Non Technical Summary
While wheat contributes significantly to dietary calories, its grain endosperm (flour) contains very low levels of provitamin A carotenoids, including β-carotene. In our previous study, we found that inhibiting the function of carotenoid β-hydroxylase 2 (HYD2) in pasta/tetraploid wheat, which mainly accumulates lutein in the grain endosperm, resulted in increased concentrations of β-carotene. To enhance β-carotene levels in the grain endosperm of bread/hexaploid wheat, we have suppressed HYD2 function in the bread wheat breeding line Triple Yellow; this breeding line was developed to produce high levels of total carotenoids, particularly lutein and zeaxanthin (both lack provitamin A activity) in the grain endosperm. In this research, we will carry out biochemical, physiological, agronomic, postharvest stability, and consumer quality evaluations of Triple Yellow and Triple Yellow/hyd2-knockout plants to facilitate their integration into the wheat breeding program and the release of these new varieties to the public. Additionally, we will investigate the role of wheat Orange (OR) proteins in regulating carotenoid accumulation and stability to further elevate β-carotene levels in wheat grains. Our goal of developing bread wheat varieties enriched in provitamin A carotenoid β-carotene seeks to address the needs of food-insecure and malnourished populations in the U.S. and alleviate vitamin A deficiency in developing countries. Our proposed research aligns with the priority areas of the NIFA program Plant Health and Production and Plant Products, specifically focusing on micronutrient biosynthesis and accumulation that are beneficial to human health and nutrition.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
204	1549	1000	50%
204	1549	1081	50%

Knowledge Area
204 - Plant Product Quality and Utility (Preharvest);

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1081 - Breeding; 1000 - Biochemistry and biophysics;

Goals / Objectives
The major goal of this research is to develop bread wheat varieties with increased levels of provitamin A carotenoid β-carotene in their grains. The objectives are to evaluate Triple Yellow, Triple Yellow/hyd2-knockout, and control (parental line of Triple Yellow) plants for biochemical, physiological, agronomic, postharvest stability, and consumer quality traits to assess their potential for integration into the wheat breeding program. The role of wheat Orange (OR) proteins in modulating carotenoid accumulation and postharvest stability will also be investigated with the goal of further enhancing β-carotene concentrations in wheat grains.

Project Methods
To accomplish these objectives, we will conduct physiological assessments for growth and development-related traits and analyze carotenoids and other metabolites in vegetative tissues and grains of Triple Yellow, Triple Yellow/hyd2-knockout, and control plants. Secondly, we will perform agronomic evaluations through multiple year/location field trials and evaluate consumer traits on grains harvested from these plants. Carotenoid concentrations in grains during storage will also be determined. Thirdly, we will investigate the molecular and biochemical properties of wheat OR proteins, and examine the impact of their ectopic expression in seeds on carotenoid levels and the formation of carotenoid sequestration structures.