Location: Children's Nutrition Research Center2019 Annual Report
Objective 1. Characterize oxalate catabolic activity in low and high oxalate plants of dietary importance such as leafy greens harvested at different stages of development. Subobjective 1A: Characterize dynamic changes in oxalic acid and calcium oxalate crystal formation and assess mineral bioavailability in low and high oxalate leafy plants at different developmental stages Subobjective 1B: Measure oxalate catabolic activity in low and high oxalate leafy plants at different developmental stages. Objective 2. Identify and characterize in a model plant system the genes and encoded proteins responsible for each step in a novel pathway of oxalate catabolism. Subobjective 2A: Isolate and biochemically characterize the putative enzymes responsible for catalyzing the remaining steps in the CoA-dependent pathway of oxalate catabolism Subobjective 2B: Assignment of each putative enzyme to the CoA-dependent pathway of oxalate catabolism. Objective 3. Determine the influence of the newly identified oxalate catabolism pathway on the nutritional composition, phytochemical profile, and production characteristics in plants of dietary importance such as leafy greens grown to different stages of maturity (microgreens to mature greens). Subobjective 3A: Manipulate oxalate catabolism in leafy greens. Subobjective 3B: Assess the impact of oxalate catabolism on leafy green growth. Subobjective 3C: Assess the impact of the manipulation of oxalate catabolism on the nutritional quality of leafy greens.
Although oxalic acid is known to impact numerous biological processes in a broad range of organisms, our understanding of the mechanisms regulating its turnover are not well understood. This is especially true in plants. To begin to fill these gaps in our knowledge we plan to first assess the oxalate catabolic activity in low and high oxalate plants of dietary importance at different stages of development.The information gained from the assessment would be of use to consumers trying to reduce dietary oxalate loads and scientists interested in gaining new insights into the mechanisms regulating oxalate metabolism in plants. We will also identify and characterize in a model plant system the genes and encoded proteins responsible for each step in the CoA-dependent pathway of oxalate catabolism. The findings obtained will contribute significantly to our understanding of oxalate turnover and will set a foundation for future investigations into oxalate metabolism in a number of organisms ranging from microbes to plants.
We completed seed bulking of the different spinach (PI169688, PI648964, PI445782, and NSL6095) and kale (Premier and Dwarf blue curled vates) varieties. An ample supply of seeds is required to conduct the oxalate, mineral, mineral bioavailability, and enzyme studies. For Objective 2, Sub-objective 2A, we completed the isolation of the candidate genes from Arabidopsis that are predicted to encode a formyl-CoA hydrolase. Each predicted formyl-CoA hydrolase gene was put into a protein expression vector to allow the production and purification of produced proteins for activity measurements. As a part of Sub-objective 2B, Arabidopsis plants that lack the potential formyl-CoA protein have been identified and seeds bulked. For Objective 3, Sub-objective 3A, we have completed construction of the Arabidopsis acyl-activating enzyme 3 over-expression construct which will allow for the generation of plants with increase oxalate degradative capacity. In regards to completing the spinach acyl-activating enzyme 3 knockout construct we have identified and isolated the spinach acyl-activating enzyme 3 gene, but still need to validate the activity of the encoded protein before completing the vector construction.