Location: Cell Wall Biology and Utilization Research
2019 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.
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 as part of enhancing sustainability of this agroecosystem. This includes improving protein/nitrogen- (N-) utilization, 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, which have significant economic and environmental impacts, we have examined two natural systems that have potential to improve N-use efficiency in dairy production systems (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-diphenolic compounds. Adapting the red clover system to forages such as alfalfa will require providing these two components, either by physically adding them or by genetically modifying forage plants to make them. We previously introduced the red clover gene for PPO into alfalfa. Working with a collaborator, we developed populations of alfalfa segregating for the PPO trait. In the summer of 2018, these were grown in field plots and provided source 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. We have begun analyses of the silage samples for protein N and non-protein N as well as other parameters related to silage quality. These will provide information with respect to 1) optimal levels of PPO substrate for protein preservation; 2) impact of the PPO system on N dynamics; 3) the mechanisms by which PPO prevents protein degradation, and; 4) to what extent the PPO system improves N-use efficiency.
The use of condensed tannin (CT)-containing forages (Objective 2) is a second approach for reducing post-harvest protein loss and improving N-use efficiency. These improvements are a result of decreased protein degradation by proteases during ensiling and during subsequent digestion in the rumen. Published studies (both in vitro and in vivo) examining impacts of CT-containing plant material on nutrition have been inconsistent. This likely is the result of poor evaluation of CT content and inadequate, if any, consideration of CT structure. We have approached remedying these deficiencies in two ways. First, we continue to modify the traditional HCl-butanol protocol for determining CT content of forages. We have previously shown that replacing water as the solvent with acetone extracts all of the CT (bound and unbound) from two Lotus species (direct assay), providing a more accurate assessment of the total CT present. We have optimized reaction conditions for this direct assay and expanded the applicability to include other forages, woody plants, foods and food by-products. In addition, a method for the HCl-butanol assay was developed to sequentially determine CT present both in the acetone/water extract (soluble) and in the resulting residue (insoluble), the sum of whose values provides a measure of total CT content. Second, we developed a technique using 2D (two-dimensional) NMR (nuclear magnetic resonance) to determine composition and structural features of purified condensed tannins. The 2D NMR spectra provide data on PC/PD (procyanidin/prodelphinidin) and cis/trans ratios, estimations of mean degrees of polymerization (mDP), and percent galloylation and A-type linkage present in the purified sample and strongly corroborates structural information determined via conventional thiolytic degradation. To supplement evaluation of CTs by 2D NMR, we used our publicly accessible (595 pageviews/25 active accounts/20 different countries) U.S. Dairy Forage Research Center Condensed Tannin NMR Database.
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 continue to add to our “library” of purified CTs (currently 28 different plant materials) containing diverse structural elements of CTs found in common forage species. Members of this library can be used in experiments to determine 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. Collaborators have used library samples to show CTs have anthelmintic activity against Ascaris suum, the most common nematode parasite of pigs worldwide, and Giardia duodenalis, a protozoan parasite, common in both ruminants and pigs. In addition, collaborators demonstrated that cranberry CTs at sub-micromolar levels can inhibit iron-mediated DNA damage.
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. In cell walls, there is evidence that xylans are closely associated with lignin and are poorly degraded by ruminal microbes. Decreasing the amount of xylans in cell walls could increase the digestibility of the total wall. Further, decreasing the formation of xylans may result in an increased cellulose or pectin content due to restricted flow of sugar nucleotides through the pathway. Because there is always a close association with lignin, xylan decreases may alter the lignification patterns and/or concentration in the cell wall. 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. Using bioinformatics, two Medicago genes were identified predicted to encode the soluble versions of UXS. Using polymerase chain reaction (PCR), we previously amplified cDNA sequence corresponding to one of these from alfalfa. We have synthesized a cDNA corresponding to the second gene since we were unable to generate it via PCR. These will be used in future research to evaluate their potential for generating modified alfalfa.
During preservation of forages by ensiling, volatile organic compound (VOC) emissions 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 (Subobjective 4.1). Fifteen liquid solutions have been selected and prepared at three application rates for laboratory-scale trials. Troubleshooting of VOC quantification methods, which will be required for these experiments, are ongoing. Fourier-transform infrared (FTIR) spectroscopy-informed identification of VOCs and emission kinetics and gas chromatography-mass spectrometry (GC-MS) profiling are both being evaluated for the upcoming experiment.
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 (Subobjective 4.1). Initial iterative selection lines show variable heritability of the high-succinate phenotype. For this reason, we are implementing 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 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 are creating microbially-distinct but nutritionally near-identical corn and alfalfa silages (Subobjective 4.2). As a first step, we have assembled a collection of silage inoculants that will be applied to forages and the resulting silages profiled for nutritional composition and fermentation parameters.
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 USDFRC, we have made substantial progress in developing model code for the crop/soil module, using hired computer science students for coding. Groups at Cornell, University of California-Davis, and University of Wisconsin-Madison have made similar progress coding the animal module, and the group at Arkansas has progressed with the manure module. We are using regular conference calls and annual meetings to maintain group cohesion and progress.
Accomplishments
1. Optimization of assays for determination of condensed tannin content. Precise measurement of condensed tannin (CT, a biochemical naturally found in many plant materials) content is required to understand their bioactive properties, particularly in ruminant nutrition where CT-containing feedstuffs are fed or used in in vitro studies. In the past, researchers have used a traditional method (HCl-Butanol) which has been shown to provide inaccurate measurement of total CT because a portion of the CT is not readily solubilized due to binding to other cellular components. ARS researchers in Madison, Wisconsin, have optimized a direct HCl-butanol-acetone-iron (HBAI) assay previously reported; showed its applicability for measuring CT content in a range of forages, woody plants, foods and food by-products; and adapted it to allow sequential measurement of solvent-extractable and insoluble CT content whose sum is total CT content. This improved method provides the scientific community of CT researchers a way to reliably and reproducibly measure the total CT content of plant material which should allow more meaningful comparison of results across laboratories and experimental systems.
Review Publications
Zeller, W.E. 2019. Activity, purification, and analysis of condensed tannins: current state of affairs and future endeavors. Crop Science. https://doi.org/10.2135/cropsci2018.05.0323.
Sikora, M.C., Hatfield, R.D., Kalscheur, K. 2019. Fermentation and chemical composition of high-moisture lucerne leaf and stem silages harvested at different stages of development using a leaf stripper. Grass and Forage Science. 74(2):254–263. https://doi.org/10.1111/gfs.12423.
Bouchez, P., Benites, V.T., Baidoo, E.E., Mortimer, J.C., Sullivan, M.L., Scheller, H.V., Eudes, A. 2019. Production of clovamide and its analogs in Saccharomyces cerevisiae and Lactococcus lactis. Letters in Applied Microbiology. https://doi.org/10.1111/lam.13190.
