Progress 02/06/18 to 02/05/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. This five-year project ended in February of 2023, and research will continue under the new project number 3042-21220-033-000D, Genetic Improvement of Sorghum for Biomass, Grain, and Disease Resistance. The following key accomplishments were achieved over the past 5 years. Lignin is a key component of plant cell walls that reduces the efficiency of bioenergy conversion and forage digestibility in livestock. In Objective 1, a transcription factor, SbMyb60 was shown to direct sorghum metabolism toward lignin deposition, which included amino acid phenylalanine and the required cofactors leading toward lignin synthesis. SbMyb60 affects biochemical pathways that also lead to lignin synthesis. The overexpression of ferulate 5-hydroxylase (F5H) and caffeoyl-CoA 3-O- methyltransferase (CCoAOMT) genes changed the cell wall composition without affecting plant yield. F5H overexpression alter the lignin composition of sorghum biomass, while CCoAOMT increased phenolic compounds within cell wall without affecting lignin. This research demonstrated new ways to change the cell wall composition of sorghum biomass, which may lead to the production of renewable chemicals from specific lignin component or related compounds. The brown midrib (bmr) mutants have long been associated with plants impaired in their ability to synthesize lignin. In sorghum, the previously characterized brown midrib loci were all shown to encode enzymes in the monolignol pathway. In Objective 1, sorghum Brown midrib 30 (Bmr30) gene was discovered to encode a flavonoid enzyme required for lignin deposition, chalcone isomerase. This discovery linked plant purple pigments and lignin through a compound called tricin, which was recently been shown to be a component of lignin in grasses like sorghum. The Bmr19 gene was shown to encode an enzyme critical for the synthesis of the cofactor s-adenosylmethionine (SAM), which is required cofactor for monolignol synthesis. The bmr12 plants were discovered to have altered roots as compared to normal sorghum. Further analyses showed that bmr12 plants were primed to respond to drought even under well- watered conditions, which increase drought tolerance. Overall, these studies demonstrated new ways to reduce lignin and improve forage, bioenergy and green chemistry utilization in the climate resilient feedstock sorghum. Sorghum grain is an important food and feed source throughout the world. However, phytate is an antinutritional phosphorus compound found in the grain. In Objective 2, sorghum mutants were identified with reduce levels of phytate. Grain from normal and waxy sorghum, a mutant affecting starch composition, was examined for its effects on the human colon microbiome using in vitro experiments. The normal sorghum starch is more resistant to digestion, which led to increased levels of bacteria beneficial to human health compared to waxy grain. Together these studies identified both viable and non-viable option to improve the nutritional value of sorghum grain for both humans and animals. Stalk pathogens can be particularly devastating to sorghum production, and identifying sources of resistance is challenging because numerous types of fungi are responsible for stalk rot diseases. It is important to assess newly developed germplasm for resistance to stalk pathogens before deployment. A potential control measure to prevent stalk rot pathogens from infecting sorghum is beneficial to soil microorganisms. In Objective 3, potentially beneficial microorganisms were applied to sorghum seeds, and then germination and seedling size were measured in two types of sterilized field soil. The seeds tested included bmr6, bmr12,the double mutant, and their normal counterparts, which were treated with three fluorescent Pseudomonas bacterial strains, two of which showed promising results in previous tests of biological control agents for stalk rot. One strain also increased seedling growth under drought conditions. A set of 16 bacterial strains that associate with sorghum roots and soil, and inhibit pathogen growth, were characterized by sequencing with next- generation technology. We determined that four of these strains had a suite of genes that could indicate superior biological control capabilities such as competitiveness with other microorganisms, plant root colonization, drought survival, and plant growth promotion. The cell wall polymer lignin has been implicated as a defense against pathogens including stalk rots, and altering its content and composition may improve sorghum for forage and bioenergy uses. In Objective 3 we tested six novel bmr mutants decreased in lignin content, incorporated into three elite commonly-used sorghum backgrounds for susceptibility to Fusarium stalk rot and charcoal rot pathogens, and determined that these lines were not more susceptible to the tested pathogens than normal lines. Two sets of mutant lines were also tested for stalk diseases under drought conditions, which did not alter their susceptible to the pathogens. In one of the three backgrounds, the normal line was highly susceptible to charcoal rot under drought conditions, but the two mutant lines were resistant. In Objective 3 we also tested lines with bmr6, bmr12 and bmr2 and their corresponding overexpression lines for responses to the two stalk diseases under adequate water and drought conditions. These were as resistant as the normal lines to the two stalk pathogens. These results demonstrate that altering lignin synthesis does not increase susceptibility to these potentially devastating pathogens of sorghum including under drought conditions, which is important information for development of forage and bioenergy sorghums. The sorghum D locus controls whether sorghum stalks are dry or juicy, which can improve post-harvest drying of forage, but may affect interactions with stalk pathogens, although no such studies have appeared in the scientific literature. For Objective 3, we examined this potential interaction using a series of lines with either functional or non-functional alleles at D and Bmr6 loci for response to Fusarium stalk rot and charcoal rot pathogens. In greenhouse assessments, there were no significant differences in the diseases tested, which indicated that stalk composition controlled at the D locus does not affect sorghum stalk disease responses. In field assessments of Fusarium stalk rot under adequate water, the juicy line was more susceptible than the dry (normal) line, but the juicy bmr6 line was as resistant as the dry bmr6 line. There was no differences between the dry and juicy lines under dryland conditions however. Together these studies indicate that in combination with lignin modified lines, manipulation of stalk moisture content for forage or bioenergy uses may not alter susceptibility to stalk pathogens in some environments, but the bmr6 in combination with the juicy stalks may increase resistant compared their dry counterpart in all environments. Fungal pathogens also infect sorghum grain, which may render it unusable as food or feed due to the presence of fungal toxins. The phenolic pigments of sorghum grain affect its end-uses, but they are also sources of antioxidants and may help defend against grain molds. In Objective 4, sorghum grain was grown in both Texas (high disease pressure) and Nebraska (moderate disease pressure) to assess the role of pigments in grain mold resistance. Fungi have been isolated and identified using morphological characteristics; molecular identification is on-going to identify strains with the potential to be pathogenic and toxigenic. Understanding the role of grain pigments is critical for controlling grain mold, because people consume both unpigmented and pigmented sorghum grain. Tannins (polyphenols) are also deposited in the outer layer of some sorghum grain. Tannins may also reduce mold infection, lines with or without tannins in a tall and a short background were grown at two locations in Nebraska over two years to determine whether tannins affect fungal infection. The results indicated that tannins can protect the grain from pathogenic and toxigenic fungi under dryland conditions, but not under irrigation. Because sorghum is often grown under marginal conditions, this result suggests that tannin plants would be superior in protecting grain against contamination with pathogenic or toxigenic fungi. ACCOMPLISHMENTS 01 Stalk rot-resistant sweet sorghum line responds to wounding with defense compounds and effective defense signaling. Charcoal rot and Fusarium stalk rot threaten sweet sorghum production, which is used to make molasses syrup, ethanol, and other products, but these diseases can reduce yield and diminish syrup quality. These pathogens enter the plant through wounds and cause stalk destruction resulting in flattened stands of plants. The best way to control these diseases is to identify sweet sorghum lines that are resistant to both diseases. ARS researchers in Lincoln, Nebraska, showed that the sweet sorghum variety M81-E was more resistant to both diseases than the sweet sorghum variety Colman. Using state-of-the-art molecular techniques, researchers identified the plant compounds and the genes induced in response to the charcoal rot infection, which showed M81-E activated sorghum defense pathways more rapidly than Colman did. These genes and pathways identified in this study are potential targets for developing new disease-resistant sweet sorghums. Discovering new mechanism to increase stalk rot resistance will increase harvestable yield and benefit both sweet sorghum producers 02 Recently identified Sorghum brown midrib lines were not susceptible to stalk rot pathogens under water deficit. In the U.S., the stalk diseases Fusarium stalk rot and charcoal rot cause significant losses of both sorghum biomass and grain due to flattened stands of plants, and are especially pervasive during flowering under limited water conditions. ARS scientists at Lincoln, Nebraska, developed brown midrib (bmr) plants in three different sorghum varieties from newly characterized alleles, which are impaired in lignin synthesis. Two lines, bmr29 and bmr31 lines were tested using a method developed to simulate drought in the greenhouse. The normal variety was highly susceptible to charcoal rot under drought conditions, but the bmr29-1 and bmr31-1 lines were as resistant to these conditions as the adequately watered lines. All other lines tested were as resistant to the pathogens as normal sorghums. These results showed that bmr29 and bmr31 can be used to develop varieties and hybrids resistant to charcoal rot under drought while improving sorghum for forage and bioenergy uses through reduced lignin. Increased stalk rot resistance combine with reduced lignin will benefit forage sorghum producers through increased harvestable yield with improved forage quality. 03 Identifying changes to the human gut microbiome in response to waxy and normal sorghum grain in mouse and in vitro models. Grain-containing mutations in the Granule Bound Starch Synthase gene also referred to as ⿿waxy⿿, have altered starch composition. This altered starch composition affects the physical properties of the starch and increases its digestibility. ARS and University of Nebraska researchers in Lincoln, Nebraska, examined the effects of normal and waxy sorghum grain on the human colon microbiome using in vitro experiments and mouse models. The normal sorghum starch was more resistant to digestion, which led to increased levels of bacteria beneficial to human health compared to waxy grain. Adding starch from normal sorghum to the waxy grain restored the growth of these beneficial bacteria in the experiments. The conclusion was waxy starch has potentially undesirable effects on the human gut microbiome. Examining the effects of plant traits on the human microbiome opens new avenues to improve sorghum for human health.
Impacts
(N/A)
Publications
- Funnell-Harris, D.L., Sattler, S.E., Toy, J.J., Oneill, P.M., Bernhardson, L.F. 2023. Response of sorghum lines carrying recently identified brown midrib (bmr) mutations to stalk rot pathogens and water deficit. Plant Pathology. https://doi.org/10.1111/ppa.13702.
- Grover, S., Shinde, S., Puri, H., Palmer, N.A., Sarath, G., Sattler, S.E., Louis, J. 2022. Dynamic regulation of phenylpropanoid pathway metabolites in modulating sorghum defense against fall armyworm. Frontiers in Plant Science. 13:1019266. https://doi.org/10.3389/fpls.2022.1019266.
- Grover, S., Puri, H., Xin, Z., Sattler, S.E., Louis, J. 2022. Genetic analysis of seed traits in Sorghum bicolor that affect the human gut microbiome. Molecular Plant-Microbe Interactions. 35(9):755-767. https:// doi.org/10.1094/MPMI-01-22-0005-R.
- Yang, Q., Van Haute, M., Korth, N., Sattler, S.E., Toy, J.J., Schnable, J. C., Benson, A.K. 2022. Genetic analysis of seed traits in Sorghum bicolor that affect the human gut microbiome. Nature Communications. 13:5641. https://doi.org/10.1038/s41467-022-33419-1.
- Cardona, J.B., Grover, S., Busta, L., Sattler, S.E., Louis, J. 2022. Sorghum cuticular waxes influence host plant selection by aphids. Planta. 257:22. https://doi.org/10.1007/s00425-022-04046-3.
- Zhang, B., Vermerris, W., Sattler, S.E., Kang, C. 2023. A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids. Nature Communications. 192(1):102-118. https://doi.org/10.1093/plphys/kiac604.
- Puri, H., Grover, S., Pingault, L., Sattler, S.E., Louis, J. 2023. Temporal transcriptomic profiling elucidates sorghum defense mechanisms against sugarcane aphids. BMC Genomics. 24:441. https://doi.org/10.1186/ s12864-023-09529-5.
- Cardona, J.B., Grover, S., Bowman, M.J., Busta, L., Sarath, G., Sattler, S. E., Louis, J. 2023. Sugars and cuticular waxes impact sugarcane aphid (melanaphis sacchari) colonization on two different developmental stages of sorghum. Plant Science. 330.Article 111646. https://doi.org/10.1016/j. plantsci.2023.111646.
- Zhang, B., Lewis, J., Kovacs, F., Sattler, S.E., Sarath, G., Kang, C. 2023. Activity of cytosolic ascorbate peroxidase (APX) from panicum virgatum against ascorbate and phenylpropanoids. International Journal of Molecular Sciences. 24.Article 1778. https://doi.org/10.3390/ijms24021778.
- Yang, Q., Vah Haute, M., Korth, N., Sattler, S.E., Rose, D., Price, J., Beede, K., Ramer-Tait, A.E., Toy, J.J. 2023. The waxy mutation in sorghum and other cereal grains reshapes the gut microbiome by reducing levels of multiple beneficial species. Gut Microbes. 15(1):2178799. https://doi.org/ 10.1080/19490976.2023.2178799.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. Lignin is a component of plant cell walls, and its presence affects the use of sorghum as a livestock forage or a bioenergy feedstock. The brown midrib (bmr) mutants of grasses are impaired in the deposition of lignin within cell walls. Objective 1: Sorghum line carrying mutations in several pigment related genes are being screened for their impact of pigment accumulation, because brown midrib 30 (bmr30) mutant was shown to be impaired in both lignin and pigment synthesis in previous fiscal years. The gene was identified and shown to encode an enzyme in flavonoid synthesis, a reddish-purple pigment of plants. The second allele of bmr30 has a leaf spotting defect that results in brown lesions and loss of leave, not observed in the first allele. The second allele was backcrossed to wild-type to separate the bmr30 mutation from other mutations in its genome, which likely caused the leaf defect. However, the leaf midrib and leaf browning traits were not separated through this backcross, hence are genetically linked. An alternative strategy to generate additional bmr30 alleles using gene editing is being developed. Efforts have been focused on bmr19 and bmr30, which affect lignin synthesis indirectly, unlike the previously characterized loci bmr2, bmr6, and bmr12 which directly affect the synthesis of lignin precursors. A series of crosses between bmr12 and bmr19 or bmr30 were performed in order to impair two different steps of lignin synthesis simultaneously, which should act additively to impair syringyl lignin (S-lignin) synthesis, one of three main types. These plants are being screened in the field to identify plants carrying both bmr mutations. Future investigations will determine effectiveness and viability of these strategies to combine bmr mutations to alter lignin content and composition for bioenergy and forage uses in sorghum and other grasses. Lines overexpressing ferulate 5-hydroxylase enzyme of lignin synthesis were combined with bmr2 to further elevate S-lignin in sorghum biomass. The biomass from two replicated studies is currently being analyzed to determine the impact of lignin content and composition. 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 that the lignin structure is improved for applications like carbon fiber in these overexpression lines. Additionally, some lines have shown increased resistance to limited water conditions. Lignin is most prevalent within cell walls of plant water conducting elements, which may explain this result. These approaches 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. Likewise, they may also lead to novel ways to increase resistance to a wide range of plant stresses. 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 that reduced phytate levels in grain, but next generation plants did not have reduced phytate levels. The seeds from the previous generation were screened for low phytate and planted in the greenhouse. Germination rates of seeds were approximately 20%, which suggests low phytate affects seed viability. The seeds produced from plants that germinated were analyzed for phytate and the low phytate trait was still segregating in progeny of these plants. Further investigation indicated that plants heterozygous for either mutation may have low phytate, which would explain the results observed. This strategy was determined to not be a viable way to reduce phytate in sorghum. Currently, we are exploring other options to develop reduced phytate sorghum, which 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 samples were scanned by Near Infrared Scanning Spectroscopy, and a calibration set is being identified to determine starch content of the grain. 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 biological control agents 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. The stalk diseases Fusarium stalk rot and charcoal rot result in significant losses of sorghum biomass in the U.S. These diseases are associated with plant lodging and impair biomass and grain harvest and are particularly insidious when water limited, especially around the time of flowering. Objective 3: We had previously developed a method to successfully simulate reduced water conditions in a greenhouse. Using this technique, we screened lines altered in lignin deposition, either through overexpression of genes related to lignin synthesis or brown midrib (bmr) mutations impairing this process for responses to these two fungal diseases under both adequate water and significantly reduced water conditions. Compounds related to synthesis of lignin can have antifungal properties, so their accumulation may slow disease progression. Plants either augmented or impaired in one of three different enzymatic steps related to lignin synthesis did not increase disease compared to normal plant. Some of these experimental lines were actually more resistant to the diseases under drought conditions. To examine the role of syringyl- lignin (S-lignin), one of three main types of lignin, a series of experimental lines, whose levels of S-lignin were altered by mutation and over-expression, were screened for responses to the two stalk diseases. Most of these lines were as resistant as their normal counterparts to the two stalk diseases. Mutants from four more recently identified bmr loci (bmr19, bmr29, bmr30 and bmr31) were screened for responses to three fungal stalk pathogens in three different grain sorghum lines. Results from the screen indicated that the bmr29 or bmr31 mutants were possibly more resistant to the stalk rot diseases, and further analyses are under way to determine their responses to the pathogens under reduced water conditions. The stalk moisture level is conferred by alleles at the D locus (⿿dry versus ⿿juicy culms), which may affect pathogen interactions, although no studies have appeared in the scientific literature. To examine this potential interaction, a series of lines with either functional or non-functional alleles at D and Bmr6 loci were examined for response to three different stalk pathogens. There were no significant differences in disease caused by all three pathogens tested on the lines, which indicated that stalk composition controlled at the D locus does not affect sorghum stalk disease responses. Currently, field assessments of these lines in response to the disease Fusarium stalk rot are being performed. Together these studies indicate that manipulating lignin or moisture content of stalks for forage or bioenergy uses does not increase susceptibility to stalk pathogens. Grain mold disease, which reduces the quality of sorghum grain, is caused by a complex of fungal pathogens from several genera. Objective 4: Previously, compounds responsible for red grain of sorghum have been shown to limit the infection of some pathogens. To examine the role grain pigment plays in resistance to mold disease, fungi were isolated from red, yellow and white grain from plants grown in Nebraska and Texas over two years. These fungi have been identified to genus level, and representative isolates are currently being characterized to species level using both morphological and molecular techniques. This study will determine whether grain color prevents or promotes infection by specific fungal pathogens. Tannins are also deposited in the outer layer of some sorghum grain, and their presence imparts bird resistance due to their bitter taste. Tannins may also reduce grain mold infection. Using lines where tannins are present or absent in the grain, fungi were isolated from whole and decorticated (outer layer removed including the tannins) from grain samples grown at two locations in Nebraska over two years to determine whether tannin affects fungal infection. Fungal inoculations of flowering plants were also performed with three common grain pathogens to assess the impact on grain weight and its appearance. Analysis of these data will indicate whether the presence of tannins in the outer layer of grain will reduced prevalence of grain mold pathogens. Together these studies examine the effects of compounds in the grain exterior layers have on grain mold prevalence. ACCOMPLISHMENTS 01 Altering lignin deposition in sorghum did not impair resistance to two stalk diseases during drought. The lignin biosynthesis pathway has a critical role in plant defense, and the sorghum stalk diseases Fusarium stalk rot and charcoal rot are major impediments to biomass yield and quality, especially during drought. Sorghum lines were developed by ARS scientists in Lincoln, Nebraska, which are either impaired or increased activities of three different enzymes involved in lignin synthesis. Lines impaired in lignin synthesis were not more prone to these diseases under adequate water, but the brown midrib (bmr) 2 and 12 plants were more resistant to the pathogen during drought than normal plants. Lines with increased activity of these three enzymes also did not have increased stalk diseases under drought conditions, but one line overexpressing the Bmr2 gene was more resistant than normal plants under adequate water conditions. Information garnered from this research will be valuable for producing sorghum hybrids for use in bioenergy production or for production of value-added chemicals for pharmaceuticals or cosmetics while still maintaining resistance to stalk diseases. 02 Sorghum Brown midrib 30 (Bmr30) gene encodes a flavonoid enzyme required lignin deposition. Energy, biofuels and renewable chemicals can be produced from plant cell walls, which are composed of three main components, cellulose, hemicellulose and lignin. The brown midrib (bmr) mutants have long been associated with plants impaired in their ability to synthesize lignin. To understand how the bmr30 mutant affects lignin synthesis and cell walls, an ARS scientist from Lincoln, Nebraska, identified the gene through next-generation sequencing, which encoded an enzyme chalcone isomerase. This enzyme is involved in flavonoid synthesis, which is responsible for red and purple pigments of plants. Bmr30 showed the interconnection between plant pigmentation and lignin through a compound called tricin, which has recently been shown to be a component of lignin in grasses like sorghum. Overall, this study demonstrates an important link between two plant biochemical pathways and provides a new way to reduce lignin in sorghum for improved forage, bioenergy and green chemistry utilization.
Impacts
(N/A)
Publications
- Funnell-Harris, D.L., Sattler, S.E., O'Neill, P.M., Toy, J.J., Bernhardson, L.F., Kilts, M., Khasin, M. 2022. Association of dhurrin levels and post- flowering non-senescence with resistance to stalk rot pathogens in Sorghum bicolor. European Journal of Plant Pathology. 163:237-254. https://doi.org/ 10.1007/s10658-022-02473-2.
- Zhang, B., Ralph, J., Davydov, D.R., Vermerris, W., Sattler, S.E., Kang, C. 2022. Characterization of three cytochrome P450 reductases from sorghum bicolor. Journal of Biological Chemistry. 298(4):1-20. https://doi.org/10. 1016/j.jbc.2022.101761.
- Grover, S., Betancurt Cardona, J., Zogli, P., Alvarez, S., Naldrett, M., Sattler, S.E., Louis, J. 2022. Reprogramming of sorghum proteome in response to sugarcane aphid infestation. Plant Science. https://doi.org/10. 1016/j.plantsci.2022.111289.
- Tetreault, H.M., Gries, T.L., Liu, S., Toy, J.J., Xin, Z., Vermerris, W., Ralph, J., Funnell-Harris, D.L., Sattler, S.E. 2021. The sorghum (Sorghum bicolor) Brown midrib 30 (Bmr30) gene encodes a chalcone isomerase required for cell wall lignification. Frontiers in Plant Biology. 12:1-18. https://doi.org/10.3389/fpls.2021.732307.
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Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Record of Any Impact of Maximized Teleworking Requirement: Maximized telework has led to a massive backlog of samples to be processed and analyzed for all four of the project objectives. Our support staff have been given additional pandemic related tasks, which have reduced the number of experiments performed. Communications among project personnel are more cumbersome and time-consuming, because scientists and staff are working remotely. Initiation of many planned projects has been delayed, because the numbers of personnel in laboratories has been limited to 25% capacity due to an ARS directive. Projects requiring two or more people working together to set-up or collect results has been delayed for over 15 months due to social distancing. Collaborating locations and institutions have been extremely delayed in performing necessary experiments and analyses for project objectives. The support staff may be unable to meet all of their goals under the mission-oriented performance element in FY21. Although publication requirements were met for FY21, ARS project and grant progress goals were mostly fully or substantially met this FY, this backlog in analyses and inability to initiate planned research will lead to the possibility of missing these goals in FY22. Scientists have missed opportunities to showcase our research at the national and international levels at conferences and symposia. COVID-19 restriction on foreign visitors in federal facilities by executive order, has significantly impacted the research involving graduate students and other foreign visitors. This restriction has strained relationships with university partners. ACCOMPLISHMENTS 01 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, NE 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. 02 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, NE 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. 03 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, NE 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.
Impacts
(N/A)
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.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Lignin is a component of the plant cell wall and its presence affects the use of sorghum as livestock forage or bioenergy feedstock. Objective 1: Candidate genes for each brown midrib (bmr) loci 29, 30, 31 and 32 were identified in previous fiscal years. The candidate gene for bmr30 was confirmed through the identification of a second mutation in this gene with the brown midrib phenotype. The three alleles of bmr32 were confirmed to be alleles of the previously described bmr19 through DNA sequencing and DNA marker analyses. Experiments are being performed to verify that the mutations identified are responsible for the changes to lignin observed in bmr29 and 31 mutant plants. The identification of the bmr30 and 32 genes validate the use of the brown midrib phenotype to discover new genes that affect lignin synthesis. The newly developed bmr29 through 32 mutant lines in three sorghum varieties were grown in the field for the second year of a two-year trial at two Nebraska locations. Field traits were measured and biomass from these lines was collected for future analyses. The biomass samples from the bmr plants have been processed, and chemical compositional analyses are currently underway to determine the effects of these mutants on lignin synthesis. The discovery of the genes underlying these bmr mutants through this approach will pave 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 from this strategy are currently underway. This approach may lead to new resources for renewable chemical applications, because the elevated phenolic compounds in the biomass may not affect plant growth and may be valuable precursors for green chemistry. 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 mutants, which are in two separate genes. Reduced phytate levels in grain were not observed in the subsequent generation for these two mutants, hence seeds from the previous generation were grown, and the seed produced are being reassessed for the low phytate trait this summer. The ability to develop reduced phytate sorghum would increase the use of sorghum in animal feed. 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 this spring, and starch content will be analyzed in fiscal year 2021. Increasing the starch concentration of grain will open new opportunities for sorghum as a livestock feed and ethanol-based biofuels. Stalk rot pathogens are destructive to sorghum, which impacts both grain and biomass yield. 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 and altering its synthesis and composition may improve sorghum for forage and bioenergy uses. Objective 3: we tested several novel bmr mutants decreased in lignin content for susceptibility to three stalk pathogens and determined that these lines were not more susceptible to the pathogen than normal lines. One line was even more resistant to charcoal rot than its normal counterpart. Likewise, lines developed for altered lignin synthesis through combining bmr lines with transgenic ones were screened for resistance to stalk rot pathogens. These lines were as resistant as normal lines to these pathogens, or had slightly increased resistance to the pathogen as compared to normal lines. Together these data demonstrate that altering lignin synthesis does not increase susceptibility to these potentially devastating pathogens of sorghum, which is important information for forage and bioenergy sorghum efforts. The D locus controls whether sorghum stalks are dry or juicy, which can improve post-harvest drying of forage. The responses of plants with either stalk type are being assessed for responses to stalk pathogens along with sugar concentration and composition. This research will determine whether stalk type affects the susceptibility to stalk pathogens, which is critical information for developing forage sorghum lines. Beneficial microbes associated with sorghum are potentially effective ways to control stalk pathogens without using fungicides. To characterize the microbial genes associated with sorghum colonization, fungal inhibition and growth promotion, the genomes of beneficial bacteria were sequenced with next-generation technology through a USDA Alternatives to Antibiotics Program funded grant. The gene identification will increase our understanding of how beneficial microbes prevent plant disease and promote plant growth. Together these projects will determine how common stalk traits affect susceptibility to stalk rots and which microbe stains may limit these diseases. Fungal pathogens also infect sorghum grain, which may render it unusable as food or feed due to the presence of fungal toxins. The grain pigments of sorghum may protect against grain mold pathogens. Objective 4: we grew sorghum with red, yellow or unpigmented grain in both Texas (high disease pressure) and Nebraska (moderate disease pressure) to assess the role of grain pigments in grain mold resistance. We are currently determining the amount of pathogens in these samples. Sorghum lines with and without tannins, a lignin like compound that may be present in the outer layer of the grain, were grown in Nebraska. This outer layer was removed through decortication in collaboration with ARS scientists in Manhattan, Kansas, and we will be able to determine how far different pathogens can grow into the grain if the tannins are present or absent. 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. Accomplishments 01 Demonstrated a lignin-related enzyme changes lignin composition of sorghum biomass. Sorghum biomass serves as an important forage crop for livestock, and it is being developed as a bioenergy crop. The ferulate 5-hydroxylase (F5H) gene encodes an enzyme involved in the synthesis of the biomass component lignin. To understand the role of this enzyme in lignin synthesis and its effect on cell wall composition, researchers in Lincoln, Nebraska, and their collaborators used biotechnology to greatly elevate expression of the F5H gene in sorghum plants. F5H alone and in combination with brown midrib 12 (bmr12) changed the lignin composition within cell walls, which was observable through the microscope. This work was published in the journal Plant Molecular Biology, which featured it on the cover of the June issue. This research demonstrated new ways to change the lignin composition of sorghum biomass, which may lead to the production of renewable chemicals that require specific lignin composition. 02 Characterization of the changes responsible for increased stalk pathogen- and drought-resistant in brown midrib 12 (bmr12) sorghum. Researchers in Lincoln, Nebraska, examined the responses of two brown midrib lines (bmr6 and bmr12) and the corresponding normal line to two stalk diseases (Fusarium stalk rot and charcoal rot) under drought stress and adequate water conditions. Although bmr12 plants are impaired in lignin synthesis, which is a cell wall component that is thought to play a role in plant defenses against both drought and pathogen attack, surprisingly bmr12 plants had less disease symptoms under drought conditions compared to normal plants or even bmr12 plants under adequate water conditions. Further analyses show that bmr12 had increased defense signals under drought conditions, which suggested these plants were already prepared for a pathogen attack. This research showed that bmr12 can effectively reduce lignin to improve forage and bioenergy sorghum, and it may even increase disease and drought resistance. The identification of genes and pathways affected in bmr12 plants may lead to the development of more climate and disease resilient sorghum hybrids.
Impacts
(N/A)
Publications
- Tetreault, H.M., Gries, T.L., Palmer, N.A., Funnell-Harris, D.L., Sarath, G., Sattler, S.E., Sato, S., Ge, Z. 2020. Overexpression of ferulate 5- hydroxylase increases syringyl units in Sorghum bicolor. Plant Molecular Biology. 103(3):269-285.
- Bolanos-Carriel, C., Wegulo, S.N., Hallen-Adams, H., Baenziger, P.S., Eskridge, K.M., Funnell-Harris, D.L. 2020. Effects of field-applied fungicides, grain moisture, and time on deoxynivalenol during postharvest storage of winter wheat grain. Canadian Journal of Plant Science. 100(3) :304-313.
- Funnell-Harris, D.L., Graybosch, R.A., O'Neill, P.M., Duray, Z.T., Wegulo, S.N. 2019. Amylose-free (⿿waxy) wheat colonization by fusarium spp. and response to fusarium head blight. Plant Disease. 103(5):972-983.
- Funnell-Harris, D.L., Sattler, S.E., O'Neill, P.M., Gries, T.L., Tetreault, H.M., Clemente, T.E. 2019. Response of sorghum enhanced in monolignol biosynthesis to stalk pathogens. Plant Disease. 103(9):2277-2287.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Lignin is a component of the plant cell wall and its presence affects the use of sorghum as livestock forage or bioenergy feedstock. In Subobjective 1A, next-generation sequencing technology was used to determine a chromosome map position for brown midrib (bmr) loci 29, 31 and 32. Candidate genes at each locus were identified for bmr29 and bmr32. The gene identified for bmr30 in the previous FY was shown to no longer be a valid candidate, so an alternative gene was identified within this chromosomal interval. Experiments are being performed to verify that the mutations identified are responsible for the changes to lignin observed in bmr29, 30 and 31 mutant plants. The identification of these bmr genes with this approach will validate the use of this technology for identifying candidate genes of other mutants in sorghum. The newly developed bmr29 through 32 mutant lines in three sorghum varieties were grown in the field for the first year of a two-year trial at two Nebraska locations. Field traits were measured and biomass from these lines was collected for future analyses. The discovery of the genes underlying these bmr mutants through this approach will pave 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. In Sub-objective 1B, lines overexpressing different genes in lignin synthesis were combined to further elevate phenolic compounds in sorghum biomass. The analyses of biomass from this strategy are currently underway. This approach may lead to new resources for renewable chemical applications, because the elevated phenolic compounds in the biomass do not affect plant growth. Phytate is a major phosphorus storage compound in seeds, but it is also an antinutrient for both animals and humans, and it causes phosphorus management problems in their waste. In Subobjective 2A, several sorghum mutants with reduced levels of phytate were identified. Further testing narrowed the focus to two mutants, which are in two separate genes. Reduced phytate levels in grain were not observed in the subsequent generation for these two mutants. Previous generation of seeds were planted to reassess the genetic transmission of this low phytate trait. The ability to develop reduced phytate sorghum would increase the use of sorghum in animal feed. Stalk rot pathogens are destructive to sorghum, particularly under environmental stresses such as drought. Sorghum with impaired lignin synthesis appears to be a source of stalk rot resistance. In Subobjective 3A, the lignin synthesis-impaired lines were shown to have increased resistance under reduced water conditions. Another set of sorghum lines, which had changes to a biosynthesis pathway to increase lignin, either had no increased susceptibility or some lines even had increased stalk rot in Subobjective 3B. There are also beneficial microorganisms in the soil that may prevent stalk rot pathogens from infecting sorghum. In Subobjective 3D, a set of microorganisms were identified that were antagonistic to stalk rot pathogens. Preliminary studies using two strains of these microorganisms increased the growth of sorghum seedlings under adequate water compared to a well-known biocontrol agent or no microorganism treatment; one strain also increased seedling growth under drought conditions. The genomes of these strains will be evaluated through DNA sequencing funded through the USDA Alternatives to Antibiotics Program with the goal of identifying the genes responsible for promoting plant growth and inhibiting fungal diseases. Biological controls are emerging tools to control fungal diseases in sorghum grain, forage and bioenergy production, which reduce use of commercial fungicides and the development of resistance to these compounds. The phenolic pigments of sorghum grain affect its end-uses, but they are also sources of antioxidants and may help defend against grain molds. In Sub-objective 4A, sorghum with red, yellow or unpigmented grain has been planted in both Texas (high disease pressure) and Nebraska (moderate disease pressure) to assess the role of grain pigments in grain mold resistance. Understanding the role of grain pigments is critical for controlling grain mold, because people consume both unpigmented and pigmented sorghum grain. Accomplishments 01 Demonstrated a lignin-related enzyme increases energy content of sorghum biomass. Sorghum biomass serves as an important forage crop for livestock, and it is being developed as a bioenergy crop. The caffeoyl- CoA 3-O-methyltransferase (CCoAOMT) gene encodes an enzyme involved in the synthesis of the biomass component lignin. To understand the role of this enzyme in lignin synthesis and its effect on cell wall composition, ARS scientists in Lincoln, Nebraska, and their collaborators used biotechnology to greatly elevate levels of the CCoAOMT enzyme in sorghum plants. The increased levels of this enzyme led to increased levels of phenolic compounds in the biomass, which increased the energy content without affecting lignin levels or plant growth. Engineering of CCoAOMT in plants provides a new way to change biomass composition and increase the energy content of biomass, which may allow the plant to store more energy for renewable chemical applications. 02 Identification of sorghum resistance factors to sugarcane aphid. The sugarcane aphid has emerged as a significant pest for sorghum in the U. S. and around the world. Sugarcane aphid resistant sorghum lines are one way to combat this pest. Through a series of experiments, ARS scientists in Lincoln, Nebraska, and their collaborators characterized the differences between a sugarcane aphid resistant sorghum line and a susceptible one to determine the factors that make sorghum susceptible or resistant to this highly destructive pest. Gene expression analysis of the entire sorghum genome identified several disease resistance/ defense genes whose expression supports a role in aphid resistance. Analyses of aphid feeding showed that the aphids spent about a quarter of the time actually feeding on resistant plants compared to the susceptible ones, which shows the resistant plants inhibit aphid feeding. A single dominant gene was responsible for aphid resistance in sorghum, which allows resistant plants to grow normally and impede aphid feeding. Together these discoveries are enabling ARS scientists to develop DNA sequence-based methods to identify the sugarcane aphid resistance gene in sorghum, which will enable sorghum breeders to rapidly create sugarcane aphid-resistant hybrids to combat this devastating pest. 03 Identification of stalk rot resistance in sorghum lines overexpressing lignin biosynthesis genes. Sorghum lines were developed with increased levels of enzymes required for lignin synthesis through biotechnology to provide chemical subunits for renewable fuel and chemical uses from biomass. ARS researchers in Lincoln, Nebraska, tested whether increased enzyme levels affected plant resistance to stalk rots, which can impair biomass production. Three lines had increased resistance to Fusarium stalk rot compared to normal plants, and only one line showed increased susceptibility to charcoal rot. The research demonstrates that changes to lignin synthesis may also increase stalk rot resistance in sorghum. Sources of stalk rot resistant sorghums are critical for the successful development of sorghum as a bioenergy crop. 04 Identification of Fusarium head blight (FHB) resistance in ⿿waxy wheat. The grain of waxy wheat lacks amylose starch, which increases shelf- life of baked goods containing waxy flour and makes starch more digestible. Mattern, the first Great Plains waxy variety is vulnerable to FHB, which produces a dangerous toxin that can make the grain unconsumable for both humans and animals. ARS scientists in Lincoln, Nebraska, developed several new waxy wheats, and screened their grain for disease symptoms and the presence of this toxin. Two new lines had noticeably less disease symptoms than Mattern. These newly-developed waxy lines are valuable materials for breeding the next-generation of Northern Great Plains waxy wheats with modified starch for specialty products. FHB resistant wheat varieties are vitally important way to tackle this destructive disease, because severe FHB outbreaks are intermittent.
Impacts
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Publications
- Jun, S., Vermerris, W., Sattler, S.E., Kang, C. 2018. Biochemical and structural analysis of substrate specificity of a phenylalanine ammonia- lyase. Plant Physiology. 174:1452-1468.
- Grover, S., Wojahn, B., Varsani, S., Sattler, S.E., Louis, J. 2019. Resistance to greenbugs in the sorghum nested association mapping population. Arthropod-Plant Interactions. 13(2):261-269.
- Tetreault, H.M., Sajjan, G., Scully, E.D., Gries, T.L., Sattler, S.E., Palmer, N.A., Sarath, G., Louis, J. 2019. Global responses of resistant and susceptible sorghum (sorghum bicolor) to sugarcane aphid (melanaphis sacchari). Frontiers in Plant Science. 10:145.Available:
- Tetreault, H.M., Scully, E.D., Gries, T.L., Palmer, N.A., Sattler, S.E., Funnell-Harris, D.L., Baird, L., Seravalli, J., Sarath, G., Clemente, T.E. 2018. Over-expression of the sorghum bicolor SbCCoAOMT alters cell wall associated hydroxycinnamoyl moieties. Plant Physiology. 13(10): e0204153. Available:
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Lignin is a component of the plant cell wall, and its presence affects the use of sorghum as livestock forage or bioenergy feedstock. In Subobjective 1A, next-generation sequencing technology was used to identify a candidate gene for the brown midrib 30 (bmr30) mutant. Experiments are being performed to verify that the mutation identified is responsible for the changes to lignin observed in bmr30 mutant plants. The identification of this bmr gene using this approach will validate the use of this technology for identifying candidate genes of bmr mutants. This will pave the way to use next-gen sequencing to discover 3 other bmr loci and aid in 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. Phytate is a major phosphorus storage compound in seeds, but it is also an antinutrient for both animals and humans and causes phosphorus management problems in their waste. In Subobjective 2A, several sorghum mutants with reduced levels of phytate were identified. Tests to determine the number of genes affected in these mutants are being performed. The ability to develop reduced phytate sorghum would increase the use of sorghum in animal feed. Stalk rot pathogens are destructive to sorghum, but sorghum with altered ability to produce lignin appears to be a source of resistance to stalk rot. There are also beneficial microorganisms in the soil that may prevent stalk rot pathogens from infecting sorghum. In Subobjective 3D, potentially beneficial microorganisms were applied to sorghum seeds, and then germination and seedling size were measured in two types of sterilized field soil. The seeds tested included brown midrib 6 (bmr6), bmr12, the double mutant and their normal counterparts, which were treated with three fluorescent Pseudomonas spp. bacterial strains, two of which showed promising results in previous test of biological controls for stalk rot. Soil type, seed type and bacterial strain did not affect germination. However, one bacterial strain caused a statistically significant reduction in the size of seedlings for most of the seed types and will therefore be eliminated from future experiments. The next set of experiments will investigate the effects of the stalk rot pathogen on sorghum when grown in the presence of the potentially beneficial microbes. This work is important because biological controls are emerging tools to control pests and pathogens in sorghum grain, forage and bioenergy production. Accomplishments 01 Identification of bmr lines with resistance to Fusarium stalk rot under field and drought conditions. Reducing lignin increases the conversion efficiency of biomass into sugars, but lignin is important for plant defenses against pathogens. Fusarium stalk rot of sorghum reduces grain and biomass yields, and the destructiveness of this disease increases under drought conditions. ARS scientists in Lincoln, Nebraska investigated how fungi that cause Fusarium stalk rot affected sorghum plants with two different mutations, bmr6 and bmr12, that impair lignin synthesis under field and controlled-drought conditions. The bmr mutant and normal plants were infected with Fusarium stalk rot at two field locations, one irrigated and the other dryland in Nebraska. None of the bmr lines had more disease than the normal sorghum lines, following infection at both locations. However, the stacked line, which contained both bmr6 and bmr12 mutations, was more resistant than the normal line under irrigated conditions. These results demonstrated that the bmr lines can withstand Fusarium stalk rot infections better than normal plants under dryland and irrigated conditions. A greenhouse test was also successfully developed to infect plants with Fusarium stalk rot under drought, which will allow ARS scientists to quickly identify sorghum lines resistant to Fusarium stalk rot under conditions where the disease can be particularly devastating. 02 Identification of a transcription factor that directs sorghum metabolism toward lignin synthesis. Lignin is the major structural component of plant cell walls (biomass), whose abundance and composition within plant cell walls affect livestock digestibility and conversion of biomass into biofuels and bioproducts. ARS scientists in Lincoln, Nebraska and Manhattan, Kansas investigated the effects of SbMyb60 on other biochemical pathways in order to better understand its role in controlling lignin synthesis, which had been previously discovered by ARS scientists. It was discovered that SbMyb60 not only affects lignin synthesis, but it also redirects plant metabolism towards lignin production. SbMyb60 increased the synthesis of the amino acid phenylalanine and other cofactors required for lignin synthesis. SbMyb60 affects biochemical pathways that also lead to lignin synthesis. Therefore, SbMyb60 is one of a few factors that can be used to manipulate the amount of lignin in sorghum and other bioenergy grasses to improve this crop for forage and bioenergy uses.
Impacts
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Publications
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