Progress 07/01/24 to 06/30/25

Outputs
Target Audience:The target audience for this research consists of consumers, wheat growers, breeders, plant scientists, professionals in the food industry, and nutritionists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided training experience for two undergraduate students who are aspired to pursue careers in academia. One of the undergraduate students has since enrolled in a master's program. It also provided training for two post-baccalaureate researchers, one of whom has been admitted to a doctoral program. The postdoctoral researcher working on the project has transitioned to a Project Scientist position. All the above-mentioned researchers have developed technical, critical thinking, and communication skills. They have met regularly with the PD and received personalized mentoring. How have the results been disseminated to communities of interest?The PD gave a lecture on "Plant metabolism associated with crop quality traits" in the graduate level course "Current topics on crop quality and safety", where she discussed the research on wheat biofortification with provitamin A. She also shared this research in a keynote presentation to prospective graduate students in the Plant Biology Graduate Group at UC Davis. Furthermore, progress on this project was presented at the Gordon Research Conference on Carotenoids. The PD is currently co-editing a special research topic collection on "Gene Editing for Biofortification: Innovations and Applications", which is pertinent to the wheat biofortification research. The PD also participated as a panelist at a career event organized by the Asian and Pacific Islander Student-Athlete Resource Group, where she spoke about her career path and research program on wheat biofortification. What do you plan to do during the next reporting period to accomplish the goals?In Year 2 of the project, we will continue the biochemical and physiological characterization of hyd2 knockout lines in both Triple Yellow and UC Amarillo backgrounds. We will also carry out seed multiplication in head rows in preparation for field trials. In parallel, we will characterize wheat lines overexpressing TaORWT and TaORHis in grains on their gene expression and metabolite levels.

Impacts
What was accomplished under these goals? Objective 1. To perform biochemical and physiological characterizations of the Triple Yellow, Triple Yellow/hyd2-knockout, and control hexaploid wheat lines. In Year 1 of the project, we identified transgene-free mutants with all three HYD2 homoeologs edited in the Triple Yellow background in the T2 generation. We conducted biochemical analysis of the Triple Yellow/hyd2-knockout plants and observed increased β-carotene levels in grains. These results showed that grain β-carotene levels in Triple Yellow can be increased by knocking out the HYD2 activity as we hypothesized. The WheatCRISPR computer program evaluates on-target editing specificity and predicts potential off-target editing for guide RNAs (gRNAs) used in CRISPR editing in wheat. Analysis with the WheatCRISPR program confirms that the gRNA we designed targets specifically the HYD2 homoeologs. When 2-4 mismatches in the HYD2 gRNA sequence are considered, WheatCRISPR predicts 3 genes (9 homoeologs) contain potential off-target regions. We amplified and sequenced these regions and confirmed that there was no off-target editing in these 9 homoeologs in the transgene-free, Triple Yellow/hyd2-knockout lines. To remove any potential off-target editing in the genome, we have also backcrossed the transgene-free, Triple Yellow/hyd2-knockout lines to Triple Yellow. While we were making progress on generating and characterizing Triple Yellow/hyd2-knockout mutants, we have also initiated CRISPR/Cas9 editing of HYD2 homoeologs in the UC Amarillo background. UC Amarillo is a hexaploid wheat breeding line recently released by the UC Davis wheat breeding program that shows increased total carotenoids in grains and favorable agronomic traits. We are enriching β-carotene in UC Amarillo grains in synergy to our efforts in Triple Yellow. Objective 2. To conduct agronomic, consumer quality, and postharvest storage evaluations of the Triple Yellow, Triple Yellow/hyd2-knockout, and control hexaploid wheat lines. This objective entails field studies and postharvest evaluations of Triple Yellow, Triple Yellow/hyd2-knockout, and control hexaploid wheat lines, which will be initiated in Year 2 of the project. There is nothing to report on this objective at this time. Objective 3. To determine the role of TaORWT and TaORHis in modulating carotenoid levels and stability. We have generated TaORHis homoeologs using TaORWT homoeologs as templates via site directed mutagenesis. The open reading frames of TaORWT and TaORHis were cloned into the Gateway pGWB5 and pGWB6 vectors for expressing N- and C-terminal green fluorescent protein (GFP) fusions of TaORWT and TaORHis. These recombinant plasmids have been transiently expressed in four-week-old Nicotiana benthamiana leaves to examine their subcellular localizations using a confocal microscope. In addition, we cloned both TaORWT and TaORHis into a plant binary vector driven by a grain-specific promoter and transformed the recombinant plasmids into tetraploid wheat. Generation of T0 plants is currently underway.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Shu Yu, Cody Bekkering, Xiaoqiong Qin, Alexander Simon, Michelle Li, Nina Isaka, Benjamin Sproul, Jorge Dubcovsky, and Li Tian (2025) Exploring carotenoid metabolism for increased beta-carotene levels in wheat grains. Poster presentation at the Gordon Research Conference on Carotenoids.