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United States Department of Agriculture

Agricultural Research Service

2013 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:
This is the final report for 3655-21000-046-00D terminated January 2013. Major problems for forages utilization are:.
1)poor digestibility of cell walls limits energy availability; and.
2)excessive protein degradation during ensiling and in the rumen, leading to poor nitrogen utilization and excessive excretion. Hydroxycinnamates (ferulates and p-coumarates) are prominent components of grasses and impact both structure and digestibility of cell walls. Ferulates cross-link cell wall carbohydrates and lignin, resulting in decreased digestibility. Current work shows that relatively small changes in ferulate cross-linking can increase digestibility without sacrificing total dry matter production. The p-coumarates are usually more abundant, but do not form significant cross-linked structures. However, they appear to play a role in altering the lignin composition and perhaps the amount of lignin that forms within the cell wall. Altering the gene expression of the enzyme, p-coumaroyl transferase, in corn appears to alter lignin composition. Balancing high digestibility with high biomass in the selection of forages requires rapid methods to measure cell wall components. Fourier transformation infrared and nuclear magnetic resonance (NMR) spectrometry are being used to create databases that link cell wall composition characteristics to fiber digestion. Carbohydrate composition of cell walls is dependent upon supplies of nucleotide sugars as building blocks for polysaccharides. Altering the gene expression that controls sugar nucleotide formation can skew composition without impacting biomass production. Decreasing protein degradation during ensiling of forages has focused on polyphenol oxidases (PPO) and their o-diphenol substrates. A red clover gene, hydroxycinnamate transferase-2 [crucial for biosynthesis of the PPO substrate, phaselic acid (caffeoyl-malate) in red clover], was expressed in alfalfa and resulted in accumulation of hydroxycinnamoyl-malate esters. Manipulation of caffeoyl-CoA O-methyltransferase and hydroxycinnamoyl-CoA transferase activities showed that these enzymes in alfalfa affect the relative accumulation of hydroxycinnamoyl-malate esters. This suggests the possibility of making useful levels of PPO substrates in forage crops such as alfalfa. Additionally, an active PPO/substrate system in perennial peanut was identified that is capable of inhibiting post-harvest protein degradation like in the red clover system. In the case of perennial peanut, one of the endogenous PPO substrates is caftaric acid. PPO grasses co-ensiled with o-diphenol grasses and fed to lambs in two consecutive years indicated improved protein-use efficiencies compared to diets with single-grass silages. A database of NMR spectra of isolated condensed and hydrolyzable tannins is being developed to rapidly assess tannin composition and structure and their relationship to protein protection. 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.

1. Genetic modification in corn lignin has value as a model for further lignin research. In forages, modification of lignin (a cell wall component that gives plants support but restricts fiber digestion by animals) could lead to increased utilization by dairy cows. Even a modest 10% increase in cell wall digestion would lead to an increase in milk and meat production valued at approximately $350 million for U.S. dairies. ARS researchers at Madison, Wisconsin have shown that corn lignin can be modified by a single-enzyme change that is not part of the normal lignin pathway. This modification changes the composition of the lignin without changing the amount of lignin, the total biomass production, or the cell wall carbohydrate composition; only lignin composition is changed by this single gene down-regulation. Such plant materials can serve as models in research because they allow scientists to study how a single genetic modification affects a single trait. This modified corn gene is now being inserted into other plants to determine how it will alter interactions between cell wall composition and function (strength, lodging, insect resistance, total biomass production, and digestibility by ruminants) in these plants.

2. Perennial peanut may protect its protein during ensiling. Ensiling is a popular method of preserving forage through natural fermentation, especially in wet humid regions where drying weather to produce good hay may be limited. A protein preservation system in perennial peanut opens the door for its wider use as a forage crop in the warm, humid southern United States. Ruminant animals such as dairy cows poorly utilize plant protein that is broken down during harvest and storage. It is estimated to cost farmers over $100 million annually to supplement rations with the needed true protein because of protein breakdown in alfalfa, a major forage crop in the U.S. However, alfalfa does not grow well in warm, humid climates. A non-ARS scientist at Texas A & M University who was studying the use of perennial peanut as a forage crop in such climates consulted with ARS scientists in Madison, Wisconsin as to why perennial peanut forage sometimes show improved protein use efficiency when fed to lambs as haylage. The ARS researchers had previously demonstrated that, in red clover, reaction of special phenolic compounds with an endogenous polyphenol oxidase (PPO) enzyme prevents protein degradation when the forage is preserved by ensiling. They also demonstrated that this same mechanism is present in a perennial peanut. This discovery of a perennial peanut’s protein preservation system creates the opportunity for more research into its use as a forage crop in southern climates. Because it is difficult to grow alfalfa in the southern United States, while perennial peanut is adapted to this climate, perennial peanut forage could be an attractive alternative forage legume that sells for the same price as alfalfa, without the transport costs that southern farmers now pay to ship alfalfa from cooler regions.

Review Publications
Verdonk, J.C., Sullivan, M.L. 2013. Artificial micro RNA (amiRNA) induced gene silencing in alfalfa (Medicago sativa). Botany. 91:117-122.

Rancour, D.M., Marita, J.M., Hatfield, R.D. 2012. Cell wall composition throughout development for the model grass Brachypodium distachyon. Frontiers in Plant Science. DOI: 10.3389/fpls.2012.00266.

Vanholme, R., Morreel, K., Darrah, C., Oyarce, P., Grabber, J.H., Ralph, J., Boerjan, W. 2012. Metabolic engineering of novel lignin in biomass crops. New Phytologist. 196:978-1000.

Sullivan, M.L., Foster, J.L. 2013. Perennial peanut (Arachis glabrata Benth.) contains polyphenol oxidase (PPO) and PPO substrates that can reduce post-harvest proteolysis. Journal of the Science of Food and Agriculture. 93(10):2421-2428.

Last Modified: 4/18/2014
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