** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** PLANT SURVIVAL DURING ENVIRONMENTAL STRESS IS NECESSARY TO FEED OUR GROWING HUMAN POPULATION, AND PLANT-MICROBE INTERACTIONS ARE CENTRAL TO PLANT STRESS SURVIVAL. OUR MODEL SPECIES, BLUE GRAMA (BOUTELOUA GRACILIS), ACCOUNTS FOR THE MAJORITY OF NET PRIMARY PRODUCTIVITY IN SEMIARID GRASSLANDS--ECOSYSTEMS PREDICTED TO BECOME WARMER AND DRIER. THE RELEASE OF C-RICH ROOT EXUDATES IS A KEY MECHANISM TO MANAGE THE ROOT MICROBIOME, ATTRACTING BENEFICIAL MICROBES AND/OR SUPPRESSING HARMFUL MICROBES TO HELP PLANTS WITHSTAND ENVIRONMENTAL STRESS. HOWEVER, ROOT EXUDATION OF C DIVERTS C FROM OTHER ESSENTIAL PLANT FUNCTIONS SUCH AS GROWTH AND METABOLIC MAINTENANCE, RESULTING IN A 'C COST.' A CRITICAL KNOWLEDGE GAP IS HOW PLANTS BALANCE THE COST-BENEFIT OF MANAGING THE ROOT MICROBIOME WITH ROOT EXUDATES UNDER DIFFERENT STRESSORS. THE GOAL OF THIS WORK IS TO DETERMINE THE EFFECT OF ENVIRONMENTAL STRESS ON: 1) THE C COST OF ROOT EXUDATES AND 2) THE ROOT MICROBIOME VIA SHIFTS IN ROOT EXUDATE COMPOSITION. USING A MANIPULATIVE GREENHOUSE EXPERIMENT, WE WILL IMPOSE 4 ENVIRONMENTAL STRESS TREATMENTS ON BLUE GRAMA: AMBIENT, HEAT, DROUGHT, HEAT+DROUGHT. TO MEASURE THE C COST OF ROOT EXUDATES (1), WE WILL QUANTIFY PLANT C ALLOCATION TO GROWTH, RESPIRATION, AND ROOT EXUDATES USING 13CO2-LABELLING AND PHYSIOLOGICAL MEASUREMENTS. TO DETERMINE HOW STRESS-INDUCED SHIFTS IN ROOT EXUDATE COMPOSITION ALTER THE ROOT MICROBIOME (2), WE WILL CHARACTERIZE ROOT EXUDATE COMPOSITION WITH METABOLOMICS AND THE ROOT MICROBIAL COMMUNITY COMPOSITION AND POTENTIAL FUNCTION WITH 16S AMPLICON SEQUENCING, METAGENOMICS, AND METATRANSCRIPTOMICS. THIS RESEARCH ADDRESSES THE AGRICULTURAL MICROBIOMES PROGRAM AREA GOAL TO EXAMINE HOW HOST-MICROBE INTERACTIONS INFLUENCE MICROBIOME COMPOSITION UNDER ENVIRONMENTAL STRESS.