Location: Children's Nutrition Research Center
2023 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 Sub-objective 1A we completed the evaluation of calcium oxalate crystal formation in different spinach (PI169688, PI1648964, PI335782, and NSL6095) and kale (Premier and Dwarf blue curled vates) varieties at 14, 24, and 44 days after germination which correspond to the micro-, baby-, and mature-spinach stages of growth, respectively, that are commonly sold to the consumer at supermarkets. This information coupled with our previous determinations of mineral and oxalate concentrations can aid consumers in making informed decisions regarding which stage of spinach to consume since oxalate inhibits the consumer’s ability to absorb calcium and also contributes to the formation of kidney stones. For Sub-objective 1B we completed expression analysis of the genes encoding the enzymes that are proposed to make up a recently discovered pathway of oxalate degradation in the different spinach varieties at the 3 stages of growth. In addition, we measured the activity of the enzyme responsible for the first step in this oxalate degradative pathway in the different spinach varieties at the three stages of growth.
For Sub-objective 2A we completed expression, purification, and characterization of a specific enzyme (formate dehydrogenase) that is capable of catalyzing the last step in the pathway of oxalate degradation. There is only one gene encoding formate dehydrogenase in both spinach and the model legume, Arabidopsis, and this enzyme was found to reside in the mitochondria in both plants. For Sub-objective 2B we completed isolation and characterization of Arabidopsis plants that lack a functional copy of each gene encoding the enzymes for each of the four proposed steps in the pathway of oxalate degradation.
For Sub-objective 3A we continue to work toward generating stable transgenic spinach plants with either an increase or decrease in their oxalate degradative capacity. Due to the technical difficulties associated with generating stable transgenic spinach plants we initiated a new approach to transiently alter expression of the oxalate degradative genes. We have proven the utility of this transient approach in spinach using a marker gene and are now moving forward with efforts to use this transient approach to decrease the oxalate degradative capacity of spinach plants.
For Sub-objective 4A, we grew 30 distinct varieties of spinach under controlled conditions and harvested plants when they had 6-8 mature leaves. This process was repeated a total of three times. Spinach tissue was sent to an ARS laboratory in Ithaca, New York, where it underwent procedures that simulate the human digestive system to estimate the amount of iron that would be absorbed if consumed by a person. Although spinach is recommended to consumers as a plant source of iron, it was determined that only about 1% of that iron is available for absorption in the body. We are currently pursuing additional studies to determine the chemical components of spinach that are preventing iron absorption into the body which is a nutrient of public concern for adolescent girls, people who can become pregnant, and pregnant people. For Sub-objective 4B, we profiled the material used in Sub-objective 4A for minerals, carotenoids (fat soluble plant pigments associated with positive health outcomes), and organic acids. For carotenoids and organic acids, we developed novel methods in our laboratory to quickly extract and quantify these compounds. We found that genetics appear to separately control carotenoid content and the efficiency that it is released for absorption by the body (bioaccessibility). We are currently crossing high and low bioaccessibility spinach cultivars to better understand the genetics that control this trait. These methods and data help scientists better understand how genetics and the environment influence the chemical components of spinach associated with health. Based on our findings we are also investigating additional classes of plant molecules that may be responsible for poor iron absorption from spinach-containing meals.
For Sub-objective 5A, we grew the same 30 varieties of spinach from Sub-objective 4A but under conditions similar to large-scale microgreen production. Microgreens are becoming progressively more popular with consumers, contain a high density of nutrients, and could potentially be used by plant breeders to screen for traits of interest without needing to wait for the plant to mature. Microgreens were harvested 14 days after germination and these grow-outs were conducted three separate times. Laboratory assays for bioaccessibility indicated that microgreens are a poor predictor of carotenoid bioaccessibility from mature leaves. These data establish benchmarks for spinach microgreen carotenoid content and bioaccessibility and show that developmental stage can drastically influence this trait. Spinach and many other leafy greens are increasingly being grown within controlled environments to ensure predictable growing conditions and reduce the distance between producers and consumers. We conducted additional experiments in mature spinach plants to explore how other environmental factors such as light quality as well as nutrient and salinity stress can affect carotenoid bioaccessibility. Both carotenoid content, bioaccessibility, and total yield can be modified to varying degrees by different environmental aspects. These findings may provide producers with information they can use to alter the nutrient density of their crops and also provide preliminary data for future studies examining how conditions associated with climate change may affect the quality of our food.
Accomplishments
