Project : USDA ARS

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Research Project: Improving Utilization of Forages in Integrated Dairy Production Systems to Enhance Sustainable Farming Systems and Food Security

Location: Cell Wall Biology and Utilization Research

2021 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
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 physical addition or by genetic modification of forages. 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 samples have been assayed for N, non-protein N, and other silage quality parameters. These data are currently being analyzed and are expected to provide information on the impacts of the PPO system on N dynamics and 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 lead to related compounds p-coumaroyl- and feruloyl-malate. We completed a study of alfalfa expressing HMT, but downregulated caffeoyl-CoA O-methyltransferase (CCOMT), which 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, including a putative red clover homolog of a recently described enzyme that directly carries out this conversion. These are currently being transformed into alfalfa plants expressing HMT and downregulated for CCOMT with the goal of further enhancing accumulation of caffeoyl-malate in alfalfa expressing HMT. 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 from publicly available data (bean) and from our own recently assembled perennial peanut transcriptome and open reading frames optimized for expression in E. coli have been synthesized for characterization of the encoded proteins. Condensed tannin (CT)-containing forages may also improve N-use efficiency (Objective 2), but published studies examining impacts of CT-containing plant material on ruminant nutrition have been inconsistent. In vitro protein precipitation studies may help predict how CTs 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 and limits the ability to draw conclusions about CT-protein interactions beyond the broad effects of CT structure (size, composition, linkage type). We have examined CT-protein precipitation in a rumen fluid analog, Goering-Van Soest (GVS) buffer, at rumen temperature and using both surrogate proteins (BSA, lysozyme) and alfalfa leaf protein (mostly Rubisco). The GVS buffer system better reflects consensus conclusions from the literature on the impacts of CT structure/composition on protein precipitation and provides 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 have conducted GVS protein precipitation studies with 32 additional purified condensed tannins incorporating a range of structural diversity with mean degrees of polymerization from 4.4 to 38, flavan-3-ol subunit composition spanning the entire practical range, procyanidin to prodelphinidin ratios ranging from 99:1 to 1:99, and cis/trans ratios ranging from 7:93 to 95:5. Results from these precipitation studies are forthcoming. 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 and is supplemented by our publicly-accessible U.S. Dairy Forage Research Center Condensed Tannin NMR Database (595 views/27 active accounts/20 different countries). New samples are added 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. The purified tannins from this library have allowed us to demonstrate 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. These have been transformed into alfalfa to evaluate gene expression and gene function, although the plants have not yet been analyzed. 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 alfalfa genes were identified predicted to encode UXS. We now have constructs corresponding to both genes to be used in plant overexpression and RNAi gene silencing studies. These approaches should provide insights into the role UXS 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 (Subobjective 4.1). Gas chromatography-mass spectrometry (GC-MS) profiling of 15 solutions’ effects on silage VOC emission is ongoing. 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 have designed and tested a continuous culture-based system to increase selective pressure for succinate producing communities. Testing of this new system was validated in a proof-of-principle experiment selecting for increased heat tolerance and testing the retention of heat-tolerance following cryopreservation. 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 a library of silage inoculants that includes commercial and laboratory-isolated strains (Subobjective 4.2). In vitro rumen digestions are planned for the coming year. 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 and feed storage modules. A collaborative USDA ARS-University of Vermont postdoc will be joining the team in the coming year.

Accomplishments
1. Generation of a new red clover reference genome. Red clover is a widely grown forage legume harvested for hay, grown in pasture for grazing, and sown as a companion crop. Like for many crops, genomic resources for red clover have greatly improved over the last decade. Unfortunately, a high-quality genomic reference sequence needed for many types of bioinformatic analyses has been lacking. ARS researchers at Madison, Wisconsin, and Clay Center, Nebraska, have generated a new reference genome for red clover using the latest sequencing technologies that generate long, accurate reads. The new reference genome is a vast improvement over the currently available genome as it is 266 times more continuous than the previously released reference genome and it takes into account the heterozygous nature of red clover’s genome. The new reference genome is expected to greatly facilitate work in gene discovery, transcriptomics, marker assisted breeding, and genome structure in red clover.

2. Recreation of clovamide biosynthesis in plants and other organisms. Clovamide, a specialized metabolite in red clover, has roles in protecting plants against stresses such as ultraviolet light, ozone, insects, and pathogens. It can also help preserve forage protein following harvest and may have potential medicinal and nutraceutical value. ARS researchers at Madison, Wisconsin, in collaboration with researchers at Lawrence Berkeley National Laboratory in Berkeley, California, and Pennsylvania State University in College Park, Pennsylvania, showed a clovamide biosynthetic pathway could be recreated in other plants and yeast. These findings could serve as a basis for producing clovamide in plants and other organisms to help preserve forage protein or serve as a source of novel medicinal and nutraceutical compounds.

3. Establishment of a nomenclature system for the comprehensive capture and delineation of condensed tannin structures. Condensed tannins (CTs), a class of compounds that accumulate in many plant species and can have important impacts in the agroecosystem (e.g. improving nitrogen use efficiency and reducing methane emissions in ruminant production systems), display an incredible diversity of structure. It has become increasingly clear that CT structure is related to CTs’ bioactive properties in the agroecosystem, and thus accurate reporting of CT structure is crucial to reporting CT impacts in biological systems, including the agroecosystem. An ARS researcher at Madison, Wisconsin, collaborating with researchers at University of Illinois-Chicago, University of Mississippi, Yeungnam University, Marquette University, and the Chinese Academy of Sciences, expanded on a previously established nomenclature system used in the USDFRC Condensed Tannin NMR database. This nomenclature descriptor system now includes additional CT structural features and covers all currently known and potential future CT structures. The new system fulfills the need for a strong and comprehensive system that eliminates ambiguity, clarifies scientific meaning, and promotes reporting CT structures with precision and quality, and will advance chemical and interdisciplinary CT research to the next level.

U.S. DEPARTMENT OF AGRICULTURE

Cell Wall Biology and Utilization Research: Madison, WI