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ARS Home » Plains Area » Lincoln, Nebraska » Wheat, Sorghum and Forage Research » Research » Research Project #424223

Research Project: Genetic Improvement of Sorghum for Non-Grain Energy Uses

Location: Wheat, Sorghum and Forage Research

2016 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. In objective 1, development of transgenic sorghum over-expressing ten genes in lignin synthesis was used to determine whether these approaches alter lignin. The ten genes over-expressed in sorghum were not shown to increase lignin within cell walls. However, over-expression of the CCoAOMT and 4CL genes increased the amount of lignin intermediates, which showed the complexity of enhancing the synthesis of this polymer within cell walls. These transgenes will be combined together through traditional plant breeding in order to overexpress pairs in the same plant. Over-expression of a transcription factor, SbMyb60, activated many of the genes involved in lignin synthesis. SbMyb60 represents a tool to modify biomass of sorghum and other grasses. Together this set of transgenic plants will be used to discover ways to alter biomass composition and unravel the complexities of lignin synthesis for bioenergy or other renewable purposes. Sorghum stalk rot fungi are particularly insidious during water-deficit conditions. Stalk rots lead to lodging and biomass losses, because fallen stalks are difficult to harvest. To determine how drought affects the susceptibility of sorghum to stalk rot diseases, a greenhouse assay under water-deficit conditions was developed in objective 2. Both stalk rot susceptible and drought susceptible sorghum lines were shown to be more sensitive to Fusarium stalk rot under water stress. An assay to determine how sorghum responds to charcoal rot under these conditions was developed. These assays are being used to determine how sorghum with altered lignin, brown midrib (bmr) and transgenic plants, respond to stalk rot diseases under water-deficit conditions. Sweet sorghum is also vulnerable to stalk rot diseases. Sweet sorghum lines with resistance to both Fusarium stalk rot and charcoal rot were identified, as well as a line highly susceptible to both of these diseases. Development of these diseases was monitored in susceptible and resistant sorghums. These experiments will provide a framework to determine the genes and the biochemical pathways involved in resistance to both stalk diseases, which will expedite the breeding of stalk rot resistant sweet sorghum lines. A leaf assay is also being developed for the destructive sorghum disease anthracnose, which uses strains of the disease isolated from field locations. This assay will be used to identify genes involved in resistance to existing anthracnose strains. Multiple oils and surfactants are currently being tested, because a chemical in the published laboratory protocol was toxic to several field isolated anthracnose strains. Together these experiments will address the major disease pressures faced by sorghum. To effect further reductions in lignin content than observed in single brown-midrib (bmr) lines, bmr2, bmr6 and bmr12 mutations were combined in the same genetic backgrounds in objective 3. Plants containing the combinations bmr2/bmr12 and bmr2/bmr6 were identified, but double bmr plants (bmr2 bmr6 or bmr2 bmr12) were shorter in stature with irregular leaves. The bmr2 combinations may alter lignin within cell walls to the extent that these changes adversely affect plant growth and development. Although these combinations may be useful in future applications, this information will be used to determine the limits to which scientists can reduce lignin content in sorghum and other grasses. 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. The stalks from these plants are being analyzed to determine how these genes and combinations affect lignin and bioenergy conversion. Sweet sorghum lines containing bmr6 and bmr12 are being developed as future bioenergy feedstocks. Challenges crossing the plants occurred due to different flowering times between bmr and sweet sorghum sources. The bmr sweet sorghum lines will be used to examine how lignin affects sugar accumulation in the stalks, and will also be useful for breeding palatable forage sorghum for cattle.

4. Accomplishments
1. Discovery of a sorghum transcription factor that controls lignin synthesis. Lignin is the major structural component of plant cell walls whose presence and composition influences the usability of plant biomass for the production of biofuels and other natural products. ARS scientists at Lincoln, Nebraska discovered a Myb transcription factor, SbMyb60 that activates synthesis of lignin in sorghum. Experiments showed that overexpression of SbMyb60 activated nine genes in the lignin biosynthesis pathway, and led to increased lignin levels in sorghum biomass. SbMyb60 is the first activator of lignin synthesis to be identified in grasses. SbMyb60 represents a tool to modify plant cell wall composition and the potential to improve biomass for renewable uses in sorghum and other bioenergy grasses.

2. Identification of sweet sorghum lines resistant to stalk rot diseases. Sweet sorghum is being evaluated for bioenergy use because it can be grown in several regions of the U.S., and the juice extracted from the stalks can be used directly to produce ethanol. However, stalk rot diseases pose serious problems on yield and quality of juice and biomass harvested from sweet sorghum. ARS scientists at Lincoln, Nebraska developed a greenhouse test to determine how sweet sorghum varieties respond to two major stalk diseases; charcoal rot and Fusarium stalk rot. ARS scientists discovered that sweet sorghum varieties ‘Rio’ and ‘M81E’ were resistant to Fusarium stalk rot and charcoal rot, while ‘Colman’ was susceptible to both diseases. In addition, the progression of both diseases over time was documented in ‘Colman.’ These experiments provide a framework for identifying genes involved in resistance to stalk diseases and for breeding stalk rot resistant sweet sorghum varieties.

5. Significant Activities that Support Special Target Populations:
This project has developed grain sorghum germplasm with white grain, which serves the special target audience of gluten-free food producers for consumers with celiac disease or other forms of gluten intolerance. This project has also demonstrated that these grain and plant color traits highly valued by food industry do not increase susceptibility to plant pathogens. In addition, the project has also developed grain sorghum germplasm with altered starch composition, which will benefit the special target audience of ethanol and food producers. Small farmers could also benefit as production will require identity preservation. The project is also developing sorghum germplasm for nutritional supplementation.

Review Publications
Scully, E.D., Gries, T.L., Funnell-Harris, D.L., Xin, Z., Kovacs, F.A., Vermerris, W., Sattler, S.E. 2016. Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum. Journal of Integrative Plant Biology. 58:136-149. doi:10.1111/jipb.12375.
Scully, E.D., Gries, T.L., Sarath, G., Palmer, N.A., Sattler, S.E., Baird, L., Serapiglia, M., Dien, B.S., Boateng, A.A., Funnell-Harris, D.L., Twigg, P., Clemente, T.E. 2016. Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in sorghum bicolor. Plant Journal. 85:378-395.
Dowd, P.F., Funnell-Harris, D.L., Sattler, S.E. 2016. Field damage of sorghum (Sorghum bicolor) with reduced lignin levels by naturally occurring insect pests and pathogens. Journal of Pest Science. doi: 10.1007/s10340-015-0728-1.
Funnell-Harris, D.L., Oneill, P.M., Sattler, S.E., Yerka, M.K. 2016. Response of sweet sorghum lines to stalk pathogens Fusarium thapsinum and Macrophomina phaseolina. Plant Disease. 100:896-903.
Anderson, W.F., Sarath, G., Edme, S.J., Casler, M.D., Mitchell, R., Tobias, C.M., Hale, A.L., Sattler, S.E., Knoll, J.E. 2016. Dedicated herbaceous biomass feedstock genetics and development. BioEnergy Research. 9:399-411.
Abd-Alla, A.M., Kariithi, H.M., Cousserans, F., Parker, N.J., Ince, I.A., Scully, E.D., Boeren, S., Geib, S.M., Mekonnen, S., Vlak, J.M., Parker, A.G., Vresyen, M.J., Bergoin, M. 2016. Comprehensive annotation of the Glossina pallidipes salivary gland hypertrophy virus from Ethiopian tsetse flies: a proteogenomics approach. Journal of General Virology. 97(4):1010-1031. doi: 10.1099/igv.0.000409.
Rinerson, C.I., Scully, E.D., Palmer, N.A., Donze-Reiner, T., Rabara, R.C., Tripathi, P., Shen, Q.J., Sattler, S.E., Rohila, J.S., Sarath, G., Rushton, P.J. 2015. The WRKY transcription factor family and senescence in switchgrass. Biomed Central (BMC) Genomics. 16:912. doi: 10.1186/s12864-015-2057-4.