE CARBON TO NITROGEN RATIO. SECOND, THE ALTERED CARBON TO NITROGEN RATIOS FAVORS THE BINDING OR INTERACTION WITH THE PII GLUTAMINE SENSING PROTEIN WHICH IN TURN ENHANCES THE PRODUCTION OF THE AMINO ACID ARGININE. THIRD, INCREASED ARGININE LEVELS THEN INDUCE THE EXPRESSION OF THE BSP GENES RESULTING IN BSP ACCUMULATION IN POPLAR BARK. THE OBJECTIVE OF THIS PROJECT IS TO TEST THE HYPOTHESIS THAT GLUTAMINE INTERACTIONS WITH THE PII SENSING PROTEIN AND ACTIVATION OF THE ARGININE BIOSYNTHETIC PATHWAY ARE COMPONENTS OF THE REGULATORY PATHWAYS THAT GOVERN SEASONAL N STORAGE IN POPLAR. SPECIFICALLY, WE WILL 1) CONSTRUCT PII VARIANTS THAT DO NOT BIND GLUTAMINE TO DETERMINE THE ROLE OF GLUTAMINE BINDING IN BSP GENE EXPRESSION, 2) TEST THE ROLE OF ARGININE BIOSYNTHESIS IN REGULATING THIS PROCESS THROUGH THE GENERATION OF FEED-BACK RESISTANT N-ACETYL GLUTAMATE KINASE (NAGK), AND 3) USE A NOVEL GENE-DOSAGE POPULATION OF POPLAR TO IDENTIFY ADDITIONAL FACTORS THAT REGULATE GLUTAMINE MEDIATED BSP GENE EXPRESSION. THIS PROJECT DIRECTLY ADDRESSES THE RESEARCH PRIORITIES OF THE PHYSIOLOGY OF AGRICULTURAL PLANTS PROGRAM (A1152). SPECIFICALLY, THIS RESEARCH FALLS WITHIN THE RESEARCH PRIORITY OF NUTRIENT UPTAKE (MACRONUTRIENTS AND/OR MICRONUTRIENTS), ASSIMILATION, ACCUMULATION, AND/OR UTILIZATION, PARTICULARLY INCREASE EFFICIENCY IN USING NITROGEN, OR PHOSPHORUS.