Location: Cell Wall Biology and Utilization Research
2022 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
For Objectives 1 and 2, we have 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. We previously identified a system of protein protection in red clover consisting of the enzyme polyphenol oxidase (PPO) and PPO-oxidizable o-diphenols. Adapting the clover system to forages like alfalfa requires providing both components, either by physical addition or by genetic modification of forages. We previously carried out small scale ensiling experiments with alfalfa expressing the PPO gene where two different levels of PPO substrate were exogenously applied. Total N, non-protein N, and other silage quality parameter data from these samples are currently being analyzed and are expected to provide information on the impacts of the PPO system on N dynamics and N-use efficiency. Additionally, transgenic alfalfa with the PPO trait have been crossed with transgenic alfalfa with the o-diphenol trait to reconstruct the complete system in alfalfa. These will be used for additional experiments to understand the benefits and limitations of the PPO system.
For Sub-objective 1.3, we continued work on optimizing production of o-diphenol PPO substrates in alfalfa, since these are not normally present. 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 lead to related compounds p-coumaroyl- and feruloyl-malate. Simultaneous downregulation of endogenous caffeoyl-CoA O-methyltransferase (CCOMT) resulted in drastically increased caffeoyl-malate levels.
We have now prepared additional plant transformation constructs to enhance expression of phenylpropanoid pathway enzymes responsible for conversion of p-coumarate to caffeate. The first attempt at plant transformation failed, potentially due to the selectable marker being used. Transformation will be reattempted, but if unsuccessful, constructs will be rebuilt with a different selectable marker. We are also pursuing other legume hydroxycinnamoyl transferases which may be superior for making PPO substrates when expressed in alfalfa. These include two activities for which we have not yet definitively identified genes: HMT from common bean (hydroxycinnamates to malate) and HTT from perennial peanut (hydroxycinnamates to tartaric acid). Candidate genes for these activities have been identified. Open reading frames optimized for expression in Escherichia coli have been synthesized and we are testing the proteins for HTT and HMT activity. To investigate how structure of these transferases effect their function, we are making detailed kinetic measurements of several transferases and also determining structure by x-ray crystallography. The x-ray crystallography work by a collaborator is behind schedule due to COVID, but has recently been resumed with three legume transferases being characterized. We also carried out in silico structure modeling as an alternative to x-ray crystallography for HHHT (hydroxycinnamates to hydroxyhexanedioic acids), a transferase for which we’ve recently determined kinetic parameters.
For Sub-objective 2.1, we continue multiple approaches to investigate the potential benefits of condensed tannin- (CT) containing forages in animal production systems, including characterization of CT structure, biochemistry, and bioactivities. We are continuing to develop 2D (two-dimensional) NMR (nuclear magnetic resonance) techniques to determine composition/structural features of purified CTs to allow investigation of the impact CT structure has on biological activity. This data provides all of the structural information available from the more conventional, but labor intensive, thiolytic degradation and hydrolytic analyses but also has the potential to easily identify interflavan bond linkage types. This data is supplemented by our publicly-accessible U.S. Dairy Forage Research Center Condensed Tannin NMR Database (595 views/27 active accounts/20 different countries). We continue to add new samples to our “library” of well-characterized, purified CTs (from 35 different plants) representing diverse CT structural elements, including those found in many forages. Samples from this library have been used for studies in protein precipitation, in vitro ammonia reduction and methane abatement during rumen digestion, and tests for anthelmintic and antibiotic activity.
While CT-containing forages can be grazed, there is also a need for stored forages. We have been working to identify which preservation methods (silage, balage, or hay) would best allow CT-containing forages to deliver appropriate levels of utilizable protein for dairy and other animal production systems (Sub-objective 2.2). For this study we are using birdsfoot trefoil that is part of a U.S. Dairy Forage Research Center breeding program. Although initial work with older plots proved problematic, new plots planted at the end of 2020 provided us with stands of desired quality and quantity. These birdsfoot trefoil stands included the popular commercial variety Norcen, along with experimental germplasm bred for low- and high-tannin content. Material harvested during the 2021 growing season allowed experimental testing of all three major preservation methods (silage, balage, hay) across a range (0 to 12 months) of storage times. The resulting stored forage samples are currently being analyzed for N and protein fractions, fiber, and CT content. The experiment will be repeated with material from the 2022 growing season.
To address poor digestibility of plant cell walls (Sub-objective 3.2) in alfalfa, we are examining the role of sugar nucleotide biosynthetic enzymes in cell wall structure and assembly. We are focusing 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. Constructs for both over-expression and silencing of these have been made and are currently being transformed into alfalfa. Analysis of the resulting plants should provide insights into the role this enzyme plays in cell wall assembly.
Volatile organic compound (VOC) emissions from fermented forage components represent a loss of energy in dairy rations and an air quality issue. We are currently evaluating a method for the mitigation of silage VOC emissions by application of aqueous solutions at the feed bunk (Sub-objective 4.1A). Gas chromatography-mass spectrometry (GC-MS) profiling was used in a subsequent trial and has provided precise and accurate estimations of VOC emissions from laboratory silage samples and treatments. Data analysis from this trial is ongoing and a manuscript detailing the work is expected in 2023.
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 are working to select silage microbial communities with high succinate production (Sub-objective 4.1B). Initial iterative selection lines show variable heritability of the high-succinate phenotype. For this reason, we have designed and tested a continuous culture-based system to increase selective pressure for succinate producing communities. In order to more efficiently screen for succinate production, we have developed a high-throughput visible and near infrared spectroscopy (VNIR) method for detecting the presence of succinate in residual growth media. Work with succinate producing isolates and silage microbiomes will continue into 2023.
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 have created microbially-distinct but nutritionally near-identical corn and alfalfa silages using the library of silage inoculants; commercial and lab-isolated (Sub-objective 4.2). In vitro rumen digestions are ongoing with the milestone expected to be completed in early 2023.
We have established a collaborative team of researchers from ARS, universities, and industry to develop a next- generation, whole-farm dairy simulation model that will have animal, manure, crop/soil, and feed storage modules (Objective 5). At the U.S Dairy Forage Research Center, we have made substantial progress in developing model code for the crop/soil and feed storage modules. The system implemented to achieve this objective leverages the expertise of subject matter experts to delineate mathematical functions of interest of the processes being modeled into pseudocode. Hired computer science students then collaboratively translate the pseudocode into Python code. Collaborator groups have made similar progress coding the animal and manure modules.
Accomplishments
1. Developed a simple and cost-effective approach for determining pH and organic acid composition, and thus quality, of silage extracts. Traditional analytical approaches for analyzing industrially and agronomically relevant silage organic acids (lactic, succinic, acetic, and propionic) can be laborious and time consuming, but are critical to assessing feed value and spoilage risk of silage. ARS researchers in Madison, Wisconsin, have demonstrated that these silage organic acids can be predicted mathematically from the way they interact with visible and near-infrared wavelengths of light. This new approach is a low-cost, high-throughput method for rapid characterization of silage water extracts. This accomplishment will primarily benefit the silage research community by reducing cost of analysis and increasing throughput, but potential further development could allow increased on-farm silage quality diagnostics, saving producers money and enabling real-time decision-making.
Review Publications
Bickhart, D.M., Koch, L.M., Smith, T.P., Riday, H., Sullivan, M.L. 2022. Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species. GigaByte. 42:1-13. https://doi.org/10.46471/gigabyte.42.
Zeba, N., Berry, T.D., Panke-Buisse, K., Whitman, T.L. 2022. Effects of physical, chemical, and biological ageing on the mineralization of pine wood biochar by a Streptomyces isolate. PLoS ONE. 17(4). Article e0265663. https://doi.org/10.1371/journal.pone.0265663.
Hariharan, J., Choudoir, M.J., Diebold, P., Panke-Buisse, K., Buckley, D.H. 2022. Streptomyces apricus sp. nov. isolated from soil. International Journal of Systematic and Evolutionary Microbiology. 72(1). https://doi.org/10.1099/ijsem.0.005178.
Huang, Q., Hu, T., Xu, Z., Jin, L., McAllister, T.A., Acharya, S., Zeller, W.E., Mueller-Harvey, I., Wang, Y. 2021. Composition and protein precipitation capacity of condensed tannins in purple prairie clover (Dalea purpurea Vent.). Frontiers in Plant Science. 12. Article 715282. https://doi.org/10.3389/fpls.2021.715282.
Andersen-Civil, A.S., Myhill, L.J., Gokgoz, N.B., Engstrom, M., Mejer, H., Zhu, L., Zeller, W.E., Salminen, J., Krych, L., Lauridsen, C., Nielsen, D.S., Thamsborg, S.M., Williams, A.R. 2022. Dietary proanthocyanidins promote localized antioxidant responses in porcine pulmonary and gastrointestinal tissues during Ascaris suum-induced type 2 inflammation. Journal of Federation of American Societies for Experimental Biology. 36(4). Article e22256. https://doi.org/10.1096/fj.202101603RR.
Sullivan, M.L., Knollenberg, B.J. 2021. Red clover HDT, a BAHD hydroxycinnamoyl-coenzyme A:L-3,4-dihydroxyphenylalanine(L-DOPA) hydroxycinnamoyl transferase that synthesizes clovamide and other N-hydroxycinnamoyl-aromatic amino acid amides. Frontiers in Plant Science. 12. Article 727461. https://doi.org/10.3389/fpls.2021.727461.
Bickhart, D.M., Kolmogorov, M., Tseng, E., Portik, D., Korobeynikov, A., Tolstoganov, I., Uritskiy, G., Liachko, I., Sullivan, S.T., Shin, S.B., Zorea, A., Andreu, V., Panke-Buisse, K., Medema, M., Mizrahi, I., Pevzner, P., Smith, T.P. 2022. Generating lineage-resolved, complete metagenome-assembled genomes from complex microbial communities. Nature Biotechnology. 40:711-719. https://doi.org/10.1038/s41587-021-01130-z.
Zeller, W.E., Mueller-Harvey, I. 2021. Challenges in analyzing bioactive proanthocyanidins. In: Dreher Reed, J., Pereira de Freitas, V.A., Quideau, S. Recent Advances in Polyphenol Research. 1st edition. Hoboken, NJ: John Wiley & Sons, Inc. p. 131-175. https://doi.org/10.1002/9781119545958.ch6.
Roldan, M.B., Cousins, G., Muetzela, S., Zeller, W.E., Fraser, K., Salminen, J., Blanc, A., Kaur, R., Richardson, K., Maher, D., Jahufer, Z., Woodfield, D., Caradus, J.R., Voisey, C.R. 2022. Condensed tannins in white clover (Trifolium repens) foliar tissues expressing the transcription factor TaMYB14-1 bind to forage protein and reduce ammonia and methane emissions in vitro. Frontiers in Plant Science. 12. Article 777354. https://doi.org/10.3389/fpls.2021.777354.
