CHANGES IN THE EPIGENOME OF PLANTS SPECIFICALLY DNA METHYLATION PATTERNS ARE NOW KNOWN TO OCCUR IN SPACEFLIGHT. THESE CHANGES ARE ONLY BEGINNING TO BE UNDERSTOOD AS FAR AS THEIR EFFECT ON PHYSIOLOGICAL ADAPTATION AND PRODUCTIVITY IN SPACEFLIGHT; HOWEVER THE FACT THAT DNA METHYLATIONS ARE ALTERED BY SPACEFLIGHT IS CLEAR AND DISRUPTION OF METHYLATION MECHANISMS AFFECTS GROWTH IN SPACE. WHAT IS NOT YET UNDERSTOOD OR EVEN APPROACHED IS THE TRANS-GENERATIONAL STABILITY OF THESE EPIGENETIC CHANGES AND WHETHER ESTABLISHING SPACEFLIGHT METHYLATION PATTERNS WITHIN SEEDS CAN IMPROVE NEXT-GENERATION ADAPTABILITY TO SPACEFLIGHT. THEREFORE THIS PROPOSAL SEEKS TO EXAMINE THE TRANSMISSION OF DNA METHYLATION PATTERNS THROUGH SEEDS PRODUCED IN PLANTS GROWN ON THE ISS. SEEDS PRODUCED ON ORBIT WILL BE USED FOR SECOND GENERATION OF SPACEFLIGHT GROWTH. THIS SECOND ISS GROW OUT COMPRISING SEEDS FROM SPACEFLIGHT-GROWN PLANTS TOGETHER WITH SEEDS FROM TERRESTRIALLY-GROWN PLANTS WILL DETERMINE WHETHER SECOND GENERATION EPIGENETIC CHANGES CONTINUE TO ACCUMULATE OR STABILIZE. THE SECOND GROW OUT WILL ALSO REVEAL ANY SPACEFLIGHT-ASSOCIATED CHANGES IN PLANT GROWTH AND DEVELOPMENT OR GENE REGULATION THAT ARE EPIGENETICALLY REGULATED BETWEEN PLANTS GROWN FROM SEEDS PRODUCED ON THE ISS AND SEEDS PRODUCED ON THE EARTH. THIS EXPERIMENT WILL DETERMINE WHETHER GROWING A GENERATION OF PLANTS IN SPACE CONFERS AN ADAPTIVE ADVANTAGE TO THE NEXT GENERATION SIMILAR TO EPIGENETIC ADAPTIVE ADVANTAGE KNOWN TO OCCUR WITH SOME TERRESTRIAL PLANT STRESSES. THIS PROJECT IS ONE OF BOTH DISCOVERY ON A GENOME-WIDE SCALE AND PRACTICAL APPLICATION TO EXPLORATION NEEDS. WE PROPOSE UNBIASED WHOLE GENOME BISULFITE SEQUENCING IN ARABIDOPSIS LEAVES ACROSS SPACEFLIGHT GENERATIONS TO DISCOVER EPIGENETIC MODIFICATION PATTERNS OVER GENERATIONS COUPLED WITH RNASEQ EXAMINATION OF THE COMPARABLE TRANSCRIPTOMES TO ASSESS SPACEFLIGHT-ASSOCIATED DNA METHYLATION ON TRANSCRIPT PROFILES ACROSS GENERATIONS. THIS WILL GREATLY ADVANCE FUNDAMENTAL UNDERSTANDING OF THE ROLE OF EPIGENETICS IN SPACEFLIGHT ADAPTATION THE PRACTICAL APPLICATIONS ARISE FROM THE FACT THAT EPIGENETIC IMPRINTING OCCURS IN PLANTS EXPOSED TO ENVIRONMENTAL STRESS. ACQUIRED DNA METHYLATION PATTERNS CAN TRANSFER TO THE NEXT GENERATION WHERE THEY CONTRIBUTE TO THE ABILITY OF THE PROGENY TO COPE WITH THE STRESS EXPERIENCED BY THE PARENTAL GENERATION. SHOULD THAT OCCUR IN SPACEFLIGHT WE WILL HAVE IDENTIFIED GENETIC ELEMENTS THAT INCREASE THE ADAPTABILITY OF PLANTS TO SPACEFLIGHT THE FIRST STEPS IN DEVELOPING SPACEFLIGHT CULTIVARS BRED TO BETTER SERVE THE ROLES OF PLANTS IN LIFE SUPPORT. THE TACTICAL OBJECTIVES OF THIS PROPOSAL ARE: 1) BISULFITE WHOLE GENOME SEQUENCING TO MAP SPACEFLIGHT-INDUCED CHANGES IN 5MCYT AT SINGLE BASE-PAIR RESOLUTION FOR LEAVES OF MATURE PLANTS TO ENRICH THE METHYLOME DATA THAT WE CURRENTLY HAVE FOR SEEDLING LEAVES AND ROOTS. 2) RNASEQ TO ASSESS TRANSCRIPTOME IN THE SAME SAMPLES AS DIFFERENTIAL METHYLATION IS LINKED TO CHANGES IN GENE EXPRESSION THAT CHARACTERIZE THE FUNCTIONAL IMPACTS OF SPACEFLIGHT ACROSS THE ENTIRE GENOME IN MATURING LEAVES. 3) COLLECTING SEEDS PRODUCED ON ORBIT AND GROWING A SECOND GENERATION ON ORBIT. THE SECOND FLIGHT GROW OUT WILL BE USED TO GATHER GROWTH DEVELOPMENT GENE EXPRESSION AND EPIGENETIC DATA ON SECOND GENERATION PLANTS ON ORBIT. DURING BOTH GROWTH CYCLES ON ORBIT IMAGES OF PLANT GROWTH WILL MONITOR DEVELOPMENT. ELUCIDATING MULTIGENERATIONAL SPACEFLIGHT METHYLOMES CONTRIBUTES TO THE FUNDAMENTAL UNDERSTANDING OF HOW BIOLOGY RESPONDS AND ADAPTS TO THE SPACEFLIGHT ENVIRONMENT AND PROVIDES INSIGHT IN THE ADAPTIVE STRATEGIES PLANTS USE IN THE FACE OF NOVEL STIMULI OUTSIDE OF THEIR EVOLUTIONARY EXPERIENCE. THE LONG-TERM GOAL OF THIS LINE OF INQUIRY IS TO UNDERSTAND HOW THE METHYLOME CONTRIBUTES TO THE ADAPTIVE STRATEGIES OF PLANTS TO SPACEFLIGHT IN ORDER TO BREED PLANTS THAT ARE SPECIFICALLY AND BETTER ADAPTED TO SPACEFLIGHT IN MICROGRAVITY.