Location: Cell Wall Biology and Utilization Research
2020 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
The overarching goal of this project is to improve utilization of forages in dairy production systems to enhance sustainability of this agroecosystem. This includes improving protein/nitrogen (N) and cell wall utilization, better approaches to forage harvest and preservation, and evaluating the impact of changes to forages and harvest management via whole-farm modeling.
To reduce post-harvest protein losses, we have examined two natural systems that have potential to improve N-use efficiency in dairy production (Objectives 1 and 2). 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 physically adding them or by genetically modifying forages to make them. With a private sector collaborator, we developed populations of transgenic PPO-expressing alfalfa segregating for the PPO trait. In 2018-2019, these were grown in field plots and provided material for small scale ensiling experiments where two different levels of PPO substrate were exogenously applied, and silage samples were collected over time up to six months post-ensiling. These are being analyzed for N and non-protein N as well as other silage quality parameters. This study will provide information with respect to impact of the PPO system on N dynamics and to what extent the PPO system improves N-use efficiency.
We continued work on making the required o-diphenol PPO substrates in alfalfa, since these are not normally present (Subobjective 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 leads not to the PPO-utilizable caffeoyl-malate, but to related compounds p-coumaroyl- and feruloyl-malate. We completed a study of alfalfa expressing HMT, but also downregulated for caffeoyl-CoA O-methyltransferase, which converts caffeoyl moieties to feruloyl moieties. In these plants, caffeoyl-malate levels are dramatically increased. We have now prepared additional plant transformation constructs to enhance expression of phenylpropanoid pathway enzymes responsible for conversion of p-coumarate to caffeate. Expression of these may further enhance accumulation of caffeoyl-malate in alfalfa expressing HMT.
Condensed tannin (CT)-containing forages are a second approach for improving N-use efficiency (Objective 2). Published studies examining impacts of CT-containing plant material on ruminant nutrition have been inconsistent. In vitro protein precipitation studies are one approach to assess CT-protein interactions and help predict how CTs might affect protein/N-utilization in the rumen. However, most published studies have been performed with surrogate proteins, such as bovine serum albumin (BSA), in standard laboratory buffers and under conditions which, beyond pH, are not reflective of the rumen environment. Thus, previous CT-protein precipitation studies may not accurately represent CT-protein interactions during rumen fermentation. This leads to difficulty in interpreting literature results on CT-protein interactions, although often consensus conclusions on the impact of CT structure (size, composition, linkage type) on CT-protein interactions can be drawn. To overcome ambiguity, we have examined CT-protein precipitation using the Goering-Van Soest (GVS) buffer system (specifically designed to mimic rumen fluid) and conducting the experiments at rumen temperature using both surrogate proteins (BSA, lysozyme) and alfalfa leaf protein (mostly Rubisco). Comparison of GVS versus non-GVS buffers shows the GVS buffer system better reflects consensus conclusions from the literature on the impacts CT structure/composition have on protein precipitation. A GVS-based assay will provide better data for selecting CT-containing forages for ruminant feeding studies or forage species for enhancing CT content via conventional breeding or genetic modification.
We continue to develop 2D (two-dimensional) NMR (nuclear magnetic resonance) techniques to determine composition/structural features of purified CTs. This data provides procyanidin/prodelphinidin and cis/trans ratios, estimations of average CT size, and percent galloylation and A-type linkages present in purified samples. Further, our NMR data strongly corroborates findings from conventional, but labor intensive, thiolytic degradation analyses. Our NMR data is supplemented by our publicly accessible U.S. Dairy Forage Research Center Condensed Tannin NMR Database (595 views/25 active accounts/20 different countries), developed from NMR data already in the literature. We continue to add to our “library” (Subobjective 2.1) of well-characterized, purified CTs (from 35 different plants) representing diverse CT structural elements, including those found in many forages. Library samples are used in experiments to understand how CT structure/composition affect CT properties, especially with respect to ruminant nutrition. We have demonstrated that larger CTs are more efficient at precipitating proteins than smaller CTs and have stronger anthelmintic activity against Ascaris suum, a parasitic nematode in animals.
An approach for improving biomass production is to alter leaf abscission (Subobjective 3.1). Alfalfa can lose up to 25% of highly digestible biomass through leaf abscission. Two potential homologs of the gene NEVERSHED, implicated in abscission in arabidoposis, were identified in Medicago truncatula. The promoters of the M. truncatula genes were cloned via PCR (polymerase change reaction) and used to make reporter gene constructs to allow confirmation of abscission zone-specific expression. Alfalfa cDNA fragments corresponding to the genes were used to make RNA interference (RNAi) silencing constructs to evaluate gene function in alfalfa.
To address poor digestibility of plant cell walls (Subobjective 3.2) in alfalfa, we are examining the role of sugar nucleotide biosynthetic enzymes in cell wall structure and assembly. We are focusing on UDP-D-xylose synthase (UXS) as a target for downregulation due to its apparent central role in cell wall sugar interconversions leading to the production of xylose and arabinose which make up poorly digested xylans. Two Medicago genes were identified predicting to encode UXS. Using PCR and in vitro synthesis we now have cDNAs corresponding to both genes to be used in plant overexpression and RNAi gene silencing studies, and codon optimized versions of both genes for expression in E. coli to characterize enzyme activities. These approaches should provide insights into the role UXS plays in cell wall assembly.
Volatile organic compound (VOC) emissions from ensiled forages represent a loss of energy in dairy rations and an air quality issue. We are currently evaluating a method for mitigating silage VOC emissions by application of aqueous solutions at the feed bunk (Subobjective 4.1). Fifteen liquid solutions have been selected and tested in a laboratory-scale trial. Fourier-transform infrared (FTIR) spectroscopy-informed identification of VOCs was limited by low resolution of individual volatiles when read together. Consequently, gas chromatography-mass spectrometry (GC-MS) profiling will be used instead, but has been delayed due to a needed software upgrade to the instrument. We expect to complete this laboratory scale pilot study in the coming year.
Altering fermentation products produced during ensiling of forages could prevent biomass losses during fermentation and improve silage utilization by dairy cattle. Succinate is a non-volatile organic acid 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 (Subobjective 4.1). 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.
There is evidence that silage microbial communities can have beneficial probiotic effects for ruminants 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 a “library” of silage inoculants derived from commercial and lab-isolated strains (Subobjective 4.2).
We have established a collaborative team of researchers from ARS, University of Wisconsin, University of California-Davis, University of Arkansas, and Cornell University 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 describing the processes being modeled into pseudocode (a "text-based" algorithmic design tool). Hired computer science students collaboratively translate the pseudocode into Python, a widely-used, high-level general-purpose programming language. Collaborating groups have made similar progress on their modules. Regular conference calls and annual meetings are used to maintain group cohesion and progress.
Accomplishments
1. Condensed tannin-rich peanut skin supplementation increases growth performance of grazing meat goats. Approximately 60,000 tons of peanut skins are produced in the United States every year as a low-value byproduct. ARS researchers in Madison, Wisconsin, and Bushland Texas, along with collaborators from Tuskegee University, the Institute of Integrated Technology in South Korea, and Fort Valley State University, have found a useful outlet for what otherwise might be a waste stream. In a controlled feeding study, grazing meat goats were supplemented with alfalfa meal pellets (control) or peanut skin pellets. The meat goats in the peanut skin pellet-supplemented group grew 38.5% faster (average daily growth) when compared to the control-supplemented group. Further, with the peanut skin diet several measures of meat quality (empty body weight, hot carcass, cold carcass, shoulder, hind shank, rack, loin and fat thickness) were higher than those of animals on the control diet. This nutritional approach allows conversion of the low-value peanut skin waste stream into increased productivity for meat goat, and possibly other ruminant, production systems, providing economic benefit to the producer while alleviating the potential environmental impact from conventional disposal of the peanut skins.
2. Protein precipitation by condensed tannins in a simulated rumen fluid buffer better reflects tannin-protein interactions. Protein precipitation by condensed tannins (CTs) in in vitro experiments provides insight into how CT structure affects function in biological systems, guidance on selection of CT-containing forages for ruminant feeding studies, and assistance in the identification of specific forages amenable to CT content modification through conventional breeding or genetic modification. However, all in vitro CT-protein interaction experiments reported in the literature thus far, conducted by a multitude of researchers, utilize varied conditions (buffer, temperature, protein) not reflective, beyond similar pH, of the rumen environment. This has led to some difficulty in interpreting the literature on CT-protein interactions, although often consensus conclusions on the impact of CT structure (size, composition, linkage type) on CT-protein interactions can be drawn from the results of multiple studies. To overcome ambiguity and obtain results most relevant to ruminant nutrition, ARS researchers in Madison, Wisconsin, have examined CT-protein precipitation by using the Goering-Van Soest (GVS) buffer system (specifically designed to mimic rumen fluid) at mammalian body temperature, and using both model proteins common in the literature (bovine serum albumen, lysozyme) but also alfalfa leaf protein (primarily Rubisco). It was found that this assay approach not only best reflects the consensus results/conclusions from the literature on the impacts CT size, composition, and structure have on protein precipitation but is likely more relevant to ruminant nutrition. Further, this approach should offer researchers better guidance when selecting CT-containing forages and amendments for ruminant feeding studies or forage species for enhancing CT content via conventional breeding or genetic modification.
3. Selected condensed tannins fail to inhibit Cryptosporidium growth in animal cells. Infections with Cryptosporidium, a microscopic parasite causing gastrointestinal and respiratory illnesses, constitute a substantial public health burden and are responsible for widespread production losses in cattle herds. Currently, there are very few therapeutic options available to treat cryptosporidiosis and no vaccines are available. Recent interest in plant bioactive compounds to mitigate the spread of anthelmintic resistance in ruminants has led to investigation of these biologics against other parasitic taxa. Condensed tannins (CTs) are plant secondary metabolites that have shown promising potential against nematode parasites but their applicability to Cryptosporidium infections are comparatively under-explored. ARS researchers in Madison, Wisconsin, in collaboration with scientists at the Norweigian Veterinary Institute, Oslo, Norway and the Norwegian Centre for Organic Agriculture, Tingvall, Norway, tested well-characterized CTs with differing chemical characteristics from five plant species in an assay utilizing Cryptosporidium-infected human tissue culture cells. Although a conventional antimicrobial drug was inhibitory, none of the CTs examined demonstrated inhibitory potential against the parasite. With the lack of inhibition by these purified CTs, caution should be exercised by researchers promoting the putative inhibitory activity of CTs contained in plants extracts.
Review Publications
Zeller, W.E., Reinhardt, L.A., Robe, J.T., Sullivan, M.L., Panke-Buisse, K. 2020. Comparison of protein precipitation ability of structurally diverse procyanidin-rich condensed tannins in two buffer systems. Journal of Agricultural and Food Chemistry. 68(7):2016-2023. https://doi.org/10.1021/acs.jafc.9b06173.
Grabber, J.H., Zeller, W.E. 2019. Direct versus sequential analysis of procyanidin- and prodelphinidin-based condensed tannins by the HCl–butanol–acetone–iron assay. Journal of Agricultural and Food Chemistry. 68,10, 2906-2916. https://doi.org/10.1021/acs.jafc.9b01307.
Min, B., McTear, K., Wang, H.H., Joakin, M., Gurung, N., Abrahamsen, F., Solaiman, S., Eun, J., Lee, J.H., Dietz, L.A., Zeller, W.E. 2019. Influence of elevated protein and tannin-rich peanut skin supplementation on growth performance, blood metabolites, carcass traits, and immune-related gene expression of grazing meat goats. Journal of Animal Physiology and Animal Nutrition. 104(1):88-100. https://doi.org/10.1111/jpn.13250.
Wiesner, S., Duff, A., Desai, A.R., Panke-Buisse, K. 2020. Increasing dairy sustainability with integrated crop-livestock farming. Sustainability. 12(3), 765. https://doi.org/10.3390/su12030765.
