Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. This is the final report for this project which terminated in December 2023. See the report for the replacement project, 5090-21500-001-000D, ⿿Identifying and Developing Strategies to Enhance Sustainability and Efficiency in Dairy Forage Production Systems for additional information. This project seeks to improve dairy system sustainability through increased utilization and value of forages. For Objectives 1 and 2, we examined two natural systems that have potential to improve nitrogen (N)- use efficiency in dairy production. Reducing protein losses just 10% could save U.S. farmers $200 to 400 million annually and reduce release of excess N into the environment. Objectives 3 and 4 focus on forage preservation and overall nutrient utilization through manipulation of forage physiology and ensiling. Finally, Objective 5 seeks to amplify our research scale and impact through participation in the development of a next-generation farm systems model. We previously identified a system of protein protection in red clover consisting of the enzyme polyphenol oxidase (PPO) and PPO-oxidizable o- diphenols. We carried out small scale ensiling experiments with alfalfa expressing the PPO gene with exogenously applied PPO substrates (Sub- objective 1.1). Contrary to preliminary results, no significant reductions in protein degradation were seen. Failure to detect protein protection in the current experiments may be due to the method of tissue maceration and additional experimental variables that increased variability between silos and ensiling conditions. In a different experiment, approximately 200 transgenic alfalfa plants were screened for the traits, and several were identified expressing both PPO and producing the PPO substrate phaselic acid. Levels of phaselic acid produced were lower than the original parental plants and proteolysis in plant extracts did not differ from wild-type, likely due to inadequate levels of phaselic acid and high assay variability. For Sub-objective 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 led to accumulation of related compounds p- coumaroyl- and feruloyl-malate. Simultaneous downregulation of endogenous caffeoyl-CoA O-methyltransferase (CCOMT) resulted in increased caffeoyl- malate levels. We prepared additional plant transformation constructs to enhance expression of phenylpropanoid pathway enzymes responsible for conversion of p-coumarate to caffeate. We successfully modified our alfalfa transformation protocol for the phosphinothricin (Basta) resistance selectable marker and generated transgenic alfalfa which are being assessed for the impact of these genetic modifications on phaselic acid accumulation. Ongoing investigation of transferase structure and kinetics may provide strategies to improve o-diphenol production by this class of enzyme. Because some Objective 1 experiments became unnecessary, we generated high-quality reference genomes for high-value forage and cover crops red clover and hairy vetch. This work yielded an improved genome for red clover, and the first such resource for hairy vetch. The new red clover genome increases contiguity 500-fold and is facilitating research and breeding in the U.S. and Europe. The vetch genome has been used to identify a seed dormancy locus, enabling breeding for soft-seeded cover crop varieties. For Sub-objective 2.1, we continue characterization of condensed tannin (CT) structure, biochemistry, and bioactivities to investigate the potential benefits of CT-containing forages in animal production. We developed 2D (two-dimensional) NMR (nuclear magnetic resonance) techniques to determine composition/structural features of purified CT to evaluate the impacts of CT structure on biological activity. NMR provides the same structural information as conventional analyses but with less labor and more diagnostic power. We also developed the publicly- accessible U.S. Dairy Forage Research Center Condensed Tannin NMR Database (595 views/27 active accounts/20 different countries). 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 developed purification techniques to allow preparation of gram quantities of CTs for these studies. Our ⿿library of well-characterized, purified CTs (from 35 different plants) represents diverse CT structural elements, including those found in many forages. Library samples have been used for studies of protein precipitation ability, in vitro ammonia reduction and methane abatement during rumen digestion, and anthelmintic and antibiotic activity. This work will continue as detailed in the replacement project (5090-21500-001-00D). In addition to work on fresh/grazing consumption, we have been working to identify which preservation methods (silage, baleage, or hay) best conserve CT-containing forages (Sub-objective 2.2). These studies include birdsfoot trefoil commercial varieties and experimental germplasm bred for low and high CT content. Material harvested during 2021 and 2022 growing seasons was preserved via the three methods (silage, baleage, hay) across a range (0 to 12 months) of storage times. The resulting stored forage samples are being analyzed for N and protein fractions, fiber, and CT content. This work will continue as detailed in the replacement project (5090-21500- 001-00D). Alfalfa can lose up to 25% of highly digestible biomass through leaf abscission (Sub-objective 3.1). Two homologs of the potential arabidopsis abscission gene NEVERSHED were identified in Medicago truncatula. M. truncatula promoters were used to make reporter gene constructs to allow confirmation of abscission zone-specific expression and alfalfa sequences were used to make RNA interference (RNAi) silencing constructs. No obvious impact on leaf abscission was detected for plants transformed with the RNAi constructs. To address poor digestibility of plant cell walls (Sub-objective 3.2) in alfalfa, we examined the role of sugar nucleotide biosynthetic enzymes in cell wall structure and assembly. We focused on an enzyme involved in cell wall sugar interconversions leading to the production of two sugars which make up poorly digested xylans. Two alfalfa genes were identified predicted to encode the enzyme. Over-expression and silencing constructs were transformed into alfalfa. Silenced plants showed substantial reductions in mRNA levels while over-expressing plants showed only modest increases in mRNA levels. These plants will be further analyzed for enzyme activity and cell wall characteristics and digestibility. Large amounts of the enzyme produced in Escherichia coli will be used for kinetics and producing polyclonal antibodies for further characterization of the enzyme. This work will continue as detailed in the replacement project (5090-21500-001-00D). For Sub-objective 3.3, we have developed a reliable method for measuring protease activity in alfalfa tissue samples. We modified a method based on a commercially available kit that uses casein as a protease substrate. We have also increased throughput of this assay, and it is now possible to screen samples in a 96-well plate format. This assay will allow us to screen alfalfa germplasm for decreased protease activity. Decreased protease activity should result in a higher proportion of whole proteins in the plant after harvest, ultimately leading to increased nitrogen use efficiency in dairy production systems. This work will continue as detailed in the replacement project (5090-21500-001-00D). Volatile organic compound (VOC) emissions from fermented forage components are a loss of energy in dairy rations and an air quality issue. We evaluated a to mitigate silage VOC emissions by application of aqueous solutions at the feed bunk (Sub-objective 4.1A). Gas chromatography-mass spectrometry (GCMS) profiling revealed high sample emission heterogeneity across replicates and no significant effect for most additive treatments. Initial evidence suggests oil-based additives may increase silage VOC emissions. 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 worked to select silage microbial communities with high succinate production (Sub-objective 4.1B). However, none of the candidate isolates produced succinate reliably in a silage environment. 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 created microbially- distinct, but nutritionally near-identical, corn and alfalfa silages using a library of silage inoculants; commercial and lab-isolated (Sub- objective 4.2). Microbial ecology insights gained via amplicon sequencing data analysis from in vitro rumen digestions are expected to be completed by the end of the fiscal year. A collaborative team of researchers from ARS, universities, and industry, has been established to develop a next generation, whole-farm dairy simulation model that will have animal, manure, crop/soil, and feed storage modules (Objective 5). We are developing and testing the crop/ soil and feed storage modules. Model development is nearing completion for the full model and existing work is being reviewed with a focus on scalability, quality control, and documentation.
Impacts
(N/A)
Publications
- Fuller, T.D., Bickhart, D.M., Koch, L.M., Kucek, L.K., Ali, S., Mangelson, H., Monteros, M.J., Hernandez, T., Smith, T.P., Riday, H., Sullivan, M.L. 2023. A reference assembly for the legume cover crop hairy vetch (Vicia villosa). GigaByte. https://doi.org/10.46471/gigabyte.98.
- Dorris, M.R., Zeller, W.E., and Bolling, B.W. 2023. 1H⿿13C HSQC⿿NMR Analysis of cranberry (Vaccinium macrocarpon) juice defines the chemical composition of juice precipitate. Journal of Agricultural and Food Chemistry. 71(28):10710-10717. https://doi.org/10.1021/acs.jafc.3c01629.
|
Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. For Objectives 1 and 2, we have examined two natural systems that have potential to improve nitrogen (N)-use efficiency in dairy production. 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. We carried out small scale ensiling experiments with alfalfa expressing the PPO gene where PPO substrate was exogenously applied. Total N, non-protein N, and other silage quality parameters from these samples were analyzed. Unfortunately, in most instances no significant reductions in protein degradation were seen due to PPO and o- diphenols in these experiments, in contrast to original proof-of-concept experiments. The failure to detect protein protection in the current experiments may be due to the method of tissue maceration and additional experimental variables that increased variability between silos and ensiling conditions. In a different experiment, transgenic alfalfa plants with the PPO trait were crossed with transgenic alfalfa with the o- diphenol trait to reconstruct the complete system. Approximately 200 plants were screened for the traits, and several were identified expressing both PPO and producing the PPO substrate phaselic acid. Unfortunately, levels of phaselic acid produced were lower than the original parental plants, possibly due to genetic background. We examined proteolysis in extracts of these plants, but could not detect significant reductions compared to wild type plants in contrast to a previous experiment in which the system was reconstituted by mixing extracts. We believe failure to detect proteolytic reduction is due to inadequate levels of phaselic acid in these plants such that any effect cannot be detected over experimental variability. This experiment will be repeated to confirm the results. Thus, for Sub-objective 1.3, we continued work on optimizing/increasing production of o-diphenol PPO substrates 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, caffeoyl-malate, but HMT expression in alfalfa lead to accumulation of related compounds p-coumaroyl- and feruloyl-malate. Simultaneous downregulation of endogenous caffeoyl-CoA O- methyltransferase (CCOMT) resulted in drastically increased caffeoyl- malate levels. We prepared additional plant transformation constructs to enhance expression of phenylpropanoid pathway enzymes responsible for conversion of p-coumarate to caffeate. We have successfully modified our alfalfa transformation protocol for the phosphinothricin (Basta) resistance selectable marker used for these constructs and anticipate producing plants and being able to assess the impact of these additional genetic modifications on phaselic acid accumulation. To investigate how structure of transferases like HMT affect their function, we have been making detailed kinetic measurements of several transferases and a collaborator is determining structure by x-ray crystallography. Our collaborator has recently been able to determine structure for HMT which may provide strategies to improve o-diphenol production by this class of enzyme. For Sub-objective 2.1, we continue multiple approaches to investigate the potential benefits of condensed tannin- (CT-) containing forages in animal production systems. This includes characterization of CT structure, biochemistry, and bioactivities. Development of 2D (two-dimensional) NMR (nuclear magnetic resonance) techniques to determine composition/ structural features of purified CTs, which allows determination of how CT structure impacts biological activity, is near completion. This approach provides the same structural information as more conventional, but labor intensive, thiolytic degradation and hydrolytic analyses but also has the potential to easily identify additional structural features such as interflavan bond linkage types. We continue to add new samples to our ⿿library of well-characterized, purified CTs (from 35 different plants) representing diverse CT structural elements, including those found in many forages. Samples from this library have been used for studies of protein precipitation ability, in vitro ammonia reduction and methane abatement during rumen digestion, and anthelmintic and antibiotic activity. While CT-containing forages can be grazed, there is also a need for stored forages. We have been working to identify which preservation methods (silage, baleage, or hay) would best allow CT-containing forages to deliver appropriate levels of utilizable protein for dairy and other animal production systems (Sub-objective 2.2). We are using birdsfoot trefoil stands that include the popular commercial variety Norcen, along with experimental germplasm bred for low and high CT content. Material harvested during the 2021 and 2022 growing seasons was preserved via the three methods (silage, baleage, hay) across a range (0 to 12 months) of storage times. The resulting stored forage samples are currently being analyzed for N and protein fractions, fiber, and CT content. To address poor digestibility of plant cell walls (Sub-objective 3.2) in alfalfa, we are examining the role of sugar nucleotide biosynthetic enzymes in cell wall structure and assembly. We are focusing on an enzyme involved in cell wall sugar interconversions leading to the production of two sugars which make up poorly digested xylans. Two alfalfa genes were identified that are predicted to encode the enzyme. Constructs for both over-expression and silencing of these have been transformed into alfalfa, and several independent transformants for each construct have been identified. In the coming year, these will be analyzed for expression, enzyme activity, and ultimately cell wall characteristics and digestibility. We have also produced the large amounts of the enzyme in Escherichia coli to be used for kinetics studies and producing polyclonal antibodies as a tool for further characterization of the in vivo role of the enzyme. For Sub-objective 3.3, we are developing and testing various methods for screening alfalfa germplasm for decreased protease activity after harvest. Breakdown of whole proteins eventually leads to nitrogen loss from the dairy production system and release of nitrogen into the environment. Alfalfa is a relatively high protein forage, but the plant⿿s proteases immediately begin to break down the proteins after harvest. We are preparing to screen alfalfa germplasm for reduced protease activity to decrease post-harvest protein breakdown in this forage. As a first step, we are testing the sensitivity of a universal protease activity assay and working to increase its throughput to screen what may be hundreds of alfalfa lines. We are also assessing assays that detect specific types of protease activity such as acid proteinase, aminopeptidase, and carboxypeptidase. If alfalfa lines with decreased protease activity are identified, they should have a higher proportion of intact proteins after harvest. Thus, we are also testing assays that will quantify the amount of intact protein compared to the amount of smaller peptides and amino acids, the products of protein degradation by proteases. 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 (Sub- objective 4.1A). Gas chromatography-mass spectrometry (GCMS) profiling revealed high sample emission heterogeneity across replicates and no significant effect for most additive treatments. Initial evidence suggests that oil-based additives may increase VOC emissions from silage. 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 (Sub-objective 4.1B). Our approach includes both selection from silage microbial isolates as well as screening known succinate-producers from the NRRL culture collection for forage fermentation potential. 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; commercial and lab-isolated (Sub- objective 4.2). Amplicon sequencing data from in vitro rumen digestions are expected to be completed by the end of the fiscal year. A collaborative team of researchers from ARS, universities, and industry, has been established to develop a next generation, whole-farm dairy simulation model that will have animal, manure, crop/soil, and feed storage modules (Objective 5). We are responsible for development and testing the crop/soil and feed storage modules. Model development is nearing completion for the full model and existing work is being reviewed with a focus on scalability, quality control, and documentation. ACCOMPLISHMENTS 01 Generation of a hairy vetch reference genome. Use of cover crops can help make agriculture more sustainable. Hairy vetch is a legume species that is used as a cover crop, and can reduce soil erosion, help bees, feed livestock, and supply nitrogen to crops like corn and tomatoes. Some traits in hairy vetch limit its use as a cover crop, such as pod shatter and hard seed. To help plant breeders overcome these challenges, ARS researchers at Madison, Wisconsin, and Clay Center, Nebraska, as well as nongovernmental organization and private sector partners generated a reference genome for hairy vetch. The new reference genome is already facilitating breeding for soft seeded varieties that will increase use of hairy vetch as a cover crop. The work is expected to greatly facilitate marker assisted breeding for other useful traits, as well as work in gene discovery, transcriptomics, and genome structure. Additionally, overcoming technical challenges associated with generating this reference genome required the application and creation of new approaches that will benefit similar genomic research on other important species. 02 Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) is a more facile approach for determining accurate condensed tannin (CT) structural information compared to chemical methods. Condensed tannins (CTs) are a family of chemical compounds present in many plants, including forage crops, that are made up of smaller subunits that can be assembled in many different ways to form CTs of highly diverse structure. CT structure is known to influence important bioactivities that these compounds have such as improving protein utilization and mitigating methane emissions in ruminant animals. ARS researchers in Madison, Wisconsin, have developed new less laborious methods to determine the composition, structure, and purity of CTs using 2D-NMR. Accuracy of the NMR method has been validated by comparison of results to those obtained through labor-intensive traditional wet chemistry approaches. Thus, these improved NMR methods for CT structure determination should find widespread use in identifying CTs responsible for desired biological activities such as improved protein utilization and mitigation of methane emissions in ruminant production systems.
Impacts
(N/A)
Publications
- Sullivan, M.L. 2022. Preparation of hydroxycinnamoyl-coenzyme A thioesters using recombinant 4-coumarate:coenzyme A ligase (4CL) for characterization of BAHD hydroxycinnamoyltransferases enzyme activities. In: Jez, Joseph, editor. Methods in Enzymology. Volume 683. Cambridge, MA: Academic Press. p.3-18.
- Sullivan, M.L. 2022. Near-real time determination of BAHD acyl-coenzyme A transferase reaction rates and kinetic parameters using Ellman's reagent. In: Jez, Joseph, editor. Methods in Enzymology. Volume 683. Cambridge, MA: Academic Press. p.19-39.
- Fanelli, A., Sullivan, M.L. 2022. Bioinformatic tools for protein structure prediction and for molecular docking applied to enzyme active site analysis. In: Jez, Joseph, editor. Methods in Enzymology. Volume 683. Cambridge, MA: Academic Press. p.41-79.
- Panke-Buisse, K. 2023. Estimation of silage VOC emission impacts of surface-applied additives by GC-MS. Atmospheric Environment: X.17.Article 100206. https://doi.org/10.1016/j.aeaoa.2023.100206.
- Woolsey, I.D., Zeller, W.E., Blomstrand, B.M., Oines, O., Enemark, H.L. 2022. Effects of selected condensed tannins on Cryptosporidium parvum growth and proliferation in HCT-8 cell cultures. Experimental Parasitology. 241. Article 108353. https://doi.org/10.1016/j.exppara.2022.108353.
- Dinkins, R.D., Hancock, J.A., Bickhart, D.M., Sullivan, M.L., Zhu, H. 2022. Expression and variation of the genes involved in rhizobium nodulation in red clover. Plants. 11(21). Article 2888. https://doi.org/10.3390/ plants11212888.
|
Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. For Objectives 1 and 2, we have examined two natural systems that have potential to improve nitrogen (N)-use efficiency in dairy production. 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. We previously carried out small scale ensiling experiments with alfalfa expressing the PPO gene where two different levels of PPO substrate were exogenously applied. Total N, non-protein N, and other silage quality parameter data from these samples are currently being analyzed and are expected to provide information on the impacts of the PPO system on N dynamics and N-use efficiency. Additionally, transgenic alfalfa with the PPO trait have been crossed with transgenic alfalfa with the o-diphenol trait to reconstruct the complete system in alfalfa. These will be used for additional experiments to understand the benefits and limitations of the PPO system. For Sub-objective 1.3, we continued work on optimizing production of o- diphenol PPO substrates in alfalfa, since these are not normally present. 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. Simultaneous downregulation of endogenous caffeoyl-CoA O-methyltransferase (CCOMT) 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. The first attempt at plant transformation failed, potentially due to the selectable marker being used. Transformation will be reattempted, but if unsuccessful, constructs will be rebuilt with a different selectable marker. 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. Open reading frames optimized for expression in Escherichia coli have been synthesized and we are testing the proteins for HTT and HMT activity. To investigate how structure of these transferases effect their function, we are making detailed kinetic measurements of several transferases and also determining structure by x- ray crystallography. The x-ray crystallography work by a collaborator is behind schedule due to COVID, but has recently been resumed with three legume transferases being characterized. We also carried out in silico structure modeling as an alternative to x-ray crystallography for HHHT (hydroxycinnamates to hydroxyhexanedioic acids), a transferase for which we⿿ve recently determined kinetic parameters. For Sub-objective 2.1, we continue multiple approaches to investigate the potential benefits of condensed tannin- (CT) containing forages in animal production systems, including characterization of CT structure, biochemistry, and bioactivities. We are continuing to develop 2D (two- dimensional) NMR (nuclear magnetic resonance) techniques to determine composition/structural features of purified CTs to allow investigation of the impact CT structure has on biological activity. This data provides all of the structural information available from the more conventional, but labor intensive, thiolytic degradation and hydrolytic analyses but also has the potential to easily identify interflavan bond linkage types. This data is supplemented by our publicly-accessible U.S. Dairy Forage Research Center Condensed Tannin NMR Database (595 views/27 active accounts/20 different countries). We continue to add new samples to our ⿿library of well-characterized, purified CTs (from 35 different plants) representing diverse CT structural elements, including those found in many forages. Samples from this library have been used for studies in protein precipitation, in vitro ammonia reduction and methane abatement during rumen digestion, and tests for anthelmintic and antibiotic activity. While CT-containing forages can be grazed, there is also a need for stored forages. We have been working to identify which preservation methods (silage, balage, or hay) would best allow CT-containing forages to deliver appropriate levels of utilizable protein for dairy and other animal production systems (Sub-objective 2.2). For this study we are using birdsfoot trefoil that is part of a U.S. Dairy Forage Research Center breeding program. Although initial work with older plots proved problematic, new plots planted at the end of 2020 provided us with stands of desired quality and quantity. These birdsfoot trefoil stands included the popular commercial variety Norcen, along with experimental germplasm bred for low- and high-tannin content. Material harvested during the 2021 growing season allowed experimental testing of all three major preservation methods (silage, balage, hay) across a range (0 to 12 months) of storage times. The resulting stored forage samples are currently being analyzed for N and protein fractions, fiber, and CT content. The experiment will be repeated with material from the 2022 growing season. To address poor digestibility of plant cell walls (Sub-objective 3.2) in alfalfa, we are examining the role of sugar nucleotide biosynthetic enzymes in cell wall structure and assembly. We are focusing on an enzyme involved in cell wall sugar interconversions leading to the production of two sugars which make up poorly digested xylans. Two alfalfa genes were identified predicted to encode the enzyme. Constructs for both over- expression and silencing of these have been made and are currently being transformed into alfalfa. Analysis of the resulting plants should provide insights into the role this enzyme 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 (Sub- objective 4.1A). Gas chromatography-mass spectrometry (GC-MS) profiling was used in a subsequent trial and has provided precise and accurate estimations of VOC emissions from laboratory silage samples and treatments. Data analysis from this trial is ongoing and a manuscript detailing the work is expected in 2023. 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 (Sub-objective 4.1B). 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. In order to more efficiently screen for succinate production, we have developed a high- throughput visible and near infrared spectroscopy (VNIR) method for detecting the presence of succinate in residual growth media. Work with succinate producing isolates and silage microbiomes will continue into 2023. 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 the library of silage inoculants; commercial and lab-isolated (Sub- objective 4.2). In vitro rumen digestions are ongoing with the milestone expected to be completed in early 2023. We have established a collaborative team of researchers from ARS, universities, and industry 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 of interest of the processes being modeled into pseudocode. Hired computer science students then collaboratively translate the pseudocode into Python code. Collaborator groups have made similar progress coding the animal and manure modules. ACCOMPLISHMENTS 01 Developed a simple and cost-effective approach for determining pH and organic acid composition, and thus quality, of silage extracts. Traditional analytical approaches for analyzing industrially and agronomically relevant silage organic acids (lactic, succinic, acetic, and propionic) can be laborious and time consuming, but are critical to assessing feed value and spoilage risk of silage. ARS researchers in Madison, Wisconsin, have demonstrated that these silage organic acids can be predicted mathematically from the way they interact with visible and near-infrared wavelengths of light. This new approach is a low-cost, high-throughput method for rapid characterization of silage water extracts. This accomplishment will primarily benefit the silage research community by reducing cost of analysis and increasing throughput, but potential further development could allow increased on- farm silage quality diagnostics, saving producers money and enabling real-time decision-making.
Impacts
(N/A)
Publications
- Bickhart, D.M., Koch, L.M., Smith, T.P., Riday, H., Sullivan, M.L. 2022. Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species. GigaByte. 42:1- 13. https://doi.org/10.46471/gigabyte.42.
- Zeba, N., Berry, T.D., Panke-Buisse, K., Whitman, T.L. 2022. Effects of physical, chemical, and biological ageing on the mineralization of pine wood biochar by a Streptomyces isolate. PLoS ONE. 17(4). Article e0265663. https://doi.org/10.1371/journal.pone.0265663.
- Hariharan, J., Choudoir, M.J., Diebold, P., Panke-Buisse, K., Buckley, D.H. 2022. Streptomyces apricus sp. nov. isolated from soil. International Journal of Systematic and Evolutionary Microbiology. 72(1). https://doi. org/10.1099/ijsem.0.005178.
- Huang, Q., Hu, T., Xu, Z., Jin, L., McAllister, T.A., Acharya, S., Zeller, W.E., Mueller-Harvey, I., Wang, Y. 2021. Composition and protein precipitation capacity of condensed tannins in purple prairie clover (Dalea purpurea Vent.). Frontiers in Plant Science. 12. Article 715282. https://doi.org/10.3389/fpls.2021.715282.
- Andersen-Civil, A.S., Myhill, L.J., Gokgoz, N.B., Engstrom, M., Mejer, H., Zhu, L., Zeller, W.E., Salminen, J., Krych, L., Lauridsen, C., Nielsen, D. S., Thamsborg, S.M., Williams, A.R. 2022. Dietary proanthocyanidins promote localized antioxidant responses in porcine pulmonary and gastrointestinal tissues during Ascaris suum-induced type 2 inflammation. Journal of Federation of American Societies for Experimental Biology. 36(4) . Article e22256. https://doi.org/10.1096/fj.202101603RR.
- Sullivan, M.L., Knollenberg, B.J. 2021. Red clover HDT, a BAHD hydroxycinnamoyl-coenzyme A:L-3,4-dihydroxyphenylalanine(L-DOPA) hydroxycinnamoyl transferase that synthesizes clovamide and other N- hydroxycinnamoyl-aromatic amino acid amides. Frontiers in Plant Science. 12. Article 727461. https://doi.org/10.3389/fpls.2021.727461.
- Bickhart, D.M., Kolmogorov, M., Tseng, E., Portik, D., Korobeynikov, A., Tolstoganov, I., Uritskiy, G., Liachko, I., Sullivan, S.T., Shin, S.B., Zorea, A., Andreu, V., Panke-Buisse, K., Medema, M., Mizrahi, I., Pevzner, P., Smith, T.P. 2022. Generating lineage-resolved, complete metagenome- assembled genomes from complex microbial communities. Nature Biotechnology. 40:711-719. https://doi.org/10.1038/s41587-021-01130-z.
- Zeller, W.E., Mueller-Harvey, I. 2021. Challenges in Analyzing Bioactive Proanthocyanidins. In: Dreher Reed, J., Pereira de Freitas,V.A., Quideau, S. Recent Advances in Polyphenol Research. 1st edition. Hoboken, NJ: John Wiley & Sons, Inc. https://doi.org/10.1002/978111954595 8.ch6 p.131-175.
- Roldan, M.B., Cousins, G., Muetzela, S., Zeller, W.E., Fraser, K., Salminen, J., Blanc, A., Kaur, R., Richardson, K., Maher, D., Jahufer, Z., Woodfield, D., Caradus, J.R., Voisey, C.R. 2022. Condensed tannins in white clover (Trifolium repens) foliar tissues expressing the transcription factor TaMYB14-1 bind to forage protein and reduce ammonia and methane emissions in vitro. Frontiers in Plant Science. 12. Article 777354. https://doi.org/10.3389/fpls.2021.777354.
|
Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Record of Any Impact of Maximized Teleworking Requirement: The agency⿿s maximized telework posture has had both positive and negative impacts on the research project. On the positive side, maximized telework has allowed scientific staff more time for data analysis and publication and dissemination of findings via manuscripts. Relatedly, there were more opportunities to share our research via conferences and meetings (both scientific and producer-oriented) since most of these have been virtual and required no travel. Finally, some approaches were pursued to work toward project milestones, for example having genes commercially synthesized instead of conventional in-house PCR/cloning approaches, which we have found to be cost- and resource-effective and will be an approach we will likely use in the future during normal operations. Unfortunately, the negative impacts of maximized telework on the research project outweigh the few positives. Many of the objectives/ subobjectives of our project plan are laboratory-oriented and so have been difficult to carry out under occupancy limits specified by the USDA COVID-19 Workplace Safety Plan (25% occupancy). Thus, progress on them has been slowed considerably. While efforts have been made to keep many aspects of projects moving forward, at present it seems likely we will have been in maximized telework posture for a minimum of 20 months before we return to normal operations. This has, of course, led to most milestones being behind schedule. While we feel we will be able to substantially catch up on many milestones upon return to normal operations, there will likely be milestones not met at the end of the project plan. This loss of momentum, especially on laboratory-oriented projects, will likely have consequences long after return to normal operations. There is also some possibility publication output will dip as the backlog of data driving publication output the last two years will soon be consumed. ACCOMPLISHMENTS 01 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. 02 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. 03 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.
Impacts
(N/A)
Publications
- Fanelli, A., Reinhardt, L.A., Matsuoka, S., Ferraz, A., Franca Silva, T.D., Hatfield, R.D., Romanel, E. 2020. Biomass composition of two new energy cane cultivars compared with their ancestral Saccharum spontaneum during internode development. Biomass and Bioenergy. 141. Article 105696. https:// doi.org/10.1016/j.biombioe.2020.105696.
- Sullivan, M.L., Green, H.A., Verdonk, J.C. 2021. Engineering alfalfa to produce 2-O-caffeoyl-L-malate (phaselic acid) for preventing post-harvest protein loss via oxidation by polyphenol oxidase. Frontiers in Plant Science. 11. Article 610399. https://doi.org/10.3389/fpls.2020.610399.
- Reeves, S.G., Somogyi, A., Zeller, W.E., Ramelot, T.A., Wrighton, K.C., Hagerman, A.E. 2020. Proanthocyanidin structural details revealed by ultrahigh resolution FT-ICR MALDI-mass spectrometry, 1H-13C HSQC NMR, and thiolysis-HPLC-DAD. Journal of Agricultural and Food Chemistry. 68(47) :14038-14048. https://doi.org/10.1021/acs.jafc.0c04877.
- Jing, S., Zeller, W.E., Ferreira, D., Zhou, B., Nam, J., Russo-Bedran, A., Chen, S., Pauli, G.F. 2020. Proanthocyanidin Block Arrays (PACBAR) for Comprehensive Capture and Delineation of Proanthocyanidin Structures. Journal of Agricultural and Food Chemistry. 68(47):13541-13549. https:// doi.org/10.1021/acs.jafc.0c05392.
- Santa-Martinez, E., Castro, C.C., Flick, A.J., Sullivan, M.L., Riday, H., Clayton, M.K., Brunet, J. 2021. Bee species visiting Medicago sativa differ in pollen deposition curves with consequences for gene flow. American Journal of Botany. 108(6):1016-1028. https://doi.org/10.1002/ajb2. 1683.
- Fanelli, A., Rancour, D.M., Sullivan, M.L., Karlen, S.D., Ralph, J., Riano- Pachon, D.M., Vicentini, R., De Frank Silva, T., Ferraz, A.L., Hatfield, R. D., Romanel, E. 2021. Overexpression of a sugarcane BAHD acyltransferase alters hydroxycinnamate content in maize cell wall. Frontiers in Plant Science. 12(549). Article 626168. https://doi.org/10.3389/fpls.2021.626168.
- Panke-Buisse, K., Cheng, L., Gan, H., Wickings, K., Petrovic, M., Kao- Kniffin, J. 2020. Root fungal endophytes and microbial extracellular enzyme activities show patterned responses in tall fescues under drought conditions . Agronomy Journal. 10(8). Article 1076. https://doi.org/10. 3390/agronomy10081076.
- Bleier, J.S., Coblentz, W.K., Kalscheur, K., Panke-Buisse, K., Brink, G.E. 2020. Evaluation of warm season annual forages for forage yield and quality in the north-central United States. Translational Animal Science. 4(3). Article txaa145. https://doi.org/10.1093/tas/txaa145.
- Narengaowa, F., Panke-Buisse, K., Wang, S., Cao, Z., Wang, Y., Yao, K., Li, S. 2020. Brisket disease is associated with lower VFA production and altered rumen microbiome in Holstein heifers. Animals. 10(9). Article 1712. https://doi.org/10.3390/ani10091712.
- Higgins, S.A., Panke-Buisse, K., Buckley, D.H. 2020. The biogeography of Streptomyces in New Zealand enabled by high-throughput sequencing of genus- specific rpoB amplicons. Environmental Microbiology Reports. 23(3):1452- 1468. https://doi.org/10.1111/1462-2920.15350.
|
Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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. 03 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.
Impacts
(N/A)
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.
- 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.
- 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.
- Wiesner, S., Duff, A., Desai, A.R., Panke-Buisse, K. 2020. Increasing dairy sustainability with integrated crop-livestock farming. Sustainability. 12(3), 765.
|
Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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.
Impacts
(N/A)
Publications
- Zeller, W.E. 2019. Activity, purification, and analysis of condensed tannins: current state of affairs and future endeavors. Crop Science.
- 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.
- 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.
|
|