Location: Children's Nutrition Research Center
2021 Annual Report
Objectives
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.
Approach
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.
Progress Report
We completed measuring the oxalate and mineral concentrations in the different spinach types (PI169688, PI648964, PI335782, and NSL6095) and kale (Premier and Dwarf blue curled vates) varieties at 14, 24, and 44 days after germination which corresponds to the micro-, baby-, and mature-leafy greens, respectively, that are commonly marketed to the general public at supermarkets. These oxalate and mineral concentration measurements will benefit the consumer since oxalate can inhibit our ability to absorb certain minerals such as calcium and contribute to the formation of kidney stones. For Objective 1, Subobjective 1B, we have grown and harvested the micro-, baby-, and mature-leafy greens and have initiated measuring the activity of a specific protein (acyl-activating enzyme 3) which is responsible for the first step in the breakdown of oxalic acid in plants.
For Objective 2, Subobjective 2A, we have identified two genes that encode the FCH (formyl-CoA hydrolase) protein proposed to be responsible for the third step in the oxalate degradation pathway. Both genes have been transferred into bacteria to produce large amounts of the FCH protein to allow for additional experimental testing. Test trials have been run to determine the conditions that result in optimal FCH production, FCH purification, and FCH activity measurements. For Objective 2, Subobjective 2B, we have completed the identification and isolation of two Arabidopsis plants that lack a functional copy of the FDH (formyl-CoA dehydrogenase) gene. The FDH gene has been proposed to encode a protein responsible for the last step in oxalate degradation. Understanding how oxalate is broken-down in plants is required before we can design rational strategies to reduce this anti-nutrient and potential toxin in the foods commonly consumed.
Accomplishments
1. Identification of a key enzyme important for oxalate breakdown in plants. Plant scientists have been avidly working to discover new strategies to reduce oxalate in plant foods since it inhibits our ability to absorb calcium present in plant foods and contributes to kidney stone formation (over 75% of all kidney stones contain oxalate). Additionally, there are certain oxalate-secreting fungal pathogens that generate oxalate as a requirement for attacking/infecting plants and causes more than $100 M in crop losses annually. Thus, reducing oxalate can help improve the nutritional quality and production of plant foods. ARS scientists in Houston, Texas, discovered an enzyme, oxalyl-CoA decarboxylase, that is responsible for a critical step in a previously unidentified process of oxalate breakdown in plants. Our research showed that this key enzyme is involved in the important process of converting oxalate into carbon dioxide and that plants lacking that enzyme could not perform this conversion and were more susceptible to oxalate-secreting fungal pathogens which lead to major crop loss. Thus, the identification and isolation of this enzyme (oxalyl-CoA decarboxylase) is an important advancement in our understanding of oxalate development and provides researchers with a new strategic tool to improve the nutritional quality of plant foods and the resistance of plants against infection from certain oxalate-secreting fungal pathogens.
