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United States Department of Agriculture

Agricultural Research Service

Research Project: OPPORTUNITIES & LIMITS TO PERTURBING FORAGE PLANT BIOCHEMISTRY, GROWTH, & DEVELOPMENT FOR IMPROVING FORAGE NUTRITIONAL BENEFITS IN DAIRY SYS
2010 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;.
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; and.
4)identify cell wall structural carbohydrate components and carbohydrate interactions that impact nutritional quality, digestion, and biomass energy conversion efficiency, utilizing rapid analytical methods to assess changes related to genetic, environmental, and physiological development in forages.


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 two-dimensional nuclear magnetic resonance (2D-NMR) and Fourier transform infrared spectroscopy (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 alter 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
A major research focus of this research project is to understand what structural features of the cell wall limit utilization and efficient energy conversion. Previous work in grasses clearly showed ferulates as a major cross-linking mechanism among cell wall polysaccharides and between polysaccharides and lignin. This was originally demonstrated using an artificially lignified cell wall model system and later confirmed in analysis of forages. The cell wall model system continues to be a useful tool to investigate how diverse shifts in lignin composition/cross-linking impact fiber digestion kinetic. Continued progress is being made toward the objectives outlined in the project plan. These include improvements in rapid methods for assessing lignin concentration and composition associated with superior fiber degradability. Such work provides direction to potential modifications in cell wall composition that would be the most fruitful for increased digestibility and energy conversion efficiency for bioenergy production. Nuclear magnetic resonance (NMR) spectrometry continues to be used to obtain chemical data for chemometric approaches in screening forages. Initial studies with several grasses successfully demonstrated the feasibility of measuring cell wall components using NMR spectrometry coupled with appropriate chemometric analyses. Collaborative efforts continue to develop chemometric approaches and extend NMR applications to capture additional information concerning cell wall phenolics and carbohydrates. Efforts to decrease leaf loss from alfalfa are well underway, and at least two genes show abscission zone specific expression. Work is underway to alter expression of these two genes in alfalfa transformants. Protein degradation during ensiling of forages remains a significant problem. Work in this area has focused on the role of polyphenoloxidases (PPO) and their o-diphenol substrates. A hydroxycinnamoyltransferase (HCT)-2 gene from red clover with a role in o-diphenol production as a substrate for PPO has been used to produce alfalfa transformants. Initial analysis indicated that small amounts of the o-diphenol are produced in the alfalfa. A feeding trial is underway using ensiled forage grasses containing PPO activity in combination forage grasses that have high levels of o-diphenol substrates. The focus of the feeding trial is to determine the impact of PPO and o-diphenol substrates on preserving protein in the ensiled material and its use by the lambs. Excessive protein degradation in the cow rumen remains a challenge to prevent excess nitrogen (N) excretion from the animal. Birdsfoot trefoil germplasm containing 1.5 to 4% condensed tannin was subjected to in situ rumen incubation, digested by gastric and intestinal proteases, and fractionated by detergents to identify optimal tannin concentrations for limiting rumen proteolysis while permitting gastrointestinal digestion of protein by ruminants. Increased tannin levels resulted in increased protein protection.


4.Accomplishments
1. Expression of a red clover gene in alfalfa results in biosynthesis of phaselic acid and related hydroxycinnamoyl-malate esters. Phaselic acid (caffeoyl-malate ester) accumulates to high levels in red clover and is key component in a natural system of post-harvest protein protection in red clover, as well as having potential roles in plant ultraviolet (UV) protection and plant defensive responses. ARS researchers at Madison, WI previously identified a novel transferase enzyme in red clover whose enzymatic activities are responsible for phaselic acid production. These researchers have transferred the gene encoding this enzyme to alfalfa, which does not normally accumulate phaselic acid. The resulting plants made small amounts of phaselic acid, along with larger amounts of the related compounds, p-coumaroyl- and feruloyl-malate. This finding strongly suggests it should be possible to make useful levels of phaselic acid in forage crops like alfalfa in order to reconstitute red clover’s natural system of protein protection. If the red clover system could be reconstituted in alfalfa, it is estimated that the improved protein/nitrogen utilization would save farmers more then $100 million annually and would substantially reduce nitrogen environmental waste from ruminant animal production systems.

2. Nuclear magnetic resonance (NMR) spectroscopy is a useful tool for rapid screening of plant fiber to determine chemical composition related to energy conversion efficiency. The fiber portion of plants (cell walls) is a complex matrix of carbohydrates, protein, and phenolic materials interwoven to provide structures to support total growth. Lignin (a phenolic polymer) is key to cell wall development and function, but is indigestible and limits the breakdown and utilization of other wall components, especially carbohydrates. NMR spectroscopy is a method of analyzing complex materials to identify specific molecular components within those materials. Researchers at the U.S. Dairy Forage Research Center in Madison, WI have used NMR spectroscopy, coupled with a cell wall “solubilization” method developed at the U.S. Dairy Forage Research Center, to characterize large numbers of forage samples for specific wall components including lignin composition and ferulates involved in cross-linking. Major cell wall carbohydrates have also been identified, and this database is currently being expanded. In conjunction with collaborators in Umea, Sweden, specific molecular information about individual forage samples were correlated with the chemical components measured in the cell walls of these same forages. These findings lay the groundwork for establishing linkages between specific molecular components and functional roles such as digestibility for ruminants or energy conversion efficiency for bioenergy production. The NMR processing method, coupled with chemometric approaches, decreases analysis time from days to hours, making this a useful screening tool for specific cell wall components.

3. Incorporation of unique molecules (polyphenols) into lignin alters its structure and changes cell wall degradation. Plants synthesize phenolic compounds that are used to make structural components of cell walls such as monolignols that are used to form lignin. Plants also make a range of other phenolic compounds, called polyphenols, that have health benefits as antioxidants. Since the polyphenols can undergo oxidation, they can be incorporated into polymers such as lignin. Their distinctive chemical properties could potentially lead to the formation of a lignin with a unique chemical structure, allowing that lignin to be more easily removed from the cell wall. Thus, the remaining cell wall carbohydrates could be more easily digested by enzymes for bioenergy or by rumen microbes for improved cow performance. To test this possibility, ARS scientists at Madison, WI artificially lignified cell walls from corn culture cells with combinations of normal lignin building blocks (monolignols), plus polyphenols from other metabolic pathways not normally associated with plant lignification. The polyphenols proved to be easily incorporated into lignin. In some cases, the modified lignin enhanced the digestibility of cell walls by rumen microbes, the organisms primarily responsible for fiber digestion in cattle and sheep. Future work will expand the types of polyphenols tested for incorporation into lignin and the subsequent impact upon cell wall degradation and utilization. This work will lead to the identification of promising new bioengineering targets to improve plant fiber utilization in natural and industrial processes.


Review Publications
Sullivan, M.L., Zarnowski, R. 2010. Red Clover Coumarate 3'-Hydroxylase (CYP98A44) is Capable of Hydroxylating P-Coumaroyl-Shikimate but not P-Coumaroyl-Malate: Implications for the Biosynthesis of Phaselic Acid. Planta. 231(2):319-328.

Sullivan, M.L. 2009. A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent With a Role in Phaselic Acid Biosynthesis. Plant Physiology. 150:1866-1879.

Lee, M.R., Tweed, J.K., Cookson, A., Sullivan, M.L. 2010. Immunogold Labelling to Localize Polyphenol Oxidase (PPO) During Wilting of Red Clover Leaf Tissue and the Effect of Removing Cellular Matrices on PPO Protection of Glycerol-Based Lipid in the Rumen. Journal of the Science of Food and Agriculture. 90(3):503-510.

Marita, J.M., Hatfield, R.D., Brink, G.E. 2010. In Vitro Proteolytic Inhibition, Polyphenol Oxidase Activity, and Soluble O-Diphenols in Grasses and Cereals. Journal of Agriculture and Food Chemistry. 58:959-966.

Digman, M.F., Shinners, K.J., Casler, M.D., Dien, B.S., Hatfield, R.D., Jung, H.G., Muck, R.E., Weimer, P.J. 2010. Optimizing On-farm Pretreatment of Perennial Grasses for Fuel Ethanol Production. Bioresource Technology. 101:5305-5314.

Last Modified: 10/1/2014
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