Location: Children's Nutrition Research Center
2022 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.
Objective 4: Establish the relationship between genetic background and mineral element bioaccessibility in spinach.
Objective 5: Evaluate carotenoid bioaccessibility as a function of spinach developmental stage.
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. Additionally, researchers will profile a genetically diverse population of spinach accessions for mineral and carotenoid bioaccessibility using an in vitro digestion approach.
Progress Report
For Objective 1, Sub-objective 1A we completed measuring the oxalate and mineral concentrations on the 3 different sets of spinach (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, stage respectively, that are commonly marketed to the general public at supermarkets. Information about oxalate and mineral concentration can aid the consumer to make informed decisions regarding their choice of leafy greens since oxalate can inhibit consumers' ability to absorb certain minerals such as calcium and may also contribute to the formation of kidney stones. For Objective 1, Sub-objective 1B we have grown and harvested the micro-, baby-, and mature-leafy greens and measured the oxalate concentrations and known oxalate degradative enzyme activities (i.e., oxalyl-CoA synthetase, oxalate oxidase, and oxalate decarboxylase). For Objective 2, Sub-objective 2A we have completed the cloning and expression of the formate dehydrogenase (FDH) gene. The FDH gene has been proposed to encode a protein that possesses an activity that is responsible for the last step in oxalate break down. As a step toward determining whether two proposed formyl CoA hydrolase (FCH) proteins participate in the break-down of oxalate we first had to establish a procedure to isolate the two different proteins from all the other proteins found in the Arabidopsis plant. As a part of Objective 2, Sub-objective 2B we established such an isolation procedure. Preliminary protein activity and gene expression measurements support a role for FCH in oxalate break-down. 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 commonly consumed plant foods. For Objective 3 we have tested different varieties of spinach for their regeneration capacity which refers to the ability to regrow an intact plant from a single cell. This regenerative ability is a crucial step in the plant transformation process. The spinach variety Viroflay appeared to show the most promise out of the multiple varieties tested. We have also tested different transformation and tissue culture procedures and have successfully introduced a marker gene into Viroflay spinach plants. We are currently trying to improve the efficiency of this procedure so it can be used to introduce our gene of interest into spinach. Such capabilities are required before attempts to modify selected spinach traits (such as oxalate concentrations) using molecular approaches can be conducted.
A new project was also established based upon a new researcher joining the center. This project began in April 2022 and thus much effort has been spent establishing and acquiring the research equipment necessary to accomplish these new research objectives. Objectives and their respective sub-objectives are expected to contribute fundamental knowledge about the relationship between plant genetics and vitamin deliverability as well as provide novel tools to plant breeders to screen plant populations more efficiently for health-promoting properties. Defining standard operating protocols and method development for Objectives 4 and 5 has been the central priority to ensure successful execution of the proposed experiments. Protocols are currently being refined to allow for consistent and robust growth of wild spinach varieties necessary for the study. Newly acquired research equipment and instrumentation are being set up and technical personnel staff are actively being trained to reliably operate and maintain these tools. Alternative strategies for assays with consumables affected by ongoing supply chain issues are being explored. It is anticipated that formal experiments will initiate at the end of this fiscal year.
Accomplishments
1. An enzyme important for oxalate breakdown is conserved in plants. Plant scientists have been avidly working to discover new strategies to reduce oxalate in plant foods. Oxalate inhibits the consumer's ability to absorb the calcium present in plant foods and can contribute to kidney stone formation (>75% of all kidney stones contain oxalate as a primary component). Additionally, certain plant pathogens produce oxalate as a requirement for infection and these oxalate-secreting pathogens are responsible for more than $100M in crop losses annually; therefore, reducing oxalate through degradation can improve the nutritional quality and production of plant foods. ARS scientists in Houston, Texas, discovered that oxalyl-CoA decarboxylase is the enzyme responsible for the second step in a previously uncharacterized pathway of oxalate break down in Arabidopsis. The researchers also found that this enzyme is present in other crop plants such as rice, maize, spinach, and tomato. These findings suggests that this enzyme and pathway of oxalate break down may be an avenue that will lead to the development of new strategies to improve the nutrition quality of plant foods and the resistance of plants against infection from certain oxalate-secreting fungal pathogens.
