1a. Objectives (from AD-416):
Obj. 1: Increase profitability, improve animal welfare & reduce manure production by improving the digestibility and energy conversion efficiency of forages in dairy rations by manipulating forage cell-wall biosynthetic pathways to lower indigestible residue formation, lower waste production, & develop more efficient tools for evaluating forage quality. Sub-Obj. 1.1: Use genetic manipulation of biosynthetic pathways (e.g., lignin, lignin modifications, lignin cross-linking, ferulate cross-linking, structural polysaccharides) to identify avenues for altering cell wall digestibility & the formation of indigestible residues. Sub-Obj. 1.2: Develop methods to provide rapid screening of plant materials for chemical characteristics related to improved energy conversion efficiency and/or other nutrient quality factors. Obj. 2: Increase profitability & reduce the amount of nitrogen-containing wastes that enter the environment by reducing protein loss during the post-harvest storage & livestock consumption of alfalfa & other forages through manipulation of forage phenolic metabolic pathways. Sub-Obj. 2.1: Gain knowledge of factors that influence accumulation of hydroxycinnamyl-conjugates (e.g., phaselic acid, clovamide, chlorogenic acid) utilizable by PPO systems in forages with respect to efficacy in post-harvest proteolytic inhibition, as well as combating abiotic stress (UV, ozone). Sub-Obj. 2.2: Determine the chemical basis for proteolytic inhibition caused by classes of tannins & polyphenol oxidase-generated o-quinones. Obj. 3: Improve forage biomass production (quantity and quality) for increased nutrient availability & novel bio-products that integrate bioenergy production with alfalfa & other forage crops to reduce input costs while improving environmental conditions. Sub-Obj. 3.1: Determine how alfalfa selected for improved stem nutritive value influences harvest management strategies & ruminant performance. Sub-Obj. 3.2: Use unique harvesting practices coupled with on-farm treatment & storage to create protein-rich fractions that produce value-added products from alfalfa. Sub-Obj. 3.3: Prevent excessive leaf loss during plant development & harvesting by acquiring additional knowledge of hydrolytic and regulatory factors involved in leave abscission in alfalfa, thus leading to gene-based strategies for improvement.
1b. Approach (from AD-416):
We will utilize a multidisciplinary approach combining plant physiology/biochemistry, chemistry, agronomy, molecular biology and genetics. Forages provide unique nutritional and environmental opportunities to improve sustainable farming systems that help ensure food security. To enhance positive characteristics of forages, work will focus on: improving cell wall digestibility under high biomass production; and capturing more plant protein in products, e.g., milk and plant bio-products, while generating less nitrogen waste. Improved utilization of cell walls can be achieved through manipulation of genes involved in biosynthesis of structural carbohydrates and lignin. Small changes in cell wall composition may lead to decreased cross-linking and increased digestibility (Objective 1). Cell wall screening methods based on nuclear magnetic resonance spectroscopy and Fourier transformed infrared spectroscopy will be used to identify chemical characteristics related to improved energy conversion efficiency. Molecular approaches will be used to modify plant biosynthetic pathways (lignification, cell wall cross-linking, structural polysaccharides) to identify avenues for altering cell wall digestibility. Efficient capture of protein nitrogen in the rumen is related to slowing protein degradation and availability of adequate digestible carbohydrate. Molecular, chemical, and biochemical approaches will be used to determine the roles of polyphenol oxidase/o-diphenols and tannins in decreasing protein degradation during ensiling and in the rumen (Objective 2). Molecular approaches will be used to alter plants for reduced protein loss during post-harvest storage and during livestock consumption of forages. A polyphenol oxidase/o-diphenol system will be inserted into alfalfa to protect proteins during ensiling. Chemical characterization of polyphenol (e.g., o-quinones and tannins) interactions with proteins will reveal mechanisms to protect proteins from degradation and provide selection criterion for forage improvement. Multiple approaches will be used to improve forage biomass production for improve animal performance and new bio-products (Objective 3). Molecular approaches will be used to down-regulate leaf abscission genes which would prevent excessive leaf loss, preserving the protein-rich fraction of alfalfa. To improve forage biomass production for increased nutrient availability and novel bio-products, field-grown alfalfa selected for increased stem digestibility will be evaluated to reveal its potential for improved animal performance. Analysis of alfalfa leaves during plant development will determine potential changes in protein and, coupled with new harvesting techniques, will lead to improved quality, as well as new bio-products to increase utilization of alfalfa in farming systems. This project plan will increase our knowledge and understanding of current limitations associated with forage utilization and provides avenues to overcome these limitations.
3. Progress Report:
A challenge that continues to face the dairy industry is the inefficient utilization of forages in dairy diets. Two major concerns are poor utilization of forage protein and incomplete digestion of cell walls (fiber). Molecular biology is being used to determine what can be altered within a plant, resulting in improved performance. Data can be obtained relatively quickly and provide direction for traditional genetic selection for improving forages. A down-regulated corn gene that controls attachment of p-coumarates to lignin is being evaluated to determine the impact upon structural aspects of the plant, as well as impact upon digestion. Cell wall carbohydrates are synthesized by a complex matrix of enzymes within plant cells. Characterization of these enzyme complexes has been difficult, and progress at the gene level has been slow. Altering the enzymes that produce the building blocks (sugar nucleotides) may alter the cell wall composition without adversely affecting total plant development. Both up- and down-regulated sugar nucleotide genes have been produced in the model grass Brachypodium. Characterization of these plants will determine if this approach leads to altered cell wall composition. Protein degradation during ensiling and rapid degradation in the rumen remains a challenge, creating wasteful use of forage protein and additional burdens on the environment. A focus remains on understanding the polyphenol oxidase (PPO)/o-diphenol system of protecting protein. To improve o-diphenol synthesis, a gene-silencing construct to silence major O-methyl transferases to increase caffeoyl molecules has been inserted into alfalfa. This is the first step in attempting to shift production of hydroxycinnamoyl-malate esters toward PPO-utilizable phaselic acid in alfalfa. Expression and silencing constructs for hydroxycinnamate transferase-3 and -4 genes from red clover will be used to evaluate whether they encode a transferase responsible for clovamide production. A silencing construct for inflorescence-deficient in abcission-2, a gene believed to be involved in alfalfa leaf abscission, will be tested. Successful isolation and characterization of condensed tannins from two sources (big trefoil and cocoa powder) was performed on a small scale. These methods will be applied to other condensed tannin-containing forages (sainfoin, birdsfoot trefoil, crownvetch, and white clover flowers). Scale-up procedures to isolate the required quantities of condensed tannins are in progress. New experimental methods, based on two-dimensional nuclear magnetic resonance spectroscopy, were developed and allow for rapid characterization of condensed tannin polymers. Laboratory synthesis of condensed tannins has been initiated to produce chemically pure molecules for characterization of tannins, and for use in model studies to understand tannin-protein interactions and their impact upon degradation of protein.