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United States Department of Agriculture

Agricultural Research Service

2008 Annual Report

1a.Objectives (from AD-416)
1) identify chemical, biochemical, and genetic factors involved in plant development that lead to altered biomass production (quantity and quality) and how changing environmental conditions alter these processes;.
2)determine the impact of genetic modifications in biosynthetic pathways upon fundamental physiological, biochemical, and anatomical development of plants to uncover key structural/functional relationships that affect forage quality, digestion, and biomass conversion efficiency; and.
3)determine the biochemical/ chemical/genetic basis for biological systems needed to inhibit degradation of forage proteins during harvest, storage and utilization to minimize nitrogen waste from dairy production systems.

1b.Approach (from AD-416)
This project is a multidisciplinary approach utilizing plant physiology/ biochemistry, chemistry, agronomy, molecular biology, and genetics. Cell wall screening methods will be developed based on 2D-NMR and FTIR applying chemometric approaches to relate specific structural/compositional information to cell wall utilization (e.g., cell wall digestion, ethanol conversion efficiencies, formation of bioproducts). Basic molecular approaches will be utilized to identify key steps in complex metabolic processes such as cell wall biosynthesis, sugar nucleotide biosynthesis and lignin biosynthesis that altered plant structure and function. Results of these experiments will provide crucial information revealing avenues for improving plant utilization and function. Combinations of agronomic and molecular approaches will be used to define the roles of polyphenols and polyphenol oxidases in the preservation of forage protein during on farm storage and degradation in the rumen. This information will lead to strategies for improved protein utilization. New strategies may include guidelines for management of crops to optimize harvest/storage conditions and development of genetic approaches to produce new plants with improved protein characteristics. Molecular techniques afford a selective approach to test for changes in metabolic pathways (e.g., cell wall biosynthetic pathways) resulting in positive or negative impacts upon digestibility and agronomic characteristics. Altering plant developmental characteristics will have to strike a balance between improved feed characteristics and resistance to environmental stresses that would alter productivity.

3.Progress Report
This new project was developed in calendar year 2007 and officially approved in March 2008. In anticipation of approval of the objectives, work was initiated in January 2008. Although this project has only been in place for seven months, significant progress has been made on initiating work on most research objectives. Lignin remains a major research focus due to the negative impact upon cell wall utilization and energy conversion efficiency. Understanding the dynamics of lignin monomer biosynthetic pathway and the role of lignin acylation continues to be a major thrust of this project. Progress on major efforts to develop and use nuclear magnetic resonance (NMR) spectrometry as a tool for generating specific chemical data for chemometrics approaches in screening forages has slowed due to a vacancy in our organic chemist position. We are developing collaborative efforts to maintain and extend NMR applications to capture additional information concerning cell wall phenolics and carbohydrates. This technique, coupled with rapid analysis methods such as near infrared (NIR) and mid-range infrared (MIR) spectrometry, will aid in the identification of forages with increased digestibility. Work has continued on using a cell wall model system to investigate how diverse shifts in lignin composition impact fiber digestion kinetics of artificially lignified cell walls incubated in vitro with rumen microflora. Such work provides direction to potential modifications in cell wall composition that would be the most fruitful for increased digestibility. Efforts to decrease leaf loss from alfalfa have been initiated, and candidate genes have been identified that may play a role in leaf abscission. Rapid protein degradation in the cow rumen and during ensiling of forages remains a significant problem. Work in this area has focused on tannins and the role of polyphenol oxidases and their o-diphenol substrates. New methods have been developed to more accurately quantify tannin levels in forages like birdsfoot trefoil and allow a better assessment of tannin impact upon protein degradation in the rumen. Work is progressing to identify the genes responsible for o-diphenol biosynthesis in red clover and how these may be utilized to produce alfalfa with a complete polyphenol oxidase/o-diphenol system that will inhibit proteolysis during ensiling. Such a system would increase protein utilization, decrease nitrogen waste, and decrease use of costly protein supplements. The work performed in this project is relevant to ARS National Program Action Plans: 215 Rangeland, Pasture and Forages; Component 3: Sustainable Harvested Forage Systems For Livestock, Bioenergy and Bioproducts. Knowledge gained from meeting these objectives will provide information useful for other National Programs, including NP 301 (Plant Genetic Resources, Genomics, and Genetics Improvement, Component 3: Genetic Improvement of Crops); NP 302 (Plant Biological and Molecular Processes, Component 2: Biological Processes that Improve Crop Productivity and Quality), and NP 307 (Bioenergy and Energy Alternatives, Component 1: Ethanol, and Component 4: Energy Crops).

1. Identification of a novel red clover hydroxycinnamoyl transferase capable of creating hydroxycinnamate malic acid esters. Red clover accumulates large amounts of the o-diphenol phasalic acid, a caffeic acid ester of malic acid that is a powerful antioxidant and part of the PPO/o-diphenol system that protects red clover protein following harvest. Important forages like alfalfa do not accumulate this compound. We have identified a novel red clover enzyme with an activity capable of creating phasalic acid or its immediate precursor, p-coumaroyl malate. This enzyme may be responsible for a key step in phasalic acid biosynthesis. If the phasalic acid biosynthetic pathway could be recreated in alfalfa and other forages, the PPO/o-diphenol protein protection system could be exploited in these, saving farmers $100 million annually and preventing release of excess nitrogen into the environment. This finding also provides important basic information to researchers who are investigating plant secondary metabolism. This accomplishment addresses NP215 Component 3: Sustainable Harvested Forage Systems for Livestock, Bioenergy and Bioproducts; Problem Statement H: Need for improved plant materials that enhance the environment while improving the economic viability harvesting and using grasses and forage legumes for livestock, bioenergy and bioproduct production.

2. Lignin is a major barrier limiting the conversion of fibrous crops into paper and ethanol and digestion of many feeds by livestock. In this study, we artificially lignified cell walls from corn (Zea mays L.) with varying levels of coniferyl ferulate, a potential building block of lignin that is readily broken down by mild chemical treatments. Adding coniferyl ferulate with other lignin precursors slightly reduced the amount of lignin formed in cell walls. Quite unexpectedly, we found that coniferyl ferulate reduced the activity of peroxidase (the enzyme responsible for lignin formation), and it interfered with the coupling of lignin to ferulates. Ferulates are small molecules that help to interconnect polymers in cell walls of grasses. As expected, coniferyl ferulate increased the extraction of cell wall lignin by up to twofold into mild alkaline solutions. Thus, this novel precursor provides the option of producing fiber under conventional cooking conditions with substantially less lignin contamination or of delignifying cell walls at lower temperatures to increase fiber yields. Coniferyl ferulate incorporation also increased sugar yields during enzymatic hydrolysis, both with and without alkaline pretreatment. Our results provide compelling evidence that bioengineering of plants to incorporate coniferyl ferulate into lignin should enhance lignocellulosic biomass saccharification and particularly, pulping for paper production. These results support NP215 Component 3, Sustainable Harvested Forage Systems, Problem Statement H. Need for improved plant materials that enhance the environment while improving the economic viability of harvesting and using grasses and forage legumes.

3. Lignin hinders the enzymatic breakdown of polysaccharides into sugars limiting the value forages for livestock and as renewable feedstocks for the bioconversion to ethanol and other products. In this study, we artificially lignified cell walls from corn (Zea mays L.) with various building blocks of lignin found in normal, mutant, and genetically engineered plants. We also manipulated the cell wall deposition of ferulate, a molecule responsible for attaching (cross-linking) polysaccharides to lignin. When the cell walls were incubated with rumen bacteria, we found that reducing both the amount of lignin formed in cell walls and its cross-linking to polysaccharides by ferulate enhanced the rate and extent of cell wall digestion. In contrast, shifts in lignin composition had no impact on cell wall degradability. Our results indicate that selection or engineering of plants for reduced lignification or ferulate-lignin cross-linking will improve fiber fermentability more than current approaches for shifting lignin composition. These results support NP215 Component 3, Sustainable Harvested Forage Systems, Problem Statement H. Need for improved plant materials that enhance the environment while improving the economic viability of harvesting and using grasses and forage legumes.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of the New MTAs (providing only)2
Number of Non-Peer Reviewed Presentations and Proceedings10
Number of Newspaper Articles and Other Presentations for Non-Science Audiences2

Review Publications
Funk, C., Grabber, J.H., Steinhart, H., Bunzell, M. 2008. Artificial Lignification of Maize Cell Walls Does Not Affect Bile Acid Adsorption. Cereal Chemistry. 85:14-18.

Funk, C., Braune, A., Grabber, J.H., Steinhart, H., Bunzell, M. 2007. Model studies of lignified fiber fermentation by human fecal microbiota and its impact on heterocyclic aromatic amine adsorption. Mutation Research. 624:41–48.

Schmitz, G.E., Sullivan, M.L., Hatfield, R.D. 2008. Three polyphenol oxidases from red clover (Trifolium pratense) differ in enzymatic activities and activation properties. Journal of Agricultural and Food Chemistry. 56(1):272-280.

Sullivan, M.L., Hatfield, R.D., Samac, D.A. 2008. Cloning of an alfalfa polyphenol oxidase gene and evaluation of its potential in preventing postharvest protein degradation. Journal of the Science of Food and Agriculture. 88(8):1406-1414.

Ralph, J., Kim, H., Lu, F., Grabber, J.H., Leple, J., Sierra, J.B., Derikvand, M.M., Jouanin, L., Boerjan, W., Lapierre, C. 2008. Identification of the structure and origin of a thioacidolysis marker compound for ferulic acid incorporation into angiosperm lignins and a pseudo-marker compound for cinnamoyl-CoA reductase. Plant Journal. 53(2):368-379.

Ralph, J., Brunow, G., Harris, P., Dixon, R.A., Schatz, P.F., Boerian, W. 2008. Lignification: Are lignins biosynthesized via simple combinatorial chemistry or via proteinaceous control and template replication? In: Daayf, F., El Hadrami, A., Adam, L., Ballance, G.M., editors. Recent Advances in Polyphenols Research. Oxford, United Kingdom: Wiley-Blackwell Publishing. p. 36-66.

Last Modified: 4/18/2014
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