Location: Wheat, Sorghum and Forage Research
2021 Annual Report
Objectives
Objective 1: Identify and manipulate the genetic and biochemical mechanisms controlling lignin deposition to develop improved sorghum germplasm for bioenergy and forage uses.
Subobjective 1A: Characterize the effects six recently identified brown midrib mutants (bmr) on phenylpropanoid metabolism and lignin deposition.
Subobjective 1B: Evaluate ways to increase lignin deposition and alter phenolic composition of biomass through overexpression of monolignol genes.
Objective 2: Identify and manipulate the genetic and biochemical mechanisms controlling starch and phosphorus composition of grain to develop novel sorghum traits for food, biofuel and livestock production.
Subobjective 2A: Identify and characterize mutants that alter phosphorus composition and reduce phytate in grain.
Subobjective 2B: Develop germplasm with altered starch composition and content in grain.
Objective 3: Identify resistance to fungal pathogens in lignin modified sorghum germplasm for development of stalk rot-resistant grain, bioenergy, and forage sorghums.
Subobjective 3A: Determine the responses of sorghum lines with six recently identified bmr mutants to stalk pathogens.
Subobjective 3B: Assess impact of the stalk pathogen Fusarium thapsinum on sorghum with altered the monolignol synthesis.
Subobjective 3C: Determine the response of sorghum stalk moisture phenotypes on stalk rot pathogens.
Subobjective 3D: Determine whether beneficial microorganisms increase protection of bmr mutants against root and stalk pathogens.
Objective 4: Identify resistance interactions between sorghum grain with novel composition and fungal pathogens for food, fuel, and feed uses.
Subobjective 4A: Determine whether pericarp pigments provide protection against grain pathogens.
Subobjective 4B: Determine whether grain tannins prevent fungal infection.
Approach
Sorghum (Sorghum bicolor) is a climate resilient crop, which is capable of providing grain and forage (biomass) to both the existing agricultural markets and the emerging bioenergy markets in the United States. To compete in these markets, compositional improvements to both sorghum grain and forage are needed and an understanding how these changes affect the fungal pathogens of sorghum. The objectives of this project will focus on the genetic, biochemical, and physiological mechanisms affecting the composition of sorghum biomass and grain. Efforts will result in sorghum with altered lignin content and/or composition of biomass, and increased starch content and reduced phytate content of grain for improved bioenergy conversion, livestock utilization and human nutrition. The impacts of fungal pathogens on sorghum with compositionally modified biomass and grain will be determined. Sorghum germplasm with desirable traits enhancing sorghum biomass and grain utilization will be developed, fully characterized, released and deposited into USDA–ARS National Plant Germplasm System (NPGS) 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. Molecular and conventional methodologies will be utilized, and the project scale will range from gene-level to field-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 value-added traits and biotic stress tolerance, and tools to assess these biological pathways and fungal pathogen responses of sorghum.
Progress Report
Lignin is a component of plant cell walls, and its presence affects the use of sorghum as a livestock forage or a bioenergy feedstock. Objective 1: The gene encoding Brown midrib (bmr) 30 was identified and shown to encode an enzyme in flavonoid synthesis, the reddish-purple pigment of plants. The activity of the enzyme in flavonoid synthesis was confirmed. The effects of bmr30 mutations on cell walls were determined, and bmr30 mutant cell walls had modestly less lignin than normal sorghum cell walls. This finding is the first link between flavonoid and lignin synthesis in sorghum. A second allele of bmr30 was identify in the previous fiscal year (FY). This allele has a lesion mimic phenotype that results in leaf lesions and senescence, which were not observed in the first allele. The second allele is being backcrossed to separate bmr30 mutation from other unlinked mutations in its genome, which likely cause the lesion mimic defect. Several crosses between different bmr mutants have been made, which carry mutations in different bmr genes. Efforts have been focused on bmr19 and bmr30, which effect lignin synthesis indirectly unlike the previously characterized loci bmr2, bmr6 and bmr12. These loci affect the synthesis of lignin precursors directly, so these mutations in combination with bmr19 or bmr30 should further impair lignin synthesis. Future investigations will determine effectiveness and viability of these approaches. The discovery of the genes underlying these bmr mutants through this approach has paved the way for the development of rational strategies to combine bmr mutants to alter lignin content and composition for bioenergy and forage uses in sorghum and other grasses. Understanding the impact of each brown midrib mutant in different varieties will lay the foundation for the development of the next generation of brown midrib hybrids that will benefit the livestock and bioenergy industries. Lines overexpressing different genes in lignin synthesis were combined together and with bmr mutants to further elevate phenolic compounds related to lignin in sorghum biomass. The analyses of biomass showed the overexpression of caffeoyl-CoA o-methyltransferase (CCoAOMT) and 4-coumarate: CoA ligase (4CL) genes combined together increase the energy concentration of biomass significantly over either line alone. Lignin from the overexpression lines was extracted and used to make carbon fiber. The carbon fiber from several of these overexpression lines had greater tensile strength than fiber from normal sorghum lignin. This promising result suggests the lignin structure is improved for application like carbon fiber in these overexpression lines. This approach may lead to new resources for renewable chemical applications, because the altered lignin and elevated phenolic compounds in the biomass may be valuable precursors for green chemistry and other applications.
Phytate is a major phosphorus storage compound in seeds, but it is also an antinutrient for both animals and humans, and its presence in animal waste leads to phosphorus management problems for surface water. Objective 2: Several sorghum mutants with reduced levels of phytate were identified in previous fiscal years. Further testing narrowed the focus to two mutations in separate genes, but the reduced phytate levels in grain were not observed in the subsequent generation for these two mutants. The seeds from the previous generation were screened for low phytate and planted in the greenhouse. Germination rates of seeds were approximately 20%, which suggest low phytate affects seed viability. The seeds produced from plants that germinated are being analyzed for phytate this summer. Investigation will focus on whether plant heterozygous for either mutation also have low phytate levels in grain, which would explain the results observed. Whether this strategy is a viable way to reduce phytate and maintain plant fitness will be determined in FY 22. The ability to develop reduced phytate sorghum would increase the use of sorghum in animal feed and alleviate phosphate management issues.
Starch is a major component of sorghum grain, and it is also the starting material for ethanol production and provides nutritional energy for both humans and livestock. Objective 2: Sorghum lines with increased starch content in the grain were identified and crossed into elite sorghum lines in previous fiscal years. The resulting lines were self-pollinated for six generations. These lines were planted in the field in 2019 and 2020, and starch content is being analyzed this summer. Increasing the starch concentration of grain will open new opportunities for sorghum in livestock feed and ethanol-based biofuels.
Stalk rot pathogens are destructive to sorghum, which impacts both grain and biomass yield. Many of the fungi involved in these diseases can inhabit stalks without causing disease, then various stresses such as drought trigger the development of stalk rot diseases. These pathogens damage stalks, which cause lodging and impede harvest. Plant resistance and biocontrols are the main strategies to protect grain and forage sorghums from these fungal pathogens. The cell wall polymer lignin has been implicated as a defense against pathogens including stalk rots; altering lignin synthesis and composition may improve sorghum for forage and bioenergy uses. Objective 3: Two different bmr mutations in three genetic backgrounds were chosen for further investigation from several bmr mutants previously evaluated. Some lines contain one bmr mutation were more resistant to all three pathogens tested, while resistance in lines containing the other mutation depended on the genetic background and the pathogen. Assays are currently being performed to assess the responses of these near-isogenic bmr lines under drought and well-watered conditions to determine if there are interactions between mutation, plant background, pathogen and water sufficiency. Sorghum plants altered in lignin synthesis by combining bmr mutants with lines overexpressing lignin-related genes were evaluated for resistance to stalk pathogens. These plants were as resistant as the normal sorghum, and some combination tended to show increased resistance. Taken together these results indicate that these alterations to lignin synthesis can result in novel lignin components, while maintaining or possibly increasing resistance to pathogens. The D locus controls whether sorghum stalks are dry or juicy; dry stalks may require less curing time after harvest. It has been presumed that drier stalks have increased resistance to pathogens, although critical experiments to address this hypothesis have not yet been reported. Near-isogenic lines at the D locus were to determine whether dry stalks provide increased resistance to pathogens. No clear differences between dry and juicy lines were found under greenhouse conditions. Stalk pathogen inoculations will be performed under field conditions in the summer of 2021 to further test this hypothesis. Biological control of stalk pathogens would provide an environmentally safe way to protect sorghum and enhance its growth. The genomes of 17 potentially beneficial bacterial isolates from sorghum roots shown to impair fungal pathogen growth and produce antibiotics were sequenced using Next-Gen technologies. Genes associated with biological controls that included root colonization, biofilm formation, iron sequestration, plant hormone production and chitin degradation were identified. Together these projects will determine how stalk traits such as lignin content and composition or juiciness affect susceptibility to stalk rots, and which microbe strains may limit these diseases. Stalk health impacts all types of sorghum grain, forage, sweet and bioenergy, hence controlling stalk rot pathogens is critical for sorghum production.
Fungal pathogens also infect sorghum grain causing grain mold, which may render the grain unusable for food or feed due to its reduced quality and the presence of fungal toxins. The pigments found in the outer layers of sorghum grain may protect against grain mold pathogens. Objective 4: Near isogenic lines varying for grain pigments, red, yellow or unpigmented (white) were grown at two locations Corpus Christi, TX and Mead, NE to determine whether pigments protect grain against high or moderate level of grain molds, respectively. The grain harvested is currently being screened for the presence of grain mold fungi. The grain grown at the Texas location was exposed to high humidity and precipitation during the growing season in 2020, so levels of toxin-producing fungi should be high. Tannins, a lignin-like compound is found in the outer layer of some sorghum grain. Near-isogenic lines differing in the presence or absence of tannins were grown in 2020, and samples were decorticated by ARS scientists in Manhattan, KS. Whole and decorticated grain are currently being screened for fungal contamination to determine how far mold pathogens penetrate into the grain from its surface in presence or absence of tannins. Understanding whether grain pigments or tannins affect grain molds is critical information for the development of food-grade and specialty sorghums where pigments may be undesirable. These components of sorghum grain may also have nutritional benefits for both humans and animals.
Accomplishments
1. Discovered genes that protect sweet sorghum against the stalk disease charcoal rot. Sweet sorghum is a source of sugars that are used to make molasses syrup, bioethanol and other value-added products. However, charcoal rot threatens sweet sorghum production causing lodging and reducing harvestable yield. Researchers at ARS in Lincoln, Nebraska, examined gene expression in a charcoal rot resistant line M81-E and a susceptible line Colman to identify sorghum genes critical for resistance to stalk rot. The analyses showed that genes involved in plant stress responses and plant defenses responded more rapidly and robustly in the charcoal rot resistance sorghum. The responses in M81-E to wounding and charcoal rot disease were stronger than those of Colman. The identification of gene expression patterns will allow researchers to screen for new sources of charcoal rot resistance, and ultimately develop better stalk rot resistant sweet sorghums through plant breeding.
2. Discovered new sources of resistance to sorghum stalk diseases. Sorghum is a drought-tolerant crop with multiple uses for both its grain and plant material. Sorghum is also vulnerable to stalk rot diseases, especially during drought conditions. Stay-green sorghums maintain green leaves longer under drought, but this trait is also associated with increased levels of a cyanide producing compound. Researchers at ARS in Lincoln, Nebraska, analyzed several stay green sorghums for resistance to two stalk diseases, cyanide production and their ability to remain green under drought conditions. Two sorghum lines were identified that had greater disease resistance, low cyanide levels and maintained green leaves. These sorghums would be excellent sources for producing sorghum hybrids resistant to stalk diseases, which also reduce the risk of plant toxicity to livestock.
3. Demonstrated brown midrib 12 plants have altered drought responses. In the U.S., sorghum biomass (stalks and leaves) is an important forage crop for livestock as well as being developed as a bioenergy crop. The brown midrib (bmr) sorghums have reduced levels of lignin, a cell wall component that makes these materials less resistant to breakdown for both livestock and bioenergy conversion. Researchers at ARS in Lincoln, Nebraska, and their collaborators examined the effects of the brown midrib 12 (bmr12) plants, under well-watered and drought conditions. The root response of bmr12 plants were altered, and gene expression analyses showed that bmr12 plants were primed to respond to drought even under well-watered conditions. Together, these findings showed that changing plant cell wall to improve bioenergy conversion may improve plant responses to drought. This research provides a basis to further investigate the roles of cell walls in perceiving the environment, which is critical for developing forage and bioenergy crops in a changing climate.
Review Publications
Saluja, M., Zhu, F., Yu, H., Walla, H., Sattler, S.E. 2021. Loss of COMT activity reduces lateral root formation and alters drought response in sorghum brown midrib (bmr) 12 mutant. New Phytologist. 229:2780-2794. https://doi.org/10.1111/nph.17051.
Zhang, B., Lewis, K.M., Abril, A., Davydov, D.R., Vermerris, W., Sattler, S.E., Kang, C. 2020. Structure and function of the cytochrome P450 monooxygenase cinnamate 4-hydroxylase from sorghum bicolor1. Plant Physiology. 183:957-973. https://doi.org/10.1104/pp.20.00406.
Bolanos-Carriel, C., Baenziger, S.P., Funnell-Harris, D.L., Hallen-Adams, H., Eskridge, K.M., Wegulo, S.N. 2020. Effects of cultivar resistance, fungicide chemical class, and fungicide application timing on Fusarium head blight in winter wheat. European Journal of Plant Pathology. 158:667-679. https://doi.org/10.1007/s10658-020-02109-3.
Bolanos-Carriel, C., Wegulo, S.N., Baenziger, S.P., Eskridge, K.M., Funnell-Harris, D.L., Mcmaster, N., Schmale III, D.G., Hallen-Adams, H.E. 2020. Tri5 gene expression analysis during postharvest storage of wheat grain from field plots treated with a triazole and a strobilurin fungicide. Canadian Journal of Plant Pathology. 42(4):547-559. https://doi.org/10.1080/07060661.2019.1700169.
