TITLE: THE USE OF MICROGRAVITY SIMULATORS FOR A MECHANISTIC UNDERSTANDING OF CYTOSKELETAL-MEDIATED REGULATION OF ROOT GROWTH DIRECTIONALITY PLANTS WILL BE A MAJOR COMPONENT OF ADVANCED LIFE SUPPORT SYSTEMS TO ENABLE NASA TO REALIZE ITS VISION OF LONG DURATION SPACE EXPLORATION THAT WILL TAKE HUMANS TO MARS AND BEYOND. HOWEVER PLANTS CAN ONLY BE EFFECTIVELY USED FOR SUCH SYSTEMS IF WE HAVE A DEEPER MECHANISTIC UNDERSTANDING ABOUT HOW THEIR DEVELOPMENT IS REGULATED BY THE SPACEFLIGHT ENVIRONMENT. RECENT OMICS STUDIES HAVE SHOWN THAT PLANTS REPROGRAM THEIR GENETIC CIRCUITRY WHEN GROWN IN THE NEAR WEIGHTLESS CONDITIONS OF SPACE. GENES INVOLVED IN CELL WALL REMODELING AND OXIDATIVE STRESS ARE MAJOR GENE CATEGORIES THAT ARE DIFFERENTIALLY REGULATED BY MICROGRAVITY. FURTHER IT HAS BEEN SHOWN THAT TRUE MICROGRAVITY IN SPACE AND SIMULATED MICROGRAVITY USING CLINOSTATS OR RANDOM POSITIONING MACHINES ELICIT ROUGHLY SIMILAR ROOT SKEWING RESPONSES IN THE MODEL PLANT ARABIDOPSIS THALIANA. ROOT SKEWING WAS MORE PRONOUNCED IN MUTANTS TO A ROOTEXPRESSED VEGETATIVE ACTIN ISOFORM. CONSISTENT WITH THE ENHANCED ROOT SKEWING OF ACTIN MUTANTS IN MICROGRAVITY IS THE OBSERVATION THAT OTHER PLANT SPECIES SUCH AS MAIZE (ZEA MAYS L.) HAVE STRONGER ROOT DIRECTIONAL GROWTH RESPONSES ON A CLINOSTAT WHEN CHEMICALS SUCH AS LATRUNCULIN B DISRUPT THE ACTIN CYTOSKELETON. BASED ON THESE OBSERVATIONS WE WILL TEST THE HYPOTHESIS THAT THE ACTIN CYTOSKELETON IS A KEY PLAYER IN DICTATING ROOT GROWTH DIRECTIONALITY IN TRUE AND SIMULATED MICROGRAVITY BY REGULATING THE PROCESS OF AUTOTROPIC ORGAN STRAIGHTENING. MOREOVER WE HYPOTHESIZE THAT A SET OF CELL WALL CROSS-LINKING (I.E. CLASS III PEROXIDASES) AND PUTATIVE ACTINREGULATORY PROTEINS (I.E. HLB1 AND MIN7/BEN1) MEDIATE ROOT HAIR ELONGATION AND DIRECTIONAL ROOT GROWTH RESPECTIVELY UNDER TRUE AND SIMULATED MICROGRAVITY. OUR HYPOTHESES WILL BE ADDRESSED THROUGH THREE SPECIFIC AIMS INCLUDING THE ANALYSIS OF CELL WALL COMPOSITION/ DISTRIBUTION IN WILD-TYPE ARABIDOPSIS ROOTS AND DETAILED CHARACTERIZATION OF SELECTED ARABIDOPSIS CELL WALL AND ACTIN MUTANTS SUBJECTED TO MICROGRAVITY SIMULATION ON A SLOW ROTATING CLINOSTAT AND RANDOM POSITION MACHINE (AIM 1 AND 3). WE WILL ALSO USE MICROGRAVITY SIMULATORS TO ADDRESS THE HYPOTHESIS THAT THE PLANT HORMONE BRASSINOLIDE REGULATES DIRECTIONAL ROOT GROWTH IN MAIZE THROUGH ITS MODULATION OF ACTIN ORGANIZATION AND DYNAMICS (AIM 2). OUR RESEARCH PLAN BUILDS ON SUCCESSES OF PREVIOUS NASA-FUNDED GROUND AND SPACEFLIGHT RESULTS AND OUTLINES A COMPREHENSIVE SET OF EXPERIMENTS INCLUDING CELL WALL IMMUNOCYTOCHEMISTRY COMPUTER-BASED ROOT GROWTH ANALYSES ACTIN QUANTIFICATION AND GENETICS. THE PROPOSED WORK ALIGNS WITH PLANT BIOLOGY ELEMENT OF THE SPACE BIOLOGY SCIENCE PLAN. RESULTS OBTAINED FROM PLANT EXPERIMENTS ON MICROGRAVITY SIMULATORS WILL ADDRESS GUIDING QUESTIONS LINKED TO THE DECADAL SURVEY RECOMMENDATIONS INCLUDING THE IMPACT OF GRAVITY ON PLANT GROWTH DEVELOPMENT AND METABOLISM (PB-1) AND MOLECULAR MECHANISMS UNDERLYING HOW PLANTS SENSE AND REACT TO GRAVITY (PB-5). WE EXPECT THAT THE PROPOSED RESEARCH WILL PROVIDE US WITH BASIC KNOWLEDGE THAT WILL GUIDE THE DEVELOPMENT OF PLANT CULTIVARS BETTER ADAPTED TO THE MICROGRAVITY ENVIRONMENT OF SPACEFLIGHT AN OUTCOME THAT WILL PROVE BENEFICIAL FOR THE DESIGN OF PLANT HABITATS ON FUTURE SPACE COLONIES AND FOR ADDRESSING GREAT AGRICULTURAL CHALLENGES ON EARTH