AS NASA PURSUES ITS GOAL OF SENDING HUMAN COLONIES TO MARS AND BEYOND THERE IS A PRESSING NEED TO BETTER UNDERSTAND HOW THE SPACEFLIGHT ENVIRONMENT INFLUENCES FUNDAMENTAL BIOLOGICAL PROCESSES. UNDERSTANDING HOW PLANTS RESPOND TO GRAVITY ON EARTH AND THE MICROGRAVITY ENVIRONMENT IN SPACE IS OF PARTICULAR INTEREST BECAUSE OF THE IMPORTANT ROLE THAT PLANTS WILL PLAY IN ADVANCED BIOREGENERATIVE LIFE SUPPORT SYSTEMS DURING LONG DURATION SPACE MISSIONS. IN RECENT YEARS NUMEROUS TRANSCRIPTOMIC DATASETS HAVE SHOWN THAT HUNDREDS OF GENES CHANGE EXPRESSION WHEN PLANTS ARE GROWN IN SPACE. HOWEVER IT HAS BEEN CHALLENGING TO TEASE APART WHICH GENES CHANGE DUE TO MICROGRAVITY FROM THOSE THAT CHANGE AS A RESULT OF GENERAL PLANT STRESS RESPONSES. THIS DIFFICULTY STEMS FROM THE FACT THAT PLANT SPACEFLIGHT GENOMICS DATASETS HAVE BEEN DERIVED ALMOST EXCLUSIVELY FROM WHOLE SEEDLINGS AND PLANT ORGANS WHICH COULD POTENTIALLY MASK OR DILUTE GENE EXPRESSION CHANGES THAT ARE OCCURRING WITHIN THE DISTINCT CELL-/TISSUE-TYPES THAT ARE RESPONSIBLE FOR GRAVITY SENSING (E.G. ROOT COLUMELLA AND STEM ENDODERMAL CELLS). THUS THE MAJOR GOAL OF THIS NASA-GROUND-BASED PROPOSAL IS TO BEGIN TO TAKE STEPS TO DEVELOP METHODOLOGY THAT WILL ENABLE SPACE GENOMIC STUDIES ON HOMOGENOUS CELL POPULATIONS. HERE WE PROPOSE TO USE LASER CAPTURE MICRODISSECTION (LCM) TO CONDUCT RNA-SEQUENCING (RNA-SEQ) ON GRAVITY SENSING COLUMELLA CELLS TO IDENTIFY GENE REGULATORY NETWORKS THAT GOVERN EARLY RESPONSES OF PLANTS TO GRAVITY. WE HYPOTHESIZE THAT GENE PROFILE SIGNATURES FROM A HOMOGENOUS POPULATION OF COLUMELLA CELLS WILL MORE CLEARLY REFLECT TRANSCRIPTOMIC CHANGES THAT ARE DIRECTLY LINKED TO MICROGRAVITY. THE RNASEQ DATA THAT WE OBTAIN FROM OUR ANALYSIS OF ROOT COLUMELLA CELLS WILL BE COMPARED TO COLUMELLA-SPECIFIC GENE MODULES WE IDENTIFIED THROUGH OUR WEIGHTED GENE CO-EXPRESSION NETWORK ANALYSIS (WGCNA) OF ARABIDOPSIS SEEDLINGS GROWN FOR 6 AND 11 DAYS ON THE INTERNATIONAL SPACE STATION (ISS) (SPECIFIC AIM 2). IN PARALLEL WE WILL REFINE LCM METHODS WITH THE GOAL OF IMPLEMENTING THEM FOR FUTURE PLANT SPACEFLIGHT GENOMICS (SPECIFIC AIM 1). THE LATTER AIM WILL INVOLVE GROUND-BASED EXPERIMENTS IN WHICH SPECIFIC STEPS IN THE LCM METHODS WILL BE MODIFIED TO MIRROR IN-ORBIT OPERATIONS. UPON SUCCESSFUL COMPLETION OF THIS PROPOSAL WE EXPECT TO GENERATE NEW TOOLS FOR CONDUCTING GENOMICS IN HOMOGENOUS PLANT CELL POPULATIONS THAT WILL ENABLE US TO BETTER DIFFERENTIATE MICROGRAVITY EFFECTS FROM MICROGRAVITY INDEPENDENT EFFECTS.