PLANTS GROWN DURING SPACEFLIGHT EXPERIENCE CONDITIONS OF MICROGRAVITY IONIZING RADIATION AND REDUCED GAS EXCHANGE. IF WE ARE TO USE PLANTS IN ADVANCED LIFE-SUPPORT SYSTEMS DURING LONG SPACE MISSIONS IT IS IMPERATIVE THAT WE UNDERSTAND HOW PLANTS RESPOND TO THESE VARYING CONDITIONS TO ENSURE THEIR PRODUCTIVITY. PLANT GROWTH ON EARTH IS SHAPED IN GREAT PART BY THE FORCE OF GRAVITY WHILE CONDITIONS OF MICROGRAVITY EXPERIENCED IN SPACE RESULT IN DRAMATIC CHANGES IN ORGAN ORIENTATION AND GROWTH. THE PLANT RESPONSE TO GRAVITY OR GRAVITROPISM IS ALSO IMPORTANT FOR THE GROWTH ANGLE OF ROOT AND SHOOT BRANCHES AND THEREFORE IT ALSO IMPINGES IN CROP ARCHITECTURE AND PRODUCTIVITY ON EARTH. THE EFFECTS OF MICROGRAVITY ON PLANT GROWTH AT THE CELLULAR LEVEL IS NOT YET FULLY CHARACTERIZED AND THE MOLECULAR MECHANISMS BY WHICH PLANTS PERCEIVE GRAVITY ARE STILL UNKNOWN. A WEALTH OF STATE-OF-THE-ART GENETIC IMAGING AND OMICS TOOLS IN THE MODEL PLANT ARABIDOPSIS THALIANA ARE RIPE TO PROVIDE THE CLUES FOR THIS ELUSIVE SYSTEM IN PLANT BIOLOGY AND NOVEL PLAYERS HAVE RECENTLY BEEN IDENTIFIED. WE PROPOSE HERE TO CHARACTERIZE THE ROLE OF TWO PROTEIN FAMILIES PREDICTED TO MEDIATE CONTACTS BETWEEN THE ENDOPLASMIC RETICULUM (ER) AND OTHER ORGANELLES AS CANDIDATES FOR IMPORTANT ROLES IN GRAVITROPISM. THESE PROTEINS WERE SELECTED BASED ON OUR PREVIOUS NASA-FUNDED RESEARCH WHERE WE CHARACTERIZED THE ROLE OF VACUOLES IN GRAVITROPISM. THIS WORK LED US TO PROPOSE THE HYPOTHESIS THAT MEMBRANE CONTACTS BETWEEN AMYLOPLASTS AND VACUOLES AND/OR ER HAVE A ROLE IN GRAVITY SENSING IN SHOOT STATOCYTES. WE PLAN TO TEST THIS HYPOTHESIS DIRECTLY BY ADDRESSING THE SPECIFIC AIMS TO 1) DETERMINE THE FUNCTION OF CANDIDATE MEMBRANE TETHERS IN MEMBRANE CONTACTS AND GRAVITY SENSING; AND 2) DETERMINE THE EFFECTS OF SPACEFLIGHT ON VACUOLE FUSION AND ER MORPHOLOGY. A REVERSE GENETIC APPROACH COMBINED TO FLUORESCENCE MICROSCOPY AND MUTANT CHARACTERIZATION WILL BE USED TO CHARACTERIZE THE ROLE OF SELECTED TETHERS. THE FLIGHT EXPERIMENT WILL USE WELL-ESTABLISHED METHODS FOR ARABIDOPSIS GROWTH IN BRIC-PDFU UNITS THAT ARE SUITABLE FOR CHEMICAL TREATMENT AND FIXATION. PLANT SUB-CELLULAR PHENOTYPES WILL BE ANALYZED AFTER SAMPLE RETURN TO EARTH. OUR GROUND-BASED RESEARCH WILL ADDRESS THE MOST IMPORTANT KNOWLEDGE GAP IN GRAVITROPISM AT THE CELLULAR LEVEL AND IN SPECIFIC CELL TYPES OF THE ARABIDOPSIS SHOOT. IN ADDITION THE PROPOSED FLIGHT EXPERIMENT WILL RESULT IN MULTIPLE OUTCOMES BEYOND DOCUMENTING THE EFFECTS OF SPACEFLIGHT ON ORGANELLE FUSION. THESE INCLUDE PROBING THE USE OF CHEMICAL INHIBITORS FOR PLANT CELL BIOLOGY EXPERIMENTATION IN SPACE AND TESTING RECENTLY DESCRIBED TISSUE CLEARING PROTOCOLS FOR VISUALIZATION OF CELLULAR ORGANELLES IN SPACE-GROWN PLANTS. OUR RESEARCH WILL TRAIN YOUNG SCIENTISTS IN SPACE BIOLOGY RESEARCH INCLUDING UNDERGRADUATE STUDENTS AND THEREFORE CONTRIBUTE TO THE DEVELOPMENT OF HUMAN RESOURCES IN STEM.