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ARS Home » Midwest Area » Madison, Wisconsin » U.S. Dairy Forage Research Center » Cell Wall Biology and Utilization Research » Research » Research Project #433936

Research Project: Removing Limitations to the Efficient Utilization of Alfalfa and Other Forages in Dairy Production, New Bio-Products, and Bioenergy to Enhance Sustainable Farming Systems and Food Security

Location: Cell Wall Biology and Utilization Research

2019 Annual Report

Obj. 1: Increase profitability, improve animal welfare & reduce manure production by improving the digestibility & energy conversion efficiency of forages in dairy rations by manipulating forage cell-wall biosynthetic pathways to lower indigestible residue formation, lower waste production, & develop more efficient tools for evaluating forage quality. Sub-Obj. 1.1: Use genetic manipulation of biosynthetic pathways (e.g., lignin, lignin modifications, lignin cross-linking, ferulate cross-linking, structural polysaccharides) to identify avenues for altering cell wall digestibility & the formation of indigestible residues. Sub-Obj. 1.2: Develop methods to provide rapid screening of plant materials for chemical characteristics related to improved energy conversion efficiency and/or other nutrient quality factors. Obj. 2: Increase profitability & reduce the amount of nitrogen-containing wastes that enter the environment by reducing protein loss during the post-harvest storage & livestock consumption of alfalfa & other forages through manipulation of forage phenolic metabolic pathways. Sub-Obj. 2.1: Gain knowledge of factors that influence accumulation of hydroxycinnamyl-conjugates (e.g., phaselic acid, clovamide, chlorogenic acid) utilizable by PPO systems in forages with respect to efficacy in post-harvest proteolytic inhibition, as well as combating abiotic stress (UV, ozone). Sub-Obj. 2.2: Determine the chemical basis for proteolytic inhibition caused by classes of tannins & polyphenol oxidase-generated o-quinones. Obj. 3: Develop novel alfalfa harvesting & management technologies & strategies that increase forage biomass quality & quantity; increase nutrient availability for dairy; decrease forage input costs in integrated dairy systems; promote novel bio-products & reduce nutrient losses (N&P) to the environment. Sub-Obj. 3.1: Determine how alfalfa selected for improved stem nutritive value influences harvest management strategies & ruminant performance. Sub-Obj. 3.2: Use unique harvesting practices coupled with on-farm treatment & storage to create protein-rich fractions that produce value-added products from alfalfa. Sub-Obj. 3.3: Prevent excessive leaf loss during plant development & harvesting by gaining more knowledge of hydrolytic and regulatory factors involved in leave abscission in alfalfa, thus leading to gene-based strategies for improvement. Sub-Obj. 3.4: Develop equipment & technologies for cost-effective separation of leaf & stem fractions of alfalfa & of perennial grasses. Sub-Obj. 3.5: Develop cost-effective, farm-scale technology for on-farm pretreatment using chemicals or enzymes (finish current project). Sub-Obj. 3.6: Optimize production of adhesive components in CBP fermentations & expand end-use formulations of adhesives containing fermentation residues. Sub-Obj. 3.7: Develop whole-farm, system-based databases & models to simulate/forecast economic & environmental impacts of implementing new & existing bioenergy practices on dairy or beef farms in the Upper Midwest. This effort will be coordinated with similar production-modeling efforts at other ARS locations.

We will utilize a multidisciplinary approach combining plant physiology/biochemistry, chemistry, agronomy, molecular biology and genetics. 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: improving cell wall digestibility under high biomass production; and capturing more plant protein in products, e.g., milk and plant bio-products, while generating less nitrogen waste. Improved utilization of cell walls can be achieved through manipulation of genes involved in biosynthesis of structural carbohydrates and lignin. Small changes in cell wall composition may lead to decreased cross-linking and increased digestibility (Objective 1). Cell wall screening methods based on nuclear magnetic resonance spectroscopy and Fourier transformed infrared spectroscopy will be used to identify chemical characteristics related to improved energy conversion efficiency. Molecular approaches will be used to modify plant biosynthetic pathways (lignification, cell wall cross-linking, structural polysaccharides) to identify avenues for altering cell wall digestibility. 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 (Objective 2). Molecular approaches will be used to alter plants for reduced protein loss during post-harvest storage and during livestock consumption of forages. A polyphenol oxidase/o-diphenol system will be inserted into alfalfa to protect proteins during ensiling. 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 forage biomass production for improve animal performance and new bio-products (Objective 3). Molecular approaches will be used to down-regulate leaf abscission genes which would prevent excessive leaf loss, preserving the protein-rich fraction of alfalfa. To improve forage biomass production for increased nutrient availability and novel bio-products, field-grown alfalfa selected for increased stem digestibility will be evaluated to reveal its potential for improved animal performance. Analysis of alfalfa leaves during plant development will determine potential changes in protein and, coupled with new harvesting techniques, will lead to improved quality, as well as new bio-products to increase utilization of alfalfa in farming systems. 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
During this 16-month bridging project, we continued to carry out research in line with the objectives of the previous project plan (5090-21000-055-00D) and laid a foundation for the objectives of the current project plan (5090-21000-071-00D). To address poor digestibility of plant cell walls (Objective 1) in alfalfa, we initiated a new project to examine 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 predicted to encode the soluble versions of UXS were identified. Using polymerase chain reaction (PCR), we were able to amplify cDNA sequence corresponding to one of these from alfalfa. We have plans to synthesize the cDNA corresponding to the second gene and utilize this information for future research. To reduce post-harvest protein losses, which have significant economic and environmental impacts, we have examined two natural systems that have potential to improve nitrogen (N) use efficiency in dairy production systems (Objective 2). A UV treatment approach was also pursued (Sub-objective 3.5). 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. 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. The samples will be analyzed 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. We also continued work on understanding how o-diphenol PPO substrates are made in red clover and other legume plants, since these are not normally present in alfalfa. 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, the caffeic acid derivative phaselic acid (PA), but HMT expression in alfalfa leads not to the PPO-utilizable PA, but to related compounds. An alternative to altering the alfalfa phenylpropanoid pathway to enhance accumulation of caffeoyl PPO substrates is to find or create a hydroxycinnamoyl-CoA transferase that favors production of caffeoyl derivatives over non-PPO substrates. We began a collaboration with researchers at Washington University in St. Louis (Missouri) to characterize hydroxycinnamoyl-CoA transferases with respect to enzymatic properties and three-dimensional structure. This will help us understand how structure of these enzymes is related to function, leading to strategies for redesigning them to make the desired PPO utilizable products. As part of this project, a hydroxycinnamoyl-CoA transferase from bean (HHHT) was produced in E. coli and purified for use in detailed kinetic studies. The use of condensed tannin (CT)-containing forages 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 page views/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. As an alternative to polyphenol-based systems to preserve forage protein after harvest, the response of alfalfa intracellular proteins to post-harvest ultraviolet (UV-C) irradiation was characterized (Sub-objective 3.5). UV rays can induce widespread intracellular damage in plants. One mechanism by which some plants mitigate negative impacts of damaged intracellular proteins is by sequestering them into large cross-linked aggregates. Cross linked and/or large proteins are associated with greater resistance to proteolysis in the silo. These experiments seem to rule out use of UV treatment in preserving forage protein, however, as no evidence of protein aggregation was observed, and irradiation actually increased protein degradation during ensiling. For preserved forages, new targets for rapid screening and estimation of fermentation potential of forages were evaluated (Sub-objective 1.2). Silage samples of varying species, dry matter, and particle size were scanned using near-infrared reflectance spectroscopy and mid-infrared reflectance spectroscopy. Spectral variation correlation with microbial biomarker quantification was sufficient to allow prediction of certain fermentation community attributes. Initial results suggest utility for these technologies as a non-destructive method for rapid evaluation of silage quality during ensiling. Development of ensiling vessels that allow repeated or continuous scanning represent the largest obstacle to method adoption.


Review Publications
Naumann, H., Sepela, R., Rezaire, A., Masih, S.E., Zeller, W.E., Reinhardt, L.A., Robe, J.T., Sullivan, M.L., Hagerman, A.E. 2018. Relationships between structures of condensed tannins from Texas legumes and methane production during in vitro rumen digestion. Molecules. 2018, 23, 2123.
Kronberg, S.L., Zeller, W.E., Waghorn, G., Grabber, J.H., Terrill, T.H., Liebig, M.A. 2018. Effects of feeding Lespedeza cuneata pellets with Medicago sativa hay to sheep: Nutritional impact, characterization and degradation of condensed tannin during digestion. Animal Feed Science And Technology. 245:41-47.