Location: Wheat, Sorghum and Forage Research
2022 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. 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
1. 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.
2. 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.
Review Publications
Funnell-Harris, D.L., Sattler, S.E., Oneill, 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.
