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The Fusarium head blight (FHB) pathogen Fusarium graminearum produces the trichothecene mycotoxin deoxynivalenol and reduces wheat yield and grain quality. Spring wheat ( Triticum aestivum ) genotype CB037 was transformed with constitutive expression (CE) constructs containing sorghum ( Sorghum bicolor ) genes encoding monolignol biosynthetic enzymes caffeoyl coenzyme A (CoA) 3- O -methyltransferase ( SbCCoAOMT ), 4-coumarate-CoA ligase ( Sb4CL ), or coumaroyl shikimate 3-hydroxylase ( SbC3'H ) or monolignol pathway transcriptional activator SbMyb60 . Spring wheats were screened for type I (resistance to initial infection, using spray inoculations) and type II (resistance to spread within the spike, using single-floret inoculations) resistances in the field (spray) and greenhouse (spray and single floret). Following field inoculations, disease index, percentage of Fusarium -damaged kernels (FDK), and deoxynivalenol measurements of CE plants were similar to or greater than those of CB037. For greenhouse inoculations, the area under the disease progress curve (AUDPC) and FDK were determined. Following screens, focus was placed on two each of SbC3'H and SbCCoAOMT CE lines because of trends toward a decreased AUDPC and FDK observed following single-floret inoculations. These four lines were as susceptible as CB037 following spray inoculations. However, single-floret inoculations showed that these CE lines had a significantly reduced AUDPC ( P < 0.01) and FDK ( P ≤ 0.02) compared with CB037, indicating improved type II resistance. None of these CE lines had increased acid detergent lignin compared with CB037, indicating that lignin concentration may not be a major factor in FHB resistance. The SbC3'H and SbCCoAOMT CE lines are valuable for investigating phenylpropanoid-based resistance to FHB., Competing Interests: The author(s) declare no conflict of interest.

Sweet sorghum ( Sorghum bicolor ) lines M81-E and Colman were previously shown to differ in responses to Fusarium thapsinum and Macrophomina phaseolina , stalk rot pathogens that can reduce the yields and quality of biomass and extracted sugars. Inoculated tissues were compared for transcriptomic, phenolic metabolite, and enzymatic activity during disease development 3 and 13 days after inoculation (DAI). At 13 DAI, M81-E had shorter mean lesion lengths than Colman when inoculated with either pathogen. Transcripts encoding monolignol biosynthetic and modification enzymes were associated with transcriptional wound (control) responses of both lines at 3 DAI. Monolignol biosynthetic genes were differentially coexpressed with transcriptional activator SbMyb76 in all Colman inoculations, but only following M. phaseolina inoculation in M81-E, suggesting that SbMyb76 is associated with lignin biosynthesis during pathogen responses. In control inoculations, defense-related genes were expressed at higher levels in M81-E than Colman. Line, treatment, and timepoint differences observed in phenolic metabolite and enzyme activities did not account for observed differences in lesions. However, generalized additive models were able to relate metabolites, but not enzyme activities, to lesion length for quantitatively modeling disease progression: in M81-E, but not Colman, sinapic acid levels positively predicted lesion length at 3 DAI when cell wall-bound syringic acid was low, soluble caffeic acid was high, and lactic acid was high, suggesting that sinapic acid may contribute to responses at 3 DAI. These results provide potential gene targets for development of sweet sorghum varieties with increased stalk rot resistance to ensure biomass and sugar quality., Competing Interests: The author(s) declare no conflict of interest.

The drought-resilient crop sorghum ( Sorghum bicolor [L.] Moench) is grown worldwide for multiple uses, including forage or potential lignocellulosic bioenergy feedstock. A major impediment to biomass yield and quality are the pathogens Fusarium thapsinum and Macrophomina phaseolina , which cause Fusarium stalk rot and charcoal rot, respectively. These fungi are more virulent with abiotic stresses such as drought. Monolignol biosynthesis plays a critical role in plant defense. The genes Brown midrib ( Bmr ) 6 , Bmr12 , and Bmr2 encode the monolignol biosynthesis enzymes cinnamyl alcohol dehydrogenase, caffeic acid O -methyltransferase, and 4-coumarate:CoA ligase, respectively. Plant stalks from lines overexpressing these genes and containing bmr mutations were screened for pathogen responses with controlled adequate or deficit watering. Additionally, near-isogenic bmr12 and wild-type lines in five backgrounds were screened for response to F. thapsinum with adequate and deficit watering. All mutant and overexpression lines were no more susceptible than corresponding wild-type under both watering conditions. The bmr2 and bmr12 lines, near-isogenic to wild-type, had significantly shorter mean lesion lengths (were more resistant) than RTx430 wild-type when inoculated with F. thapsinum under water deficit. Additionally, bmr2 plants grown under water deficit had significantly smaller mean lesions when inoculated with M. phaseolina than under adequate-water conditions. When well-watered, bmr12 in cultivar Wheatland and one of two Bmr2 overexpression lines in RTx430 had shorter mean lesion lengths than corresponding wild-type lines. This research demonstrates that modifying monolignol biosynthesis for increased usability may not impair plant defenses but can even enhance resistance to stalk pathogens under drought conditions., Competing Interests: The author(s) declare no conflict of interest.

In sorghum [Sorghum bicolor (L.) Moench] the Maturity (Ma1, Ma2, Ma3, Ma4, Ma5, Ma6) and Dwarf (Dw1, Dw2, Dw3, Dw4) loci, encode genes controlling flowering time and plant height, respectively, which are critical for designing sorghum ideotypes for a maturity timeframe and a harvest method. Publicly available whole-genome resequencing data from 860 sorghum accessions was analyzed in silico to identify genomic variants at 8 of these loci (Ma1, Ma2, Ma3, Ma5, Ma6, Dw1, Dw2, Dw3) to identify novel loss of function alleles and previously characterized ones in sorghum germplasm. From ~ 33 million SNPs and ~ 4.4 million InDels, 1445 gene variants were identified within these 8 genes then evaluated for predicted effect on the corresponding encoded proteins, which included newly identified mutations (4 nonsense, 15 frameshift, 28 missense). Likewise, most accessions analyzed contained predicted loss of function alleles (425 ma1, 22 ma2, 40 ma3, 74 ma5, 414 ma6, 289 dw1, 268 dw2 and 45 dw3) at multiple loci, but 146 and 463 accessions had no predicted ma or dw mutant alleles, respectively. The ma and dw alleles within these sorghum accessions represent a valuable source for manipulating flowering time and plant height to develop the full range of sorghum types: grain, sweet and forage/biomass., (© 2023. Springer Nature Limited.)

In sorghum ( Sorghum bicolor ) and other C 4 grasses, brown midrib ( bmr ) mutants have long been associated with plants impaired in their ability to synthesize lignin. The brown midrib 30 ( Bmr30 ) gene, identified using a bulk segregant analysis and next-generation sequencing, was determined to encode a chalcone isomerase (CHI). Two independent mutations within this gene confirmed that loss of its function was responsible for the brown leaf midrib phenotype and reduced lignin concentration. Loss of the Bmr30 gene function, as shown by histochemical staining of leaf midrib and stalk sections, resulted in altered cell wall composition. In the bmr30 mutants, CHI activity was drastically reduced, and the accumulation of total flavonoids and total anthocyanins was impaired, which is consistent with its function in flavonoid biosynthesis. The level of the flavone lignin monomer tricin was reduced 20-fold in the stem relative to wild type, and to undetectable levels in the leaf tissue of the mutants. The bmr30 mutant, therefore, harbors a mutation in a phenylpropanoid biosynthetic gene that is key to the interconnection between flavonoids and monolignols, both of which are utilized for lignin synthesis in the grasses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tetreault, Gries, Liu, Toy, Xin, Vermerris, Ralph, Funnell-Harris and Sattler.)

Background: As effects of global climate change intensify, the interaction of biotic and abiotic stresses increasingly threatens current agricultural practices. The secondary cell wall is a vanguard of resistance to these stresses. Fusarium thapsinum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot) cause internal damage to the stalks of the drought tolerant C4 grass, sorghum (Sorghum bicolor (L.) Moench), resulting in reduced transpiration, reduced photosynthesis, and increased lodging, severely reducing yields. Drought can magnify these losses. Two null alleles in monolignol biosynthesis of sorghum (brown midrib 6-ref, bmr6-ref; cinnamyl alcohol dehydrogenase, CAD; and bmr12-ref; caffeic acid O-methyltransferase, COMT) were used to investigate the interaction of water limitation with F. thapsinum or M. phaseolina infection., Results: The bmr12 plants inoculated with either of these pathogens had increased levels of salicylic acid (SA) and jasmonic acid (JA) across both watering conditions and significantly reduced lesion sizes under water limitation compared to adequate watering, which suggested that drought may prime induction of pathogen resistance. RNA-Seq analysis revealed coexpressed genes associated with pathogen infection. The defense response included phytohormone signal transduction pathways, primary and secondary cell wall biosynthetic genes, and genes encoding components of the spliceosome and proteasome., Conclusion: Alterations in the composition of the secondary cell wall affect immunity by influencing phenolic composition and phytohormone signaling, leading to the action of defense pathways. Some of these pathways appear to be activated or enhanced by drought. Secondary metabolite biosynthesis and modification in SA and JA signal transduction may be involved in priming a stronger defense response in water-limited bmr12 plants., (© 2021. The Author(s).)

Ferulate 5-hydroxylase (F5H) of the monolignol pathway catalyzes the hydroxylation of coniferyl alcohol, coniferaldehyde and ferulic acid to produce 5-hydroxyconiferyl moieties, which lead to the formation of sinapic acid and syringyl (S) lignin monomers. In contrast, guaiacyl (G) lignin, the other major type of lignin monomer, is derived from polymerization of coniferyl alcohol. In this study, the effects of manipulating S-lignin biosynthesis in sorghum (Sorghum bicolor) were evaluated. Overexpression of sorghum F5H (SbF5H), under the control of the CaMV 35S promoter, increased both S-lignin levels and the ratio of S/G lignin, while plant growth and development remained relatively unaffected. Maüle staining of stalk and leaf midrib sections from SbF5H overexpression lines indicated that the lignin composition was altered. Ectopic expression of SbF5H did not affect the gene expression of other monolignol pathway genes. In addition, brown midrib 12-ref (bmr12-ref), a nonsense mutation in the sorghum caffeic acid O-methyltransferase (COMT) was combined with 35S::SbF5H through cross-pollination to examine effects on lignin synthesis. The stover composition from bmr12 35S::SbF5H plants more closely resembled bmr12 stover than 35S::SbF5H or wild-type (WT) stover; S-lignin and total lignin concentrations were decreased relative to WT or 35S::SbF5H. Likewise, expression of upstream monolignol biosynthetic genes was increased in both bmr12 and bmr12 35S::SbF5H relative to WT or 35S::SbF5H. Overall, these results indicated that overexpression of SbF5H did not compensate for the loss of COMT activity. KEY MESSAGE: Overexpression of F5H in sorghum increases S-lignin without increasing total lignin content or affecting plant growth, but it cannot compensate for the loss of COMT activity in monolignol synthesis.

To increase phenylpropanoid constituents and energy content in the versatile C 4 grass sorghum ( Sorghum bicolor [L.] Moench), sorghum genes for proteins related to monolignol biosynthesis were overexpressed: SbMyb60 (transcriptional activator), SbPAL (phenylalanine ammonia lyase), SbCCoAOMT (caffeoyl coenzyme A [CoA] 3- O -methyltransferase), Bmr2 (4-coumarate:CoA ligase), and SbC3H (coumaroyl shikimate 3-hydroxylase). Overexpression lines were evaluated for responses to stalk pathogens under greenhouse and field conditions. Greenhouse-grown plants were inoculated with Fusarium thapsinum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot), which cause yield-reducing diseases. F. thapsinum -inoculated overexpression plants had mean lesion lengths not significantly different than wild-type, except for significantly smaller lesions on two of three SbMyb60 and one of two SbCCoAOMT lines. M. phaseolina -inoculated overexpression lines had lesions not significantly different from wild-type except one SbPAL line (of two lines studied) with mean lesion lengths significantly larger. Field-grown SbMyb60 and SbCCoAOMT overexpression plants were inoculated with F. thapsinum . Mean lesions of SbMyb60 lines were similar to wild-type, but one SbCCoAOMT had larger lesions, whereas the other line was not significantly different than wild-type. Because overexpression of SbMyb60 , Bmr2 , or SbC3H may not render sorghum more susceptible to stalk rots, these lines may provide sources for development of sorghum with increased phenylpropanoid concentrations.

Hexaploid waxy wheat ( Triticum aestivum L.) has null mutations in Wx genes and grain lacking amylose with increased digestibility and usability for specialty foods. The waxy cultivar Mattern is susceptible to Fusarium head blight (FHB) caused by Fusarium graminearum species complex, which produces the mycotoxin deoxynivalenol (DON). In experiment 1, conducted during low natural FHB, grain from waxy breeding lines, Mattern, and wild-type breeding lines and cultivars were assessed for Fusarium infection and DON concentration. Nine Fusarium species and species complexes were detected from internally infected (disinfested) grain; F. graminearum infections were not different between waxy and wild-type. Surface- and internally infected grain (nondisinfested) had greater numbers of Fusarium isolates across waxy versus wild-type, but F. graminearum -like infections were similar; however, DON levels were higher in waxy . In experiment 2, conducted during a timely epidemic, disease severity, Fusarium -damaged kernels (FDK), and DON were assessed for waxy breeding lines, Mattern, and wild-type cultivars. Disease severity and FDK were not significantly different from wild-type, but DON was higher in waxy than wild-type lines. Across both experiments, waxy breeding lines, Plant Introductions 677876 and 677877, responded similarly to FHB as moderately resistant wild-type cultivar Overland, showing promise for breeding advanced waxy cultivars with reduced FHB susceptibility.

Sorghum (Sorghum bicolor) is a drought tolerant crop, which is being developed as a bioenergy feedstock. The monolignol biosynthesis pathway is a major focus for altering the abundance and composition of lignin. Caffeoyl coenzyme-A O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase that methylates caffeoyl-CoA to generate feruloyl-CoA, an intermediate required for the biosynthesis of both G- and S-lignin. SbCCoAOMT was overexpressed to assess the impact of increasing the amount of this enzyme on biomass composition. SbCCoAOMT overexpression increased both soluble and cell wall-bound (esterified) ferulic and sinapic acids, however lignin concentration and its composition (S/G ratio) remained unaffected. This increased deposition of hydroxycinnamic acids in these lines led to an increase in total energy content of the stover. In stalk and leaf midribs, the increased histochemical staining and autofluorescence in the cell walls of the SbCCoAOMT overexpression lines also indicate increased phenolic deposition within cell walls, which is consistent with the chemical analyses of soluble and wall-bound hydroxycinnamic acids. The growth and development of overexpression lines were similar to wild-type plants. Likewise, RNA-seq and metabolite profiling showed that global gene expression and metabolite levels in overexpression lines were also relatively similar to wild-type plants. Our results demonstrate that SbCCoAOMT overexpression significantly altered cell wall composition through increases in cell wall associated hydroxycinnamic acids without altering lignin concentration or affecting plant growth and development., Competing Interests: The authors have declared that no competing interests exist.

Few transcription factors have been identified in C 4 grasses that either positively or negatively regulate monolignol biosynthesis. Previously, the overexpression of SbMyb60 in sorghum (Sorghum bicolor) has been shown to induce monolignol biosynthesis, which leads to elevated lignin deposition and altered cell wall composition. To determine how SbMyb60 overexpression impacts other metabolic pathways, RNA-Seq and metabolite profiling were performed on stalks and leaves. 35S::SbMyb60 was associated with the transcriptional activation of genes involved in aromatic amino acid, S-adenosyl methionine (SAM) and folate biosynthetic pathways. The high coexpression values between SbMyb60 and genes assigned to these pathways indicate that SbMyb60 may directly induce their expression. In addition, 35S::SbMyb60 altered the expression of genes involved in nitrogen (N) assimilation and carbon (C) metabolism, which may redirect C and N towards monolignol biosynthesis. Genes linked to UDP-sugar biosynthesis and cellulose synthesis were also induced, which is consistent with the observed increase in cellulose deposition in the internodes of 35S::SbMyb60 plants. However, SbMyb60 showed low coexpression values with these genes and is not likely to be a direct regulator of cell wall polysaccharide biosynthesis. These findings indicate that SbMyb60 can activate pathways beyond monolignol biosynthesis, including those that synthesize the substrates and cofactors required for lignin biosynthesis., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Several Fusarium spp. cause sorghum (Sorghum bicolor) grain mold, resulting in deterioration and mycotoxin production in the field and during storage. Fungal isolates from the air (2005 to 2006) and from leaves and grain from wild-type and brown midrib (bmr)-6 and bmr12 plants (2002 to 2003) were collected from two locations. Compared with the wild type, bmr plants have reduced lignin content, altered cell wall composition, and different levels of phenolic intermediates. Multilocus maximum-likelihood analysis identified two Fusarium thapsinum operational taxonomic units (OTU). One was identified at greater frequency in grain and leaves of bmr and wild-type plants but was infrequently detected in air. Nine F. graminearum OTU were identified: one was detected at low levels in grain and leaves while the rest were only detected in air. Wright's F statistic (F ST ) indicated that Fusarium air populations differentiated between locations during crop anthesis but did not differ during vegetative growth, grain development, and maturity. F ST also indicated that Fusarium populations from wild-type grain were differentiated from those in bmr6 or bmr12 grain at one location but, at the second location, populations from wild-type and bmr6 grain were more similar. Thus, impairing monolignol biosynthesis substantially effected Fusarium populations but environment had a strong influence.

Sweet sorghum (Sorghum bicolor (L.) Moench) has potential for bioenergy. It is adapted to a variety of U.S. locations and the extracted juice can be directly fermented into ethanol. However, little research on fungal stalk rots, diseases that pose serious constraints for yield and quality of juice and biomass, has been reported. A greenhouse bioassay was designed to assess charcoal rot (Macrophomina phaseolina) and Fusarium stalk rot (Fusarium thapsinum) in plants at maturity, the developmental stage at which these diseases are manifested. Multiple plantings of a susceptible grain line, RTx430, were used as a control for variation in flowering times among sweet sorghum lines. Lesion length measurements in inoculated peduncles were used to quantify disease severity. Sweet sorghum lines 'Rio' and 'M81E' exhibited resistance to F. thapsinum and M. phaseolina, respectively; and, in contrast, 'Colman' sorghum exhibited susceptibility to both pathogens. Lesion development over time in Colman was monitored. These results will enhance molecular and biochemical analyses of responses to pathogens, and breeding stalk-rot-resistant sweet sorghum lines.

The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses, the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example, the Bmr6 gene in sorghum (Sorghum bicolor) has been previously shown to encode cinnamyl alcohol dehydrogenase (CAD), which catalyzes the final step of the monolignol biosynthesis pathway. Mutations in this gene have been shown to reduce the abundance of lignin, enhance digestibility, and improve saccharification efficiencies and ethanol yields. Nine sorghum lines harboring five different bmr6 alleles were identified in an EMS-mutagenized TILLING population. DNA sequencing of Bmr6 revealed that the majority of the mutations impacted evolutionarily conserved amino acids while three-dimensional structural modeling predicted that all of these alleles interfered with the enzyme's ability to bind with its NADPH cofactor. All of the new alleles reduced in vitro CAD activity levels and enhanced glucose yields following saccharification. Further, many of these lines were associated with higher reductions in acid detergent lignin compared to lines harboring the previously characterized bmr6-ref allele. These bmr6 lines represent new breeding tools for manipulating biomass composition to enhance forage and feedstock quality., (© 2015 Institute of Botany, Chinese Academy of Sciences.)

The phenylpropanoid biosynthetic pathway that generates lignin subunits represents a significant target for altering the abundance and composition of lignin. The global regulators of phenylpropanoid metabolism may include MYB transcription factors, whose expression levels have been correlated with changes in secondary cell wall composition and the levels of several other aromatic compounds, including anthocyanins and flavonoids. While transcription factors correlated with downregulation of the phenylpropanoid biosynthesis pathway have been identified in several grass species, few transcription factors linked to activation of this pathway have been identified in C4 grasses, some of which are being developed as dedicated bioenergy feedstocks. In this study we investigated the role of SbMyb60 in lignin biosynthesis in sorghum (Sorghum bicolor), which is a drought-tolerant, high-yielding biomass crop. Ectopic expression of this transcription factor in sorghum was associated with higher expression levels of genes involved in monolignol biosynthesis, and led to higher abundances of syringyl lignin, significant compositional changes to the lignin polymer and increased lignin concentration in biomass. Moreover, transgenic plants constitutively overexpressing SbMyb60 also displayed ectopic lignification in leaf midribs and elevated concentrations of soluble phenolic compounds in biomass. Results indicate that overexpression of SbMyb60 is associated with activation of monolignol biosynthesis in sorghum. SbMyb60 represents a target for modification of plant cell wall composition, with the potential to improve biomass for renewable uses., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Loss of function mutations in waxy, encoding granule bound starch synthase (GBSS) that synthesizes amylose, results in starch granules containing mostly amylopectin. Low amylose grain with altered starch properties has increased usability for feed, food, and grain-based ethanol. In sorghum, two classes of waxy (wx) alleles had been characterized for absence or presence of GBSS: wx(a) (GBSS(-)) and wx(b) (GBSS(+), with reduced activity). Field-grown grain of wild-type; waxy, GBSS(-); and waxy, GBSS(+) plant introduction accessions were screened for fungal infection. Overall, results showed that waxy grains were not more susceptible than wild-type. GBSS(-) and wild-type grain had similar infection levels. However, height was a factor with waxy, GBSS(+) lines: short accessions (wx(b) allele) were more susceptible than tall accessions (undescribed allele). In greenhouse experiments, grain from accessions and near-isogenic wx(a), wx(b), and wild-type lines were inoculated with Alternaria sp., Fusarium thapsinum, and Curvularia sorghina to analyze germination and seedling fitness. As a group, waxy lines were not more susceptible to these pathogens than wild-type, supporting field evaluations. After C. sorghina and F. thapsinum inoculations most waxy and wild-type lines had reduced emergence, survival, and seedling weights. These results are valuable for developing waxy hybrids with resistance to grain-infecting fungi.

Sorghum lines were bred for reduced lignin for cellulosic bioenergy uses, through the incorporation of brown midrib (bmr)6 or -12 into two backgrounds (RTx430 and Wheatland) as either single or double-mutant lines. When these lines were assessed for resistance to Fusarium thapsinum stalk rot, a cause of lodging, they were as resistant to F. thapsinum as the near-isogenic wild type. Peduncles of newly identified bmr lines from an ethyl-methanesulfonate-mutagenized population, inoculated with F. thapsinum, were as resistant as the wild-type line, BTx623. One bmr line (1107) had significantly smaller mean lesion lengths than BTx623, suggesting that a mutation is associated with reduced susceptibility. Growing F. thapsinum on medium with ferulic, vanillic, sinapic, syringic, and caffeic acids (phenolic compounds derived from the lignin pathway and elevated in different bmr lines) indicated that F. thapsinum was tolerant to these compounds. When eight other sorghum fungi were tested for response to the presence of these compounds, ferulic acid inhibited these fungi. Most of the phenolics inhibited F. verticillioides and F. proliferatum. Accumulation of phenolic metabolites in bmr plants may inhibit growth of some sorghum pathogens, while other factors such as aromatic phytoalexins or salicylic acid may be involved in resistance to F. thapsinum.

Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6, and bmr12, and, in addition, six bmr mutants were identified that were not allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Overall, most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles., (Copyright © 2014 Sattler et al.)

Lignin is a ubiquitous polymer present in cell walls of all vascular plants, where it rigidifies and strengthens the cell wall structure through covalent cross-linkages to cell wall polysaccharides. The presence of lignin makes the cell wall recalcitrant to conversion into fermentable sugars for bioenergy uses. Therefore, reducing lignin content and modifying its linkages have become major targets for bioenergy feedstock development through either biotechnology or traditional plant breeding. In addition, lignin synthesis has long been implicated as an important plant defense mechanism against pathogens, because lignin synthesis is often induced at the site of pathogen attack. This article explores the impact of lignin modifications on the susceptibility of a range of plant species to their associated pathogens, and the implications for development of feedstocks for the second-generation biofuels industry. Surprisingly, there are some instances where plants modified in lignin synthesis may display increased resistance to associated pathogens, which is explored in this article.

Mold diseases, caused by fungal complexes including Alternaria, Cochliobolus, and Fusarium species, limit sorghum grain production. Media were tested by plating Fusarium thapsinum, Alternaria sp., and Curvularia lunata, individually and competitively. Dichloran chloramphenicol rose bengal (DRBC) and modified V8 juice (ModV8) agars, found to be useful, were compared with commonly used agar media, dichloran chloramphenicol peptone (DCPA) and pentachloronitrobenzene (PCNB). Radial growth, starting with mycelia or single-conidia and hyphal tips, demonstrated an effect of media. For isolation of grain fungi, DRBC and ModV8 were similar or superior to DCPA and PCNB. When seedlings were inoculated with conidia of C. lunata, Alternaria sp., F. thapsinum, or mixtures, the percentage of root infection ranged from 28% to 77%. For mixed inoculations, shoot weights, lesion lengths, and percentage of root infections were similar to F. thapsinum inoculations; most colonies recovered from roots were F. thapsinum. For Alternaria grain isolates, 5 morphological types, including Alternaria alternata, were distinguished by colony morphologies and conidial dimensions. Sequence analysis using a portion of the endo-polygalacturonase gene was able to further distinguish isolates. Cochliobolus isolates were identified morphologically as C. lunata, Curvularia sorghina, and Bipolaris sorghicola. Multiple molecular genotypes were apparent from rRNA internal transcribed spacer region sequences from Cochliobolus grain isolates.

Sorghum grain, valuable for feed, food, and bioenergy, can be colonized by several Fusarium spp.; therefore, it was of interest to identify possible sources of conidia. Analysis of air and soil samples provided evidence for the presence of propagules from Fusarium genotypes that may cause grain infections. Soil population estimates of members of the Gibberella fujikuroi species complex, that includes sorghum pathogens and other Fusarium spp., suggested that adequate inoculum for systemic infections was present. Conidia in air samples within two sorghum fields were collected by passive trapping for 2 years. Subsampled Fusarium isolates indicated that numbers of G. fujikuroi increased from anthesis through maturity, which coincides with grain development stages vulnerable to Fusarium spp. Genotyping using translation elongation factor 1-α gene sequences revealed that spore trap isolates included members of G. fujikuroi that are sorghum pathogens: Fusarium thapsinum, F. verticillioides, F. proliferatum, and F. andiyazi. Also detected were F. graminearum, F. subglutinans, and several F. incarnatum-F. equiseti species complex haplotypes that colonize sorghum asymptomatically. All commonly found grain colonizers were detected from air samples in this study.

To improve sorghum for bioenergy and forage uses, brown midrib (bmr)6 and -12 near-isogenic genotypes were developed in different sorghum backgrounds. The bmr6 and bmr12 grain had significantly reduced colonization by members of the Gibberella fujikuroi species complex compared with the wild type, as detected on two semiselective media. Fusarium spp. were identified using sequence analysis of a portion of the translation elongation factor (TEF) 1-alpha gene. The pathogens Fusarium thapsinum, F. proliferatum, and F. verticillioides, G. fujikuroi members, were commonly recovered. Other frequently isolated Fusarium spp. likely colonize sorghum asymptomatically. The chi(2) analyses showed that the ratios of Fusarium spp. colonizing bmr12 grain were significantly different from the wild type, indicating that bmr12 affects colonization by Fusarium spp. One F. incarnatum-F. equiseti species complex (FIESC) genotype, commonly isolated from wild-type and bmr6 grain, was not detected in bmr12 grain. Phylogenetic analysis suggested that this FIESC genotype represents a previously unreported TEF haplotype. When peduncles of wild-type and near-isogenic bmr plants were inoculated with F. thapsinum, F. verticillioides, or Alternaria alternata, the resulting mean lesion lengths were significantly reduced relative to the wild type in one or both bmr mutants. This indicates that impairing lignin biosynthesis results in reduced colonization by Fusarium spp. and A. alternata.

In sorghum, brown midrib (bmr) 6 and 12 impair the last two steps of monolignol synthesis. bmr genes were introduced into grain sorghum to improve the digestibility of lignocellulosic tissues for grazing or bioenergy uses following grain harvest. Near-isogenic grain sorghum hybrids (AWheatland x RTx430) were developed containing bmr6, bmr12, and the bmr6 bmr12 double mutant (stacked), and their impacts were assessed in a two-year field study. The bmr genes did not significantly impact grain or lignocellulosic tissue yield. Lignocellulosic tissue from bmr6, bmr12, and stacked hybrids had reduced lignin content and increased in vitro dry matter digestibility (IVDMD) compared to those of the wild type (WT). The lignin content of the stacked lignocellulosic tissue was further reduced compared to that of bmr6 or bmr12. Surprisingly, bmr12 modestly increased carbohydrates in lignocellulosic tissue, and bmr6 increased fiber and lignin content in grain. These data indicate that bmr6 and bmr12 have broader effects on plant composition than merely lignin content, which has promising implications for both livestock utilization and bioenergy conversion.

Brown midrib6 (bmr6) affects phenylpropanoid metabolism, resulting in reduced lignin concentrations and altered lignin composition in sorghum (Sorghum bicolor). Recently, bmr6 plants were shown to have limited cinnamyl alcohol dehydrogenase activity (CAD; EC 1.1.1.195), the enzyme that catalyzes the conversion of hydroxycinnamoyl aldehydes (monolignals) to monolignols. A candidate gene approach was taken to identify Bmr6. Two CAD genes (Sb02g024190 and Sb04g005950) were identified in the sorghum genome based on similarity to known CAD genes and through DNA sequencing a nonsense mutation was discovered in Sb04g005950 that results in a truncated protein lacking the NADPH-binding and C-terminal catalytic domains. Immunoblotting confirmed that the Bmr6 protein was absent in protein extracts from bmr6 plants. Phylogenetic analysis indicated that Bmr6 is a member of an evolutionarily conserved group of CAD proteins, which function in lignin biosynthesis. In addition, Bmr6 is distinct from the other CAD-like proteins in sorghum, including SbCAD4 (Sb02g024190). Although both Bmr6 and SbCAD4 are expressed in sorghum internodes, an examination of enzymatic activity of recombinant Bmr6 and SbCAD4 showed that Bmr6 had 1 to 2 orders of magnitude greater activity for monolignol substrates. Modeling of Bmr6 and SbCAD4 protein structures showed differences in the amino acid composition of the active site that could explain the difference in enzyme activity. These differences include His-57, which is unique to Bmr6 and other grass CADs. In summary, Bmr6 encodes the major CAD protein involved in lignin synthesis in sorghum, and the bmr6 mutant is a null allele.