2013 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.
This five-year project started March of 2013, which is the renewal of the former project number 5440-21220-027-00D, Enhancement of Sorghum for Bioenergy, Feed, and Food Value. For Objective 1, the sorghum genes involved in the monolignol biochemical pathway have been expressed in E. coli and the proteins purified to raise antibodies for protein detection. Lead events (lines) for the overexpression of three genes involved in lignin synthesis and one gene that regulates this process were identified based on lines that showed the highest levels of the targeted gene product. Transgenic lines with over-expression of the sucrose synthase gene have been isolated to determine whether this enzyme is critical to sugar accumulation in sweet sorghum. Procedures were developed to screen sorghum lines for susceptibility to Fusarium stalk rot pathogen. bmr lines that have reduced lignin content were screened for disease susceptibility. In addition, transgenic lines that overexpress a regulatory gene in lignin biosynthesis were screened also for disease susceptibility. These results indicate that modifying lignin content of sorghum did not negatively affect response to the Fusarium stalk rot pathogen. To monitor the growth of the disease Anthracnose in sorghum, twelve C. sublineolum fungi and the molecular tools to express the green fluorescent protein marker in the pathogen were obtained. To identify sweet sorghum lines with resistance to stalk rot pathogens, six sweet sorghum cultivars are presently being screened for response to the diseases Fusarium stalk rot and charcoal rot. The effects of combining bmr2 and bmr6 on lignin content and composition are being investigated by developing the strains with these genes. The crosses have been completed, and DNA markers have been developed to screen the F2 generation. To develop sorghum with novel lignin composition, lead transgenic lines over-expressing two lignin biosynthetic genes, F5H and CCR, have been identified. Sweet sorghum lines containing bmr6 and bmr1 which should reduce biomass lignin concentration are being developed. The F1 generation is growing in the greenhouse for the additional rounds of backcrossing.
Reducing lignin content of bioenergy sorghum does not increase its susceptibility to stalk rot. Stalk rot can significantly decrease biomass yields, because it contributes to lodging. Sorghum brown midrib (bmr) lines have reduced lignin content, which has been shown to improve bioenergy conversion to ethanol. A critical question is whether decreased lignin content in sorghum affects disease susceptibility. A series of bmr mutant lines and the normal counterparts were screened by ARS scientists from Lincoln, NE for susceptibility to the fungal pathogen Fusarium which causes stalk rot disease. None of the lines showed increased susceptibility to the disease, and two bmr mutant lines were more resistant to this disease as compared to their normal counterparts. These results indicate that bmr traits do not increase the susceptibility of sorghum to stalk rot diseases. In addition, the increase resistance observed in some bmr lines is driving research toward the identification of biochemical compounds that inhibit these fungal diseases.
Determination of the molecular structure of a sorghum lignin biosynthesis enzyme. Understanding lignin synthesis is critically important for developing plants with altered biomass composition to be used with emerging bioenergy conversion technologies to produce liquid fuels. The sorghum enzyme hydroxycinnamoyltransferase (SbHCT) is a key enzyme that participates in an early step of lignin synthesis. The structure of this enzyme was determined to understand how the enzyme functions in lignin synthesis. The structure of SbHCT was similar to the structure of other enzymes found in plants. The observations of ARS scientists from Lincoln NE and collaborators explain how SbHCT and other enzymes that share similar structural features can participate in different biochemical pathways in different plant species. Knowledge of this protein structure will enable future research on modifying lignin content and composition of sorghum and other crops for bioenergy.