Location: Wheat, Sorghum and Forage Research2017 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 two genes in lignin synthesis, an enzyme, CCoAOMT or a transcription factor, SbMyb60 changed the chemical composition of plant cell walls. However, the amounts of lignin polymers were only increased in transgenic SbMyb60 sorghum, but the amounts of lignin intermediates were increased when either gene was over-expressed. To unravel the complexity of enhancing the synthesis of lignin within cell walls and to understand the limits to lignin synthesis, plant gene expression was globally analyzed in transgenic sorghum that overexpressed SbMyb60 or CCoAOMT. These experiments showed that SbMyb60 overexpression altered the expression of about a quarter of the genes in sorghum. In contrast, CCoAOMT overexpression altered the expression of only a handful of genes. CCoAOMT overexpression did not reduce the plant size, unlike SbMyb60 overexpression. The differences in gene expression between CCoAOMT and SbMyb60 overexpression are being analyzed to determine the minimal set of genes needed to increase lignin. Together these 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 pathogens are numerous, which makes identifying sources of resistance challenging. Previously, bmr lines, which have reduced lignin content, were shown to have increased resistance to one Fusarium stalk rot pathogen compared to normal sorghum plants. Two additional Fusarium species and a charcoal rot species were also screened on these lines, and bmr lines showed no increased susceptibility to these pathogens as compared with normal sorghum in objective 2. Some bmr lines had increased resistance to one of the Fusarium stalk pathogens and the charcoal rot pathogen as compared with their normal counterparts. To identify the potential causes of this resistance, the Fusarium stalk rot pathogen was grown on artificial media containing soluble stalk extracts of bmr and normal plants. The fungus grew better on the media containing bmr extracts than normal extracts, which suggests that the factor responsible for increased resistance was not present in these stalk extracts. Stalk rot assays with a Fusarium stalk rot pathogen and the charcoal rot pathogen were completed with lines that over-expressed genes in lignin synthesis in order to increase energy content of the biomass. Of the over-expression lines tested, only one of the two PAL over-expression lines was more susceptible to the pathogens than normal sorghum. Lines over-expressing CCoAOMT generally appeared more resistant to fungal pathogens compare to their normal counterparts. In lines over-expressing a Myb transcription factor, the lines with highest expression of this Myb also had significantly increased resistance. Because stalk rots, especially charcoal rots, are more destructive under conditions of drought, a greenhouse assay was developed to test the charcoal rot pathogen under simulated drought conditions. To assess the vulnerability of bmr lines in the field to populations of Fusarium pathogens, Fusarium species from grain and leaves of bmr6, bmr12 and wild-type lines were analyzed. Analyses of fungal spores trapped from air throughout the growing season at two locations showed differences in Fusarium spp. between the locations early in the growing season, but there were essentially no differences in airborne populations at later stages when grain was vulnerable to infection. Airborne populations included not only sorghum pathogens but also corn and wheat pathogens. An improved greenhouse method for assessing lines for responses of sorghum to the fungal disease anthracnose was identified and the inoculation protocol was redesigned. Together these experimental tools will improve our ability to identify pathogen resistance in sorghum, and address the potential of altering the lignin pathway as a new way to increase plant defense against fungal pathogens. To develop sorghum plants with novel lignin composition for bioenergy conversion evaluation, transgenic lines over-expressing ferulate 5-hydroxylase (F5H) a lignin biosynthetic gene, were crossed in objective 3 with bmr12, which is impaired in the synthesis of one type of lignin. The stalks contained a novel lignin, which is not normally found. The presence of this lignin does not affect plant growth or development. The plant material will be evaluated for its impact on bioenergy conversion. To develop plants containing new ways to impair lignin synthesis, six bmr mutations from four recently identified bmr loci were introduced in three sorghum varieties to be evaluated for their impact on lignin and plant performance. Together these mutants are new tools to impair lignin synthesis and improve sorghum biomass for bioenergy conversion. The bmr6 and bmr12 mutations were used to develop reduced lignin sudan grasses to be evaluated as improved forage for cattle.
1. Characterization of the sorghum Cinnamoyl-CoA reductase (CCR), a key enzyme in lignin synthesis. Sorghum biomass (stalks and leaves) serves as an important forage crop for livestock. In addition, sorghum is being developed as a bioenergy crop for advanced or second generation biofuels production. Advanced biofuels are derived from the breakdown of the cellulose and hemicellulose components of biomass into sugars, and their subsequent conversion into biofuel molecules. A third biomass component, lignin, impedes breakdown of biomass in either livestock digestive systems or bioenergy conversion processes. Cinnamoyl-CoA reductase (CCR) gene encodes an enzyme involved in the synthesis of lignin. ARS scientists in Lincoln, Nebraska together with scientists from Washington State University examined how this enzyme makes precursors to lignin. In sorghum, two classes of CCR enzymes were discovered, which have different roles in lignin synthesis. The second class of CCR enzymes is involved in making a specific type of lignin associated with plant defenses against pathogens. Collectively, this research gives a new perspective on the dual functions of this enzyme in lignin synthesis, and may lead to ways to protect plants from pathogens or insects. In addition, this research provides new ways to alter biomass composition of sorghum and other grasses for improved bioenergy conversion.
2. Identification of bmr lines with resistance to stalk diseases. Reducing lignin increases conversion efficiency of biomass into sugars, but lignin is important for plant defenses against pathogens. ARS scientists in Lincoln, Nebraska investigated how fungi that cause the stalk diseases Fusarium stalk rot and charcoal rot affected sorghum plants with two different mutations, bmr6 and bmr12, that impair lignin synthesis in cell walls. Plants with either bmr mutation or both mutations had similar responses to the pathogens as normal plants. Some of the bmr plants were more resistant than the normal plants to the stalk rot pathogen Fusarium proliferatum and to the charcoal rot pathogen Macrophomina phaseolina. Surprisingly, another stalk rot pathogen, Fusarium thapsinum grew faster on artificial media containing the soluble extracts from bmr6 stalks than when grown on extracts from normal stalks. Therefore, other factors contribute to increased resistance in bmr6 plants than compounds that can be extracted from stalks.
3. Several fungi can cause sorghum grain mold, which reduces grain quality in the field and during storage. Fusarium fungi were isolated from air and from brown midrib (bmr; plants with reduced lignin) and normal sorghum from two fields in Nebraska, one under irrigation and the other rainfed. ARS scientists at Lincoln, Nebraska analyzed DNA sequences of these fungi and identified a common sorghum pathogen on the plants, but this fungus was only found at low levels in air. In contrast, numerous Fusarium pathogens of wheat and corn were identified from air samples, but only a few of these fungi were found at low levels on sorghum plants. Analysis of air samples during the growing season showed that Fusarium populations early in the growing season were different from the populations present at grain development and harvest. Fusarium populations found in grain from normal and bmr plants at the irrigated field were different. However, Fusarium populations found in bmr and normal grain grown at the rainfed field were very similar. These results showed that bmr affected Fusarium populations in plants, but environment also strongly influenced Fusarium populations.
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