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 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 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
Lignin and cell wall cross-linking is a major research focus due to the negative impact upon cell wall utilization and energy conversion efficiency. The artificially lignified cell wall model system continues to be a useful tool to investigate how diverse shifts in lignin composition/cross-linking impact fiber digestion kinetic. Substantial progress has been made toward the objectives set forth in our project. These include improvements in assessing lignin concentration, lignin cross-linking via ferulates, and lignin compositional/structural characteristics 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. Nuclear magnetic resonance (NMR) spectrometry was used to generate specific chemical data for chemometrics approaches in screening forages. Collaborative efforts are in place to develop chemometrics approaches and extend NMR applications to capture additional information concerning cell wall phenolics and carbohydrates. Efforts to decrease leaf loss from alfalfa have been initiated. Promoters for several genes thought to be involved in leaf abscission have been cloned from Medicago truncatula, fused to the beta-glucuronidase reporter gene, and transformed into alfalfa. 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. A transferase HCT2 from red clover has been identified and initially characterized, indicating a role in o-diphenol production as a substrate for PPO. An improved assay for condensed tannins in forages underwent final refinement and evaluation to improve detection and quantification, leading the way to better understanding of tannin roles in protein protection.
1. Identification of a key enzyme in the production of antioxidant phaselic acid (o-diphenol) in red clover: Phaselic acid (caffeoyl-malate ester) accumulates to high levels in red clover and is a key component in a natural system of post-harvest protein protection in red clover, as well as having potential roles in plant UV protection and plant defensive responses. A novel transferase enzyme was identified in red clover whose enzymatic properties are consistent with a role in phaselic acid production. Reduction in levels of this enzyme in red clover resulted in a reduction in phaselic acid and related compounds to nearly undetectable levels in red clover plants. This finding indicated this transferase enzyme is crucial for phaselic acid production in red clover and defines a new pathway for the synthesis of this class of compounds in plants. Identification of this important red clover enzyme is a key first step in producing phaselic acid in other 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 than 100 million dollars annually and would substantially reduce nitrogen emissions from ruminant animal production systems.
2. Rapid screening of plant fiber for energy conversion efficiency using nuclear magnetic resonance (NMR) spectroscopy: Cell walls (the fiber portion of plants) are a complex matrix of carbohydrates, protein, and phenolic materials interwoven to provide strength to support total growth. Lignin (a phenolic polymer) is key to cell wall development and function, but is indigestible and hinders 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. Using a cell wall “solubilization” method developed at the U.S. Dairy Forage Research Center, a processing method was developed to characterize large numbers of forage samples for specific wall components including lignin composition and phenolics involved in cross-linking such as ferulic acid. Major cell wall carbohydrates have also been identified, and this database is being expanded. With specific molecular information about the individual samples, correlations can be established between specific molecular components and functional roles such as digestibility for ruminants or energy conversion efficiency for bioenergy production.
3. Ensiling of forages with polyphenol oxidase activity to decrease protein degradation and improve the nutrient value of the silage: Forage grasses are often harvested and preserved as silage in the more humid regions of agriculture production around the world. Great efforts and dollars are often expended to prevent excessive protein degradation during this process. Red clover is forage with high protein levels and requires little effort to preserve this protein (>85%) during the ensiling process. Red clover protein preservation is due to the presence of polyphenol oxidase (PPO) and appropriate o-diphenols that together inhibit protein degradation by proteases. Temperate forages orchard grass (Dactylis glomerata L.), smooth bromegrass (Bromus inermis Leyss), ryegrass (Lolium perenne L.), and meadow fescue (Festuca pratensis) also contain PPO activity but, unlike red clover, tend to have much higher levels of protein degradation during ensiling. This appears to be due to the lack of appropriate o-diphenol substrates. When chlorogenic acid (an o-diphenol found in many common plants including coffee, dried plums, and the forage tall fescue) is added during the ensiling process, protein degradation is inhibited. Co-ensiling experiments with macerated orchard grass and tall fescue produced silage with reduced protein degradation. This would suggest that adding extracts with chlorogenic acid or co-ensiling two forages that complement each other (one with PPO activity and the other with chlorogenic acid) would produce a superior silage. This would provide an economic advantage to farmers in terms of decreased cost for additional protein supplements and, more importantly, would decrease nitrogen waste to the environment.
4. Use of unique natural molecules to substitute as lignin building blocks and alter cell wall digestibility: Lignin, a component of plant fiber, is the major barrier limiting the digestion of many feeds by ruminant livestock and the conversion of fibrous plant biomass into ethanol. To identify promising new avenues for lignin bioengineering, ARS scientists have artificially lignified cell walls from corn with various combinations of normal lignin building blocks (monolignols), plus a wide variety of molecules from metabolic pathways not normally associated with plant lignification. These new molecules proved to be easily incorporated into lignin as alternative building blocks. In some cases, the monolignol substitutes greatly enhanced the digestibility of cell walls by rumen bacteria, the organisms primarily responsible for fiber digestion in cattle and sheep. Ongoing work involves characterizing the enzymatic breakdown of intact and chemically pretreated cell walls lignified by various monolignol substitutes. These and subsequent studies will identify promising bioengineering targets for improving plant fiber utilization in natural and industrial processes.Lee, M.R., Tweed, J.K., Scollan, N.D., Sullivan, M.L. 2008. Ruminal Micro-Organisms do not Adapt to Increase Utilization of Poly-Phenol Oxidase Protected Red Clover Protein and Glycerol-Based Lipid. Journal of the Science of Food and Agriculture. 88(14):2479-2485.