Location: Cell Wall Biology and Utilization Research
2024 Annual Report
Objectives
Objective 1: Develop or adapt poly-phenol systems in forage legumes for improved N use efficiency in dairy production systems.
Subobjective 1.1: Evaluate efficacy of the polyphenol oxidase (PPO)/o-diphenol system on preserving true protein during ensiling and improving N-use efficiency.
Subobjective 1.2: Determine the chemical basis for proteolytic inhibition caused by PPO-generated o-quinones.
Subobjective 1.3: Develop strategies to produce optimal levels of PPO substrates in alfalfa.
Objective 2: Develop or adapt tannin systems in forage legumes for improved N use efficiency in dairy production systems.
Subobjective 2.1: Determine the chemical basis for protection of protein during rumen digestion and providing elevated levels of escape protein into the hindgut by condensed tannins (CTs).
Subobjective 2.2: Analyze the effects of harvesting and storage methods on active CT content and protein preservation.
Objective 3: Improve forage digestibility and nutrient utilization efficiency in the cow through physiological modifications.
Subobjective 3.1: Prevent excessive leaf loss during plant development and harvesting by identifying genetic factors involved in leaf abscission in alfalfa providing a foundation for gene-based strategies for improvement.
Subobjective 3.2: Use genetic manipulation of sugar nucleotide biosynthetic pathways to identify avenues for altering cell wall structural polysaccharides and matrix interactions.
Subobjective 3.3: Explore alfalfa physiological mechanisms to enhance the utility of alfalfa as a cattle feed and other uses.
Objective 4: Improve forage silage quality and preservation to lessen forage losses, improve nutrition value for the cow, enhance soil ecology, and reduce environmental impacts for integrated dairy systems.
Subobjective 4.1: Incorporate non-traditional silage additives (novel forages, inoculants, concentrates) and management strategies to reduce forage loss and improve relative nutrition in the animal.
Subobjective 4.2: Leverage computational and sequencing technologies to elucidate connections between plant, silo, and animal microbiomes.
Objective 5: Develop system-based models to assess the productivity, efficiency, and environmental impact of dairy forage production.
Develop a whole-farm dairy simulation model that can be used to assess the impact of forage crop modifications and management on farm-scale nutrient cycling, farm crop and milk productivity, and environmental impacts.
Approach
Will utilize a multidisciplinary approach combining plant physiology/biochemistry, chemistry, agronomy, microbiology, molecular biology and genetics, and computer modeling. Forages provide unique nutritional and environmental opportunities to improve sustainable farming systems that help ensure food security. To enhance positive characteristics of forages, work will focus on capturing more plant protein in products, i.e., milk and plant bio-products, while generating less nitrogen waste; improving the amount of digestible cell wall biomass; and developing approaches to best maintain and optimize nutritional quality after harvest and during storage. We will also evaluate impacts of forage improvements and management by whole farm modeling. Efficient capture of protein nitrogen in the rumen is related to slowing protein degradation and availability of adequate digestible carbohydrate. Molecular, chemical, and biochemical approaches will be used to determine the roles of polyphenol oxidase/o-diphenols and tannins in decreasing protein degradation during ensiling and in the rumen (Objectives 1 and 2). Molecular approaches will be used to introduce a polyphenol oxidase/o-diphenol system into alfalfa to protect proteins during ensiling, including optimizing biochemical pathways in alfalfa to produce the o-diphenol PPO substrates. Chemical characterization of polyphenol (e.g., o-quinones and tannins) interactions with proteins will reveal mechanisms to protect proteins from degradation and provide selection criterion for forage improvement. Multiple approaches will be used to improve production of digestible forage biomass (especially carbohydrate) for improved animal performance (Objective 3). Molecular approaches will be used to down-regulate leaf abscission genes which would prevent excessive leaf loss, preserving a highly digestible fraction of alfalfa. The role of sugar nucleotide biosynthetic pathways in cell wall assembly and their influence on digestibility will be evaluated using molecular biology and biochemistry techniques. Approaches to maintain and optimize nutrition of preserved forages will be investigated using engineering, microbiological, and genomic approaches (Objective 4). Novel silage additives to prevent nutrient losses (for example via volatile organic compounds) will be investigated at lab and farm scales; microbial selection will be used to improve silage fermentation profiles, which could have impacts on greenhouse gas emissions; and metagenomics will be used to examine the complex interactions of field, silo, and rumen microbiomes. In order to better assess how changes in forages and forage management/storage impact the whole farm/agroecosystem, better whole-farm computer models will be developed with collaborators inside and outside of ARS (Objective 5). This project plan will increase our knowledge and understanding of current limitations associated with forage utilization and provides avenues to overcome these limitations.
Progress Report
This is the final report for this project which terminated in December 2023. See the report for the replacement project, 5090-21500-001-000D, “Identifying and Developing Strategies to Enhance Sustainability and Efficiency in Dairy Forage Production Systems” for additional information.
This project seeks to improve dairy system sustainability through increased utilization and value of forages. For Objectives 1 and 2, we examined two natural systems that have potential to improve nitrogen (N)-use efficiency in dairy production. Reducing protein losses just 10% could save U.S. farmers $200 to 400 million annually and reduce release of excess N into the environment. Objectives 3 and 4 focus on forage preservation and overall nutrient utilization through manipulation of forage physiology and ensiling. Finally, Objective 5 seeks to amplify our research scale and impact through participation in the development of a next-generation farm systems model.
We previously identified a system of protein protection in red clover consisting of the enzyme polyphenol oxidase (PPO) and PPO-oxidizable o-diphenols. We carried out small scale ensiling experiments with alfalfa expressing the PPO gene with exogenously applied PPO substrates (Sub-objective 1.1). Contrary to preliminary results, no significant reductions in protein degradation were seen. Failure to detect protein protection in the current experiments may be due to the method of tissue maceration and additional experimental variables that increased variability between silos and ensiling conditions. In a different experiment, approximately 200 transgenic alfalfa plants were screened for the traits, and several were identified expressing both PPO and producing the PPO substrate phaselic acid. Levels of phaselic acid produced were lower than the original parental plants and proteolysis in plant extracts did not differ from wild-type, likely due to inadequate levels of phaselic acid and high assay variability.
For Sub-objective 1.3, we previously identified an enzyme and its gene (HMT [hydroxycinnamoyl-CoA:malate transferase]) involved in making one of the major o-diphenolic compounds in red clover, caffeoyl-malate, but HMT expression in alfalfa led to accumulation of related compounds p-coumaroyl- and feruloyl-malate. Simultaneous downregulation of endogenous caffeoyl-CoA O-methyltransferase (CCOMT) resulted in increased caffeoyl-malate levels. We prepared additional plant transformation constructs to enhance expression of phenylpropanoid pathway enzymes responsible for conversion of p-coumarate to caffeate. We successfully modified our alfalfa transformation protocol for the phosphinothricin (Basta) resistance selectable marker and generated transgenic alfalfa which are being assessed for the impact of these genetic modifications on phaselic acid accumulation. Ongoing investigation of transferase structure and kinetics may provide strategies to improve o-diphenol production by this class of enzyme.
Because some Objective 1 experiments became unnecessary, we generated high-quality reference genomes for high-value forage and cover crops red clover and hairy vetch. This work yielded an improved genome for red clover, and the first such resource for hairy vetch. The new red clover genome increases contiguity 500-fold and is facilitating research and breeding in the U.S. and Europe. The vetch genome has been used to identify a seed dormancy locus, enabling breeding for soft-seeded cover crop varieties.
For Sub-objective 2.1, we continue characterization of condensed tannin (CT) structure, biochemistry, and bioactivities to investigate the potential benefits of CT-containing forages in animal production. We developed 2D (two-dimensional) NMR (nuclear magnetic resonance) techniques to determine composition/structural features of purified CT to evaluate the impacts of CT structure on biological activity. NMR provides the same structural information as conventional analyses but with less labor and more diagnostic power. We also developed the publicly-accessible U.S. Dairy Forage Research Center Condensed Tannin NMR Database (595 views/27 active accounts/20 different countries). A major hurdle in understanding CT effects on dairy nutrition and their underlying mechanisms is the availability of sufficient amounts of well-characterized, purified CTs for lab studies. We developed purification techniques to allow preparation of gram quantities of CTs for these studies. Our “library” of well-characterized, purified CTs (from 35 different plants) represents diverse CT structural elements, including those found in many forages. Library samples have been used for studies of protein precipitation ability, in vitro ammonia reduction and methane abatement during rumen digestion, and anthelmintic and antibiotic activity. This work will continue as detailed in the replacement project (5090-21500-001-00D).
In addition to work on fresh/grazing consumption, we have been working to identify which preservation methods (silage, baleage, or hay) best conserve CT-containing forages (Sub-objective 2.2). These studies include birdsfoot trefoil commercial varieties and experimental germplasm bred for low and high CT content. Material harvested during 2021 and 2022 growing seasons was preserved via the three methods (silage, baleage, hay) across a range (0 to 12 months) of storage times. The resulting stored forage samples are being analyzed for N and protein fractions, fiber, and CT content. This work will continue as detailed in the replacement project (5090-21500- 001-00D).
Alfalfa can lose up to 25% of highly digestible biomass through leaf abscission (Sub-objective 3.1). Two homologs of the potential arabidopsis abscission gene NEVERSHED were identified in Medicago truncatula. M. truncatula promoters were used to make reporter gene constructs to allow confirmation of abscission zone-specific expression and alfalfa sequences were used to make RNA interference (RNAi) silencing constructs. No obvious impact on leaf abscission was detected for plants transformed with the RNAi constructs.
To address poor digestibility of plant cell walls (Sub-objective 3.2) in alfalfa, we examined the role of sugar nucleotide biosynthetic enzymes in cell wall structure and assembly. We focused on an enzyme involved in cell wall sugar interconversions leading to the production of two sugars which make up poorly digested xylans. Two alfalfa genes were identified predicted to encode the enzyme. Over-expression and silencing constructs were transformed into alfalfa. Silenced plants showed substantial reductions in mRNA levels while over-expressing plants showed only modest increases in mRNA levels. These plants will be further analyzed for enzyme activity and cell wall characteristics and digestibility. Large amounts of the enzyme produced in Escherichia coli will be used for kinetics and producing polyclonal antibodies for further characterization of the enzyme. This work will continue as detailed in the replacement project (5090-21500-001-00D).
For Sub-objective 3.3, we have developed a reliable method for measuring protease activity in alfalfa tissue samples. We modified a method based on a commercially available kit that uses casein as a protease substrate. We have also increased throughput of this assay, and it is now possible to screen samples in a 96-well plate format. This assay will allow us to screen alfalfa germplasm for decreased protease activity. Decreased protease activity should result in a higher proportion of whole proteins in the plant after harvest, ultimately leading to increased nitrogen use efficiency in dairy production systems. This work will continue as detailed in the replacement project (5090-21500-001-00D).
Volatile organic compound (VOC) emissions from fermented forage components are a loss of energy in dairy rations and an air quality issue. We evaluated a to mitigate silage VOC emissions by application of aqueous solutions at the feed bunk (Sub-objective 4.1A). Gas chromatography-mass spectrometry (GCMS) profiling revealed high sample emission heterogeneity across replicates and no significant effect for most additive treatments. Initial evidence suggests oil-based additives may increase silage VOC emissions. Altering fermentation products produced during ensiling of forages could prevent losses during fermentation and improve utilization by dairy cattle. Succinate is a non-volatile organic acid of agronomic and industrial utility that is used efficiently in the rumen and could reduce production of the greenhouse gas methane in dairy animals. We worked to select silage microbial communities with high succinate production (Sub-objective 4.1B). However, none of the candidate isolates produced succinate reliably in a silage environment.
There is evidence that silage microbial communities can have beneficial probiotic effects for ruminant animals consuming silage. However, the effects of microbial communities from different silages on the rumen microbiome are difficult to distinguish from the effects of nutritional differences of the silages. To address this, we created microbially-distinct, but nutritionally near-identical, corn and alfalfa silages using a library of silage inoculants; commercial and lab-isolated (Sub-objective 4.2). Microbial ecology insights gained via amplicon sequencing data analysis from in vitro rumen digestions are expected to be completed by the end of the fiscal year.
A collaborative team of researchers from ARS, universities, and industry, has been established to develop a next generation, whole-farm dairy simulation model that will have animal, manure, crop/soil, and feed storage modules (Objective 5). We are developing and testing the crop/soil and feed storage modules. Model development is nearing completion for the full model and existing work is being reviewed with a focus on scalability, quality control, and documentation.
Accomplishments
Review Publications
Fuller, T.D., Bickhart, D.M., Koch, L.M., Kucek, L.K., Ali, S., Mangelson, H., Monteros, M.J., Hernandez, T., Smith, T.P., Riday, H., Sullivan, M.L. 2023. A reference assembly for the legume cover crop hairy vetch (Vicia villosa). GigaByte. https://doi.org/10.46471/gigabyte.98.
Dorris, M.R., Zeller, W.E., and Bolling, B.W. 2023. 1H–13C HSQC–NMR Analysis of cranberry (Vaccinium macrocarpon) juice defines the chemical composition of juice precipitate. Journal of Agricultural and Food Chemistry. 71(28):10710-10717. https://doi.org/10.1021/acs.jafc.3c01629.
