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.
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.