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
Our basic research program focuses on two major problems in dairy production: 1) poor degradation of cell walls (fiber) limits available energy to animals; and 2) excessive protein breakdown during ensiling and in the rumen leads to poor protein utilization by dairy cows. The cross-linked nature of cell walls decreases efficient breakdown of cell wall carbohydrate to energy, whether by dairy cows or by an enzyme-based process for bioenergy production. Ferulates are a major cross-link among cell wall carbohydrates, and between carbohydrates and lignin in grasses. Current work shows that relatively small changes in ferulate cross-linking can increase digestibility without sacrificing total dry matter. Formation of artificial lignins with normal lignin building blocks (i.e., monolignols) with phenolics that are normally used for the synthesis of tannins and pigments in plants enhances fiber degradability without adversely affecting formation of lignin in cell walls. Increased pressure to develop forages with high digestibility and high biomass prompts a need for rapid methods to measure cross-linking components. Databases based on Fourier transformation infrared (FTIR) and two-dimensional nuclear magnetic resonance (NMR) spectrometry are being developed to provide information on cell wall composition linked with superior fiber degradability. New methods are being developed to mine these databases for specific chemical information (chemometrics) to identify potential changes in cell wall composition that would increase digestibility. Efforts to decrease leaf loss from alfalfa are progressing, and genes related to leaf abscission have been identified. Transformed plants down-regulated for a regulatory factor and a cellulase involved in leaf abscission have been generated, and assays for their effects on abscission are being developed. Protein degradation during ensiling of forages is still a problem. Work in this area has focused on the role of polyphenoloxidases (PPO) and their o-diphenol substrates. PPO grasses were co-ensiled with o-diphenol grasses and fed to lambs; results indicated improved protein-use efficiencies compared to single-grass silages. Hydroxycinnamoyltransferase (HCT)-2 gene from red clover, when expressed in alfalfa, resulted in accumulation of hydroxycinnamoyl-malate esters not normally present in alfalfa, including low levels of phaselic acid (an o-diphenol substrate for PPO). These experiments identified other potentially important enzymes in the biosynthetic pathway, suggesting it should be possible to make useful levels of PPO substrates in forages such as alfalfa. Initial analysis indicated that small amounts of the o-diphenol are produced in alfalfa. Various tannins isolated in 2011 will be applied to mechanically macerated alfalfa in 2012 to assess how tannin composition and structure influence the pregastric and gastrointestinal degradability of protein.
1. Alfalfa can produce a new phenolic that is an important component of a natural system to decrease protein breakdown during ensiling. This phenolic, 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. Red clover protein breakdown is decreased during storage, providing animals with intact protein that is more efficiently used by the dairy cow. This phenolic may also be important for plant ultraviolet (UV) and ozone protection and plant defensive responses. ARS researchers in Madison, Wisconsin identified a novel enzyme in red clover responsible for phaselic acid production. These scientists transferred the gene that encodes this enzyme to alfalfa, which does not normally produce phaselic acid. The modified alfalfa made phaselic acid, but in limited amounts. Because the enzyme for phaselic acid production uses some of the same components that are in the lignin pathway, down-regulation of key enzymes in the pathway would result in increased production of the caffeoyl portion of phaselic acid. The resulting plants had substantially higher levels of phaselic acid compared to plants expressing only the red clover transferase. These results suggested that, with added optimization, useful levels of phaselic acid can be made in forage crops such as alfalfa. If the red clover system of protein protection can be reconstituted in alfalfa, it is estimated that the improved protein/nitrogen utilization would save farmers more then $100 million annually through fewer purchases of supplemental protein. In addition, substantially less nitrogen waste from ruminant animal systems would end up in the environment. Plants with higher levels of phaselic acid may also be more resistant to abiotic (e.g., UV, ozone) and biotic stresses (insects and plant pathogens).
2. Unique lignins result in increased cell wall digestion. ARS scientists in Madison, Wisconsin found that common phenolic compounds that are typically identified as anti-oxidants (providing health benefits to humans) can be incorporated into lignin. When lignins form with these anti-oxidant phenols, along with the normal lignin building blocks, they produce lignified cell walls with increased digestibility. Targeting these compounds to the cell wall may provide new avenues for producing lignins that are less inhibitory toward fiber digestion. To identify suitable lignin building blocks, we artificially lignified maize cell walls with normal lignin building blocks plus various phenolic compounds. These mixtures formed wall-bound lignins at levels similar to normal lignins. However, their incorporation increased 48-h in vitro ruminal fiber digestibility by 20 to 33%, relative to normally lignified controls. The results suggested that several of these anti-oxidant phenols are promising lignin bioengineering targets for improving the digestibility of cell walls. Improving cell wall digestibility increases the energy conversion efficiency of forages by dairy cows, producing more milk and less waste.
3. Small changes in cell wall cross-linking can increase digestibility without decreasing biomass production. Cell walls of plants are held together by a complex matrix of cross-linked components, much in the same way as concrete walls. In grasses such as corn, cell walls are cross-linked by substances called ferulates that tie carbohydrates together and to lignin. ARS scientists in Madison, Wisconsin and St. Paul, Minnesota evaluated the chemical composition of corn plants produced using a system of natural mutation that affects the genetic makeup of a plant (called a transposon system) to create mutants with low ferulates (seedling ferulate mutants [sfe]). Detailed analysis of cell wall components indicated minor changes in carbohydrates and lignin. For the mutant corn (sfe) compared to the non-mutant line, there was a decrease in ferulates involved in forming cell wall cross-links both to carbohydrates and to lignin. This decrease was evident in both the ferulate monomers and ferulate dimers attached to lignin. Changes in the cross-linked nature of the cell wall were modest (25 to 35%). However, the changes in the cross-linked nature of the corn cell walls resulted in increased digestibility and animal performance without a loss in total biomass production. This work indicates that minor changes in key cell wall components, especially those involved in cross-linking cell wall matrices, can have a significant impact upon digestibility of the plant. Such plants have more efficient energy conversion in the dairy cow rumen, producing more milk and less manure waste.
4. Co-ensiling grasses can produce silage mixtures with improved animal performance. Cool-season grasses such as orchard grass and smooth brome grass contain high amounts of an enzyme, polyphenoloxidase (PPO). This is the same type of enzyme that causes browning in many fruits like apples and bananas. It is also the enzyme responsible for inhibiting protein breakdown in red clover silage. A key part of this natural protein protection system in red clover is the phenolic compound that the PPO enzyme uses as a substrate. Even though orchard grass and brome grass have high levels of the PPO enzyme, they lack phenol substrates. However, grasses like tall fescue and timothy contain high levels of phenols that could be used by PPO, but have low levels of PPO enzyme. To take advantage of the PPO/phenol system for protecting protein during ensiling, ARS scientists in Madison, Wisconsin produced co-ensiled grass combinations, pairing a PPO grass with a phenol grass (e.g., orchard grass with tall fescue). Silage bales were opened after 6-8 weeks and fed to young lambs. Control silages consisted of monocultures of the respective grasses. Feeding trials indicated that total protein utilization by lambs fed combinations of a PPO grass with a phenol grass significantly improved (20-30%) over the individual control silages. Less nitrogen was excreted in the urine, indicating an increased utilization of protein by the lambs. This was the result of decreased protein breakdown in the silages that contained a PPO grass and a phenol grass combination. These data suggest that PPO/phenol systems can help prevent excessive protein degradation during ensiling, leading to improved animal performance. This would be an economical advantage to farmers due to a decreased need for protein supplements and decreased nitrogen waste excreted to the environment.
Sullivan, M.L., Zarnowski, R. 2011. Red clover HCT2, a hydroxycinnamoyl-coenzyme A:malate hydroxycinnamoyl transferase, plays a crucial role in biosynthesis of phaselic acid and other hydroxycinnamoyl-malate esters in vivo. Plant Physiology. 155:1060-1067.