** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** OUR LONG-TERM GOAL IS TO DEVELOP STRATEGIES FOR ENGINEERING OF PLANTS ENRICHED IN NUTRITIONALLY AVAILABLE VITAMIN B2. THE MAIN DIETARY SOURCES OF VITAMIN B2 IN WESTERN DIETS ARE MILK AND DAIRY PRODUCTS, FOLLOWED BY FORTIFIED CEREALS, MEAT, AND FATTY FISH. PLANTS ARE POORER SOURCES OF THIS VITAMIN, TYPICALLY CONTAINING AROUND 5-30% THE AMOUNT COMPARED TO DAIRY PRODUCTS AND MEAT. THE RECOMMENDED DIETARY ALLOWANCE (RDA) FOR ADULT MEN AND WOMEN IS 1.3 MG AND 1.1 MG DAILY OF VITAMIN B2, RESPECTIVELY, SO TO SATISFY THE RDA BY EATING PLANT-BASED FOODS, AN AVERAGE WOMAN WOULD NEED TO EAT DAILY ABOUT 2 LBS OF BROCCOLI, 5 LBS OF TOMATOES, OR 15 LBS OF POTATOES. VITAMIN B2-ENRICHED PLANTS ARE EXPECTED TO OFFER A CRITICAL SOLUTION TO THE POOR DIETARY INTAKE OF THIS ESSENTIAL NUTRIENT IN HUMANS, REDUCING DEPENDENCY ON MEAT AND DAIRY CONSUMPTION, AND PROVIDING AN OVERALL HEALTHIER DIET, THUS BENEFITING THE SOCIETY. LIKE HUMANS, MANY FARMED ANIMALS NEED VITAMIN B2 IN THEIR DIETS. AS A RESULT, NEARLY 80% OF THE TOTAL COMMERCIAL PRODUCTION OF RIBOFLAVIN IS NOW USED TO SUPPLEMENT ANIMAL FEED. AN ALTERNATIVE TO THIS EXCESSIVE SUPPLEMENTATION CAN THUS BE FEEDING ENGINEERED PLANTS ENRICHED IN VITAMIN B2. WE HERE PROPOSE TO GENERATE SUCH PLANTS BY INCREASING THE BIOSYNTHESIS OF THE B2 VITAMERS RIBOFLAVIN, FMN, AND FAD.TO ACCOMPLISH OUR ENGINEERING GOAL, WE WILL TAKE ADVANTAGE OF A MUTATION IN ONE OF THE ENZYMES ON THE VITAMIN B2 BIOSYNTHESIS PATHWAY IN PLANTS. THIS MUTATION CAUSES A HUGE INCREASE IN VITAMIN B2 ACCUMULATION IN SEEDS OF THE MODEL PLANT SPECIES ARABIDOPSIS. WE WILL DETERMINE IF THE SAME MUTATION CAN BE USED TO INCREASE VITAMIN B2 ACCUMULATION IN SEEDS OF THE CROP SPECIES SOYBEAN AND PENNYCRESS. SOYBEAN IS ONE OF THE MOST IMPORTANT CROPS GROWN IN THE US ON A LARGE SCALE, AND IS AN EXCELLENT MODEL SPECIES FOR OTHER LEGUME CROPS. PENNYCRESS (THLASPI ARVENSE) IS BEING DEVELOPED FOR USE AS A ROTATION CROP FOR CORN AND SOYBEANS, TO BE GROWN IN THE CORN BELT AND OTHER SUITABLE AREAS IN THE LATE FALL, WINTER, AND EARLY SPRING WHEN THE OTHER CROPS CANNOT BE GROWN. THE CREATION OF CROPS WITH ENHANCED VITAMIN B2 CONTENTS IS EXPECTED TO PROVIDE MEANS FOR IMPROVING NUTRITIONAL UPTAKE OF THIS VITAMIN IN HUMANS, AND TO OFFER AN ALTERNATIVE TO SUPPLEMENTING ANIMAL FEED WITH RIBOFLAVIN.