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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
2012 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 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:
Two major problems exist in forages utilization:.
1)inefficient degradation of cell walls limits energy availability; and.
2)excessive protein breakdown during ensiling and in the rumen leads to poor protein utilization and excessive nitrogen excretion. Ferulates are a major cross-link among cell wall carbohydrates, between carbohydrates and lignin in grasses, and a major factor limiting wall degradation. Selection of forages with high digestibility and high biomass requires rapid methods to measure cross-linking components. Fourier transformation infrared (FTIR) and nuclear magnetic resonance (NMR) spectrometry are being used to develop databases linking key cell wall composition characteristics to fiber degradability. Preventing leaf loss from alfalfa during growth would increase total biomass production. Leaf abscission genes have been identified, and plants have been generated with a down-regulated regulatory factor and others with a down-regulated cellulase active in leaf abscission. Decreasing protein degradation during ensiling of forages has focused on the role of polyphenol oxidases (PPO) and their o-diphenol substrates. We previously identified a red clover gene, hydroxycinnamate transferase-2 (HCT2), crucial for biosynthesis of the PPO substrate, phaselic acid (caffeoyl-malate), in red clover. Expression of HCT2 in alfalfa results in accumulation of hydroxycinnamoyl-malate esters including low levels of phaselic acid. We manipulated additional up- and downstream activities of caffeoyl-CoA o-methyltransferase and hydroxycinnamoyl-CoA transferase and showed that alterations in these enzyme levels in alfalfa affect the relative accumulation of different hydroxycinnamoyl-malate esters. This suggests it should be possible to make useful levels of PPO substrates in forage crops such as alfalfa through additional pathway manipulations. PPO grasses were co-ensiled with o-diphenol grasses and fed to lambs in two consecutive years. Overall results indicated lambs fed PPO grasses that were co-ensiled with an o-diphenol-containing grass improved protein-use efficiencies compared to diets with single-grass silages. Thus, PPO-o-diphenol systems can lead to increased nitrogen use efficiencies in ruminant animals. Various isolated tannins applied to mechanically macerated alfalfa were used to assess how tannin composition and structure influence the degradability of protein. A database of NMR spectra of isolated condensed and hydrolyzable tannins is being developed to relate tannin composition and structure to protein protection.


4.Accomplishments
1. Polyphenol oxidase improves protein utilization by ruminants. During ensiling, excessive protein degradation results in inefficient protein use and excessive nitrogen excretion when the silage is fed to ruminants. Polyphenol oxidase ([PPO] what causes browning in fruits like apples) can decrease protein degradation in plant extracts when combined with the right phenolic. ARS scientists in Madison, Wisconsin co-ensiled grasses, pairing a PPO grass with a high-phenolic grass (e.g., orchardgrass with tall fescue). Feeding trials with lambs indicated that total protein utilization by the lambs fed co-ensiled PPO grass with a phenolic grass improved 10-20% over control silages. Less nitrogen was excreted in the urine, indicating an increased utilization of protein. On average, at least $100 million is spent annually to supplement protein losses during ensiling. Incorporating a PPO/phenolic system during ensiling would create a $10-20 million economic advantage to dairy farmers each year due to a decreased need for protein supplements, and would also decrease nitrogen waste excretion into the environment.

2. New synthesis of natural compounds aids in the study of protein protection in forage plants. Red clover accumulates the phenolic, phaselic acid, in high levels. It is a key substrate for the enzyme, polyphenol oxidase (PPO), which is a natural system for post-harvest protein protection. ARS scientists in Madison, Wisconsin developed a streamlined laboratory synthesis for phaselic acid and related phenolics. These compounds are being used to investigate the biological pathway for phaselic acid production in alfalfa. Readily available phenolics related to PPO systems allow rapid advancement of knowledge that will lead to successful incorporation of a PPO/phenolic protein protection system in alfalfa. At present, this information is most useful to other scientists working on new avenues for silage treatment which would lead to a reduction in the cost of additional protein supplements and substantially less nitrogen waste from ruminant animal systems that would end up in the environment.


Review Publications
Wang, Z., Li, R., Xu, J., Marita, J.M., Hatfield, R.D., Qu, R., Cheng, J.J. 2012. Sodium hydroxide pretreatment of genetically modified switchgrass for improved enzymatic release of sugars. Bioresource Technology. 110:364-370.

Lee, M.R., Tweed, J.K., Sullivan, M.L. 2013. Oxidation of ortho-diphenols in red clover with and without polyphenol oxidase (PPO) activity and their role in PPO activation and inactivation. Grass and Forage Science. 68(1):83-92.

Sullivan, M.L., Zeller, W.E. 2012. Efficacy of various naturally occurring caffeic acid derivatives in preventing post-harvest protein losses in forages. Journal of the Science of Food and Agriculture. 93:219-226.

Last Modified: 9/23/2014
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