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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

2018 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
During this one-year bridging project, we continued to carry out research in line with the objectives of the previous project plan (5090-21000-055-00D) and further build upon the work completed under that plan. To address poor digestibility of plant cell walls (Objective 1) we collaborated with scientists from Universidade de Sao Paulo to alter lignin composition and content. Two genes from sugar cane were used to modify cell walls of corn through both increased and decreased expression. Both genes encode transferase enzymes involved in incorporation of the phenolic compound p-coumaric acid (pCA) into the cell wall matrix. One attaches pCA directly to lignin (pCAT) and the other (AT-10) attaches pCA to glucuronoarabinoxylans (GAX). In corn, increased expression of AT-10 resulted in significant increases of pCA attached to GAX, but did not appear to have an impact upon the amount of pCA attached to lignin or lignin content. Decreased expression of AT-10 had no impact on pCA on GAX or on lignin. Decreased expression of pCAT decreased pCA on lignin but did not impact lignin content. Increased expression of pCAT in maize has not been fully analyzed. 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). 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 private industry collaborator, we developed populations of alfalfa segregating for the PPO trait. These will be grown in field plots to provide source material for small scale ensiling experiments using exogenously applied PPO substrate to determine: 1) optimal levels of PPO substrate; 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 determine the three-dimensional structure of HMT and related transferases. 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. A preliminary structure for HMT was determined and is currently being refined. 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 the ensiling process and during the rumen digestion. 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 and have shown that replacing water as the solvent with acetone extracts all of the CT (bound and unbound) of two Lotus species, providing a more accurate assessment of the total CT present. We further validated this assay on a variety of species including woody plants. 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 provides 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 have updated our publicly accessible (589 pageviews/21 active accounts/8 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 26 different plant materials) containing 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, it was shown that cranberry CTs can inhibit iron-mediated DNA damage at sub-micromolar levels. Alternative management strategies could provide better ways of utilizing forages (Objective 3). Capturing the optimal nutrient value of alfalfa for dairy production requires frequent harvest at early developmental stages, creating an economic burden on producers. Harvesting alfalfa leaves separate from stems creates two feedstocks: a highly digestible, high protein leaf fraction and a high fiber, low protein stem fraction. We tested the stability of nutrient components during separate ensiling of alfalfa leaves and stems compared to conventional whole plant ensiling. The leaf fraction was obtained using a prototype leaf stripper, which allows separate harvest of leaves and stems. Early bud, 10-20% bloom, and > 50% bloom stages of alfalfa were harvested using the leaf stripper. Leaf and stem fractions were chopped and ensiled immediately after harvest into 0.5-L minisilos. Whole plant silages were made following typical on farm procedures (wilting to 32-35% dry matter). Changes in nutritional value were analyzed for up to 140 days of ensiling. Leaf material harvested at different developmental stages showed little difference in nutrient value at harvest. There was a slow decrease in nutritive value over the entire period of ensiling, but more available protein was retained than the whole plant silage. Stems were less stable over 140 days, likely due to poor initial fermentation, but stem silage maintained good nutritional feed value for dry cow or heifer diets. This shows alfalfa can be harvested at higher maturity/less frequent harvest intervals but maintain a high value protein-rich leaf fraction for dairy production. Improving forage preservation and storage would enhance forage feed value and prevent losses. Forages in humid regions are commonly preserved by ensiling. Proper forage preservation is critical to the maintaining dietary protein and nutrition in the feed. Previous work identified deleterious/beneficial microbes and outlined best management practices for minimizing poor outcomes, but recent advances in DNA sequencing and data analysis now allow exploration of the wider microbial communities of ensiled forages. These new tools should facilitate understanding the effects of silage additives on the microbial community and identifying potential new silage inoculants. This genomics-based approach was used to profile alfalfa and corn silage communities and optimize new laboratory workflows for microbial investigations of silages. This included the alteration of silo scale and downstream assays to accommodate smaller, more numerous samples for enhanced detection of microbial treatment effects. It is expected this work will serve as a foundation for future studies to enhance ensiling practices. We have also begun to investigate the susceptibility of silage microbial communities to ecosystem-level selection and the potential of “microbiome breeding” to influence silage community function. Preliminary work suggests selection of the microbial community can alter the products produced by the community during forage fermentation: alterations in the concentrations of acetic, propionic, and succinic acid in alfalfa silages following selection were most significant.

4. Accomplishments
1. Cranberry condensed tannins inhibit iron-mediated plasmid DNA damage. The occurrence of cancer is related to initial DNA damage by reactive oxygen species generated in cells. ARS researchers in Madison, Wisconsin, in collaboration with researchers at Clemson University, have determined that condensed tannins purified from cranberries can inhibit iron-mediated DNA damage at sub-micromolar levels. The inhibition by cranberry condensed tannins was similar to that of epigallocatechin gallate (a major component of green tea) and tannic acid. These findings provide a possible mechanism for anti-cancer activities of human consumed foods and beverages containing flavanols and condensed tannins previously documented in the literature.

2. Condensed tannins have size-dependent anti-parasitic effects against Ascaris suum and Giardia duodenalis. Parasitic infections of the intestine are very common in livestock production, resulting in poor animal health and reduced productivity, and some parasites can also be transmitted to humans. Although drug treatments are available, these can be costly and there is an increasing threat of parasites developing drug resistance. ARS researchers in Madison, Wisconsin, in collaboration with researchers at the University of Copenhagen, have shown that condensed tannins, naturally present in many plant species, have anthelmintic activity against Ascaris suum, the most common nematode parasite of pigs worldwide, and also Giardia duodenalis, a protozoan parasite that is common in both ruminants and pigs. Through the use of in vitro assays it was demonstrated that anti-parasitic activity is associated with a high degree of polymerization (i.e. a larger size) of the tannin molecules. Tannins derived from cranberry (Vaccinium macrocarpon) and white clover (Trifolium repens) flowers are particularly effective. These results suggest the possibility of treating parasitic infections using tannin-containing plants or supplementing feeds with tannin-containing additives. Such tannin-based treatments could reduce the use of pharmaceuticals and avoid the generation of drug resistant strains of these parasites.

3. Extent of methane mitigation during in vitro rumen digestion related to the antioxidant activity of the forage. The presence of condensed tannins in forages, or added to total mixed rations, has been shown to reduce methanogenesis (the production of methane [a potent greenhouse gas]) during ruminant digestion and consequently also improves energy use efficiency by the animal. To elucidate the mechanism of reduced methanogenesis by condensed tannin-containing forages, ARS researchers in Madison, Wisconsin, in collaboration with researchers at the University of Missouri and Miami University (Ohio), used a variety of techniques to determine the structure and composition of purified condensed tannins from nine warm-season perennial legumes. These data were then used in an attempt to correlate condensed tannin structure with their ability to inhibit methane production. Although no correlation between tannin structure and inhibition of methanogenesis was seen, inhibition of methanogenesis showed a strong, non-linear correlation with the antioxidant activity (of the condensed tannins and possibly other metabolites present) of the plant material. This finding suggests the antioxidant activity of the condensed tannin-containing forages plays the primary role in reducing methanogenesis in ruminants and could provide a new approach to understanding methane production during ruminant digestion. Assessing the antioxidant activity of forages and feedstuffs consumed by ruminants could provide a predictive model for methane production during ruminant digestion.

Review Publications
Naumann, H.D., Tedeschi, L.O., Zeller, W.E., Huntley, N.F. 2017. The role of condensed tannins in animal production: advances, limitations and future directions. Brazilian Journal of Animal Science. 46:929-949.

Brown, R.H., Mueller-Harvey, I., Zeller, W.E., Reinhardt, L.A., Stringano, E., Gea, A., Drake, C., Ropiak, H.M., Fryganas, C., Ramsay, A., Hardcastle, E.E. 2017. Facile purification of milligram to gram quantities of condensed tannins according to mean degree of polymerization and flavan-3-ol subunit composition. Journal of Agricultural and Food Chemistry. 65:8072-8082.

Kim, H., Padmakshan, D., Li, Y., Rencoret, J., Hatfield, R.D., Ralph, J. 2017. Characterization and elimination of undesirable protein residues in plant cell wall materials for enhancing lignin analysis by solution-state nuclear magnetic resonance spectroscopy. Biomacromolecules. 18(12): 4184-4195. doi: 10.1021/acs.biomac.7b01223.