Location: Wheat, Sorghum and Forage Research2014 Annual Report
1a. Objectives (from AD-416):
The long-term objectives of this project are to develop improved sorghum (Sorghum bicolor) germplasm (Figure 2) for biofuels production by 1) determining genetic, biochemical, and physiological mechanisms controlling traits beneficial to bioenergy conversion technologies (saccharification & fermentation, pyrolysis and combustion), 2) develop and release improved germplasm with these modified traits, and 3) determine the impact of fungal pathogens on sorghum with these modified traits. Over the next five years, the following specific objectives will be addressed: Objective 1: Determine and manipulate the genetic, biochemical and physiological mechanisms controlling the biological pathways involved in non-grain energy sorghum germplasm. Sub-objective 1.A: Determine the effects of six newly identified brown midrib (bmr) mutants on lignin synthesis and the monolignol biosynthetic pathway. Sub-objective 1.B: Develop transgenic lines over-expressing genes in monolignol biosynthesis to determine their impact on lignin content and composition. Sub-objective 1.C: Identify strategies for increasing the sugar content of sweet sorghum juice and improving its biomass composition for thermal conversion. Objective 2: Determine the impact of fungal pathogens on non-grain energy sorghum germplasm, and determine mechanisms of resistance to sorghum pathogens. Sub-objective 2.A: Determine the response of sorghum with modified lignin biosynthesis pathways to stalk pathogens. Sub-objective 2.B: Determine whether modifications in the lignin biosynthetic pathway affect growth of the pathogen Colletotrichum sublineolum within sorghum leaves. Sub-objective 2.C: Identify sweet sorghum parental lines with resistance to stalk pathogens. Objective 3: Develop and evaluate germplasm to improve sorghum for non-grain energy uses. Sub-objective 3.A: Determine the effects of brown midrib (low lignin) mutations alone or in combination on bioenergy conversion via saccharification and fermentation. Sub-objective 3.B: Develop sorghum lines with novel lignin composition and determine the effects on bioenergy conversion. Sub-objective 3.C: Develop sweet sorghum germplasm incorporating bmr6 and bmr12 to reduce lignin and determine the effects of these alleles on end-use quality.
1b. Approach (from AD-416):
The overall objective of this project is to improve sorghum (Sorghum biocolor) as an energy crop with the focus on the biomass or non-grain components of the plant. The project will conduct basic studies on the genetic, biochemical, and physiological mechanisms affecting the composition of sorghum biomass and its conversion to liquid fuels. The focus will be on decreasing or increasing lignin content and/or modifying its composition and on increasing sugar content and yield for juice extraction. Low lignin is desirable for the saccharification and fermentation conversion process while high lignin concentration is desirable for conversion via pyrolysis. The impacts of fungal pathogens on sorghum with compositionally modified biomass will be determined. Germplasm with desirable genes affecting the conversion of sorghum biomass to energy will be developed, fully characterized, and released and deposited in Germplasm Resources Information Network (GRIN) for use by public and private sector plant breeders for developing improved hybrids and cultivars. The project consists of three integrated components: germplasm development, molecular biology, and plant pathology (Figure 1). Molecular and conventional methodologies will be utilized, and the project scale will range from field-level to gene-level. The project also has extensive formal and informal collaborations enhancing our ability to conduct this research. Anticipated products include improved sorghum germplasm for the sorghum seed industry with enhanced energy traits and biotic stress tolerance, and tools to assess the biological pathways that impact bioenergy traits and fungal pathogen responses of sorghum.
3. Progress Report:
Lignin, a key component of plant cell walls, can affect the efficiency of bioenergy conversion. Antibodies against the lignin pathway enzymes were developed to detect these enzymes in plant tissues, and will be used to determine the levels of the lignin pathway enzymes various sorghum lines under development. The brown midrib (bmr) mutants of sorghum reduce lignin. A series of new bmr mutant plants was evaluated for bioenergy uses in a two-year field study. To increase lignin content of sorghum for thermo-bioenergy conversion, transgenic plants over-expressing enzymes involved in lignin synthesis were developed. These plants will be evaluated for their bioenergy potential in following years. A defect in the Bmr2 gene, which encodes an enzyme in lignin synthesis, was not detected in sweet sorghum plants in contrast to a previous report. Therefore, the reduced lignin content observed in sweet sorghum lines was not due to defects in this enzyme. Transgenic lines over-expressing the sucrose synthase gene have been isolated to determine whether this enzyme is critical to sugar accumulation in sweet sorghum. To determine the impact of reduced lignin content on stalk diseases, bmr lines were screened for susceptibility to four different stalk pathogens. In all cases, the bmr lines were as resistant as the normal sorghum lines. Sweet sorghum lines were also screened for their susceptibility to the two main stalk diseases of sorghum. Two sweet sorghum lines were identified as resistant to both stalk rot pathogens, and will be useful for breeding sweet sorghum with resistance to stalk diseases. One sweet sorghum line was highly susceptible to both pathogens and is being used to study disease progression. To determine the effects of increased lignin content on pathogen responses, transgenic lines that over-express enzymes in lignin biosynthesis were screened for disease susceptibility. Results indicated that modifying lignin content of sorghum did not change the response to the stalk rot pathogen. DNA markers for individual pathogen isolates were used to confirm presence of the pathogen in plant tissues. A greenhouse assay is being developed to determine susceptibility of young plants to isolates of the leaf disease anthracnose, and DNA markers are being developed to track the movement of the pathogen in infected leaf tissue. To effect further reductions in lignin content than observed in single bmr lines, bmr2, bmr6 and bmr12 were combined in the same genetic backgrounds. Plants containing the combinations bmr2/bmr12 and bmr2/bmr6 were identified, and effects on lignin content of these combinations will be determined during the next field season. To develop sorghum plants with novel lignin composition for bioenergy conversion evaluation, transgenic lines over-expressing ferulate 5-hydroxylase (F5H) and cinnamoyl-CoA reductase (CCR), two lignin biosynthetic genes, were crossed with two bmr lines. Once these lines are developed, the effects on lignin content and bioenergy conversion will be evaluated. Sweet sorghum lines containing bmr6 and bmr12, which should reduce lignin concentrations, are being developed as a future bioenergy feedstock. Despite some challenges crossing the plants due to different flowering times, the second generation crosses have been made to incorporate bmr6 and bmr12 into sweet sorghum lines. The sweet sorghum lines generated will be evaluated for effects of reduced lignin on bioenergy and agronomic traits.
1. Structure and function of an essential enzyme in lignin synthesis. Understanding lignin synthesis is critically important for developing plants with altered biomass composition to be used with emerging bioenergy conversion technologies. The sorghum caffeic O-methyltransferase (SbCOMT) is a key enzyme that participates in the last steps of lignin synthesis. The structure of this enzyme was determined to understand how the enzyme functions. The structure of SbCOMT was similar to the structure of other O-methyltransferase enzymes found in plants. Our observations explain how SbCOMT and other enzymes that share similar structural features can participate in different biochemical pathways in different plant species. Knowledge of this protein structure will direct future research on modifying lignin content and composition of sorghum for bioenergy.
2. Low-lignin (bmr12) grain sorghum leaves and stalks are valuable cattle feed. Grain sorghum is an important crop for rainfed production systems in the U.S. Plants with the brown midrib (bmr) trait have less lignin in their leaves and stalks. While bmr12 plants yielded approximately 6% less grain than sorghum without this mutation, leaves and stalks of bmr12 plants are more digestible by cattle. The steers grazing on bmr residue for two months had increased body weight relative to their counterparts, which translated to an estimated $53 increase in net return per acre compared to normal sorghum. These results indicate that this bmr12 sorghum hybrid is an effective dual-purpose sorghum crop for both grain production and cattle grazing.