ENGINEERING SOYBEANS AND PENNYCRESS FOR INCREASED VITAMIN B2 CONTENTS USING A MUTANT RIBOFLAVIN KINASE/FMN HYDROLASE

Our long-term goal is to develop strategies for engineering of plants enriched in nutritionally available riboflavin (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 micronutrient 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% (~2400 tons per year) 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. The creation of such plants 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. Toward that goal, we here propose the following objectives:Objective 1. Test strategies for increasing vitamin B2 contents in soybean and pennycress seeds by overexpressing the Melinoe mutant form of the bifunctional enzyme riboflavin kinase/FMN hydrolase from Arabidopsis. We here propose to produce and analyze transgenic soybean and pennycress plants that overexpress under control of appropriate seed promoters the Melinoe mutant form of the AtFMN/FHy enzyme from Arabidopsis, with or without knocking down in seeds the native FMN/FHy enzymes from soybean and pennycress. The PI Roje lab will also verify the enzymatic activities for the putative FMN/FHy enzymes from soybean and pennycress, which have not yet been cloned and biochemically characterized.Objective 2. Determine whether the Melinoe mutation causes the increase in riboflavin production because of a change in the AtFMN/FHy enzymatic activity, or because of a change in regulation of the riboflavin biosynthesis pathway, or both. To accomplish this aim, we here propose to (1) attempt rescuing the Melinoe mutant with the riboflavin kinase domain of FMN/FHy, (2) test whether overexpression of the Melinoe FMN hydrolase domain confers the same phenotype to the wild type plants as the full-length Melinoe mutant protein, (3) look for changes in the transcript abundance and enzyme activity of the enzymes on the riboflavin biosynthesis pathway in siliques and mature seeds in the Melinoe mutant vs. wild type plants, and (4) look for proteins that interact with the wild type and Melinoe forms of AtFMN/FHy in siliques and seeds.We propose to conduct the Objective 1 in 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. It is transformable and its whole genome sequence is available. 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. It is easily transformable, and represents an excellent model species for other Brassica crops such as Canola and Camelina. It is likely that the results of this study will be transferrable to other major crops, as genomic evidence suggests that flavin metabolism is highly conserved among angiosperms, including major staple crops such as corn, rice, and potatoes.