Despite its general resilience, sorghum (Sorghum bicolor) remains vulnerable to biotic stresses, including infestation by the sugarcane aphid (SCA; Melanaphis sacchari), which is a critical pest of this crop. Ferulate-5-hydroxylase (F5H), an enzyme involved in monolignol biosynthesis in sorghum, acts upstream of caffeic acid O-methyltransferase (COMT), which is encoded by the Brown midrib12 (Bmr12) gene. Loss of COMT activity enhances SCA resistance by increasing levels of the auxin conjugate indole-3-acetic acid-aspartate (IAA-Asp). To assess the role of F5H in sorghum defense against SCA, we compared F5H-overexpressing (OE) lines with RTx430 wild-type (WT) plants. Aphid bioassays and feeding behavior analysis using an electrical penetration graph (EPG) system showed that aphids prefer to colonize and feed more on F5H-OE plants than on the WT. Biochemical analyses indicated similar lignin levels between lines, but F5H-OE plants had lower basal flavonoids. Moreover, SCA infestation suppressed auxin signal transduction genes that restrict aphid proliferation. Interestingly, exogenous application of IAA-Asp restored SCA resistance in F5H-OE plants to WT levels. While the bmr12 mutant displayed resistance to SCA, the cross between F5H-OE and bmr12 resulted in significant attenuation of the bmr12-conferred resistance. Taken together, these findings reveal that F5H-OE amplifies sorghum susceptibility to SCA by disrupting auxin and flavonoid-mediated defenses, while reaffirming that the loss of Bmr12 function confers strong aphid resistance.

Lignins in plant biomass are carbon-negative aromatic biopolymers which hold tremendous potential as multipurpose resources for sustainable bioeconomy, limited only by their chemical heterogeneity. Plant lignified tissues, such as sapwood and seed coats, vary in colors within and between species, indicating that specific lignin topochemistries determine the different colors. Yet, the responsible lignin chromogen(s) are unknown. We developed chemical imaging using UV-Vis microspectroscopy to link lignin color to topochemistry in isolates and plant samples. Using synthetic and technical lignins, we identified the different stable chromogens as homomeric lignin substructures varying in size, unit chemistry and interunit linkages. We controlled the accumulation of specific lignin chromogens using genetic engineering to similarly stain lignified tissues from different plant species. We established plant tissue engineering to cast plant tissues with pre-determined color by adjusting lignin topochemistries. We proved that biotechnological manipulation of the identified lignin chromogens predictably and stably stains lignified plant tissues.

Hyperspectral imaging has been used to determine plant stress status. However, the biological interpretation of the spectral changes remain less explored. This can be addressed by building associations between stress-induced biochemical changes and variations in spectral reflectance. To this end, we tested spectral response of sorghum brown midrib (bmr) mutants under varying water stress levels using hyperspectral imaging (650-1650 nm). The bmr mutants have reduced lignin concentrations in their vegetative tissue which was reflected as spectral differences. Under water stress, the spectral signatures diverged more between the wildtype and mutants compared to control conditions. The genotype-dependent variation in spectral trends under water limitation was associated with differential sensitivity of the genotypes to water-limitation induced changes in energy density. We show that the energy density and relative water content of the plant tissue can be estimated accurately from spectral reflectance. To reduce the computational load, LASSO was used to obtain 22 wavelengths across the camera spectral range (650-1650 nm) in dried samples, to accurately predict energy density comparable to PLSR estimates. The reported wavelengths represent a useful screening tool for fast and reliable calorimetric estimations in bioenergy breeding programs.

The Waxy locus ( Wx ) encodes the granule‐bound starch synthase enzyme, which synthesizes amylose, the long‐chain, unbranched starch polymer. Loss of function alleles of Wx result in the endosperm having a waxy appearance, and contain nearly all amylopectin, the highly branched starch polymer. A critical question for commercialization of waxy grain sorghum [ Sorghum bicolor (L.) Moench] for applications ranging from food industry to bioenergy crop production is whether hybrids deleteriously affect agronomic traits. A set of six waxy sorghum A ‐lines was crossed with a set of six R ‐lines developed by USDA‐ARS in Lincoln, NE. The hybrids were grown in two field sites under dryland and irrigated conditions. Both agronomic and grain composition traits were determined. The amylose concentration from all waxy hybrid grain was around 5% compared to wild‐type hybrid checks, which had concentrations around 35%. In general, seedling emergence, test weight, and 100‐seed weight were lower for wx hybrids than the commercial check. The grain yields for wx hybrid combinations with A lines PI 670136 and PI 672150 (Wheatland wx ) or R ‐line PI 672152 (RTx430 wx ) were over 8000 kg ha −1 for several combinations. The A line PI 670136 in combination with five of the R ‐lines evaluated produced white sorghum grain on tan plants that could be milled into food grade white sorghum flour. The waxy hybrids in this study illustrate the potential to commercially produce wx grain sorghum for a range of applications.

Sorghum (Sorghum bicolor), one of the world's most important monocot crops, suffers severe yield losses due to attack by a polyphagous insect pest, fall armyworm (FAW; Spodoptera frugiperda). Here, we show that the Brown midrib 12 (Bmr12) gene, which encodes the caffeic acid O-methyltransferase (COMT) enzyme, promotes sorghum defense against FAW. Loss of Bmr12 function resulted in increased susceptibility, but enhanced resistance to FAW was observed in Bmr12-overexpression (OE) plants compared with wild-type (RTx430) plants. Although COMT is associated with modulating lignin levels, FAW infestation resulted in comparable lignin levels between bmr12 and Bmr12-OE sorghum plants. On the contrary, evidence presented here indicates that FAW feeding induced the accumulation of flavonoids, which was previously shown to have a negative impact on FAW growth and survival in Bmr12-OE plants compared with bmr12 and RTx430 plants. Furthermore, a combination of phytohormone profiling and transcriptomic analysis uncovered that COMT-mediated resistance to FAW depends on jasmonic acid (JA) and oxidative stress-associated pathways. Exogenous application of FAW oral secretions stimulated flavonoid accumulation in Bmr12-OE plants compared with bmr12 and RTx430 plants, indicating that COMT has an essential function in perceiving FAW oral cues. Taken together, the critical role of COMT in sorghum defense against FAW hinges upon the interplay between JA and its derivatives and hydrogen peroxide, which potentially helps to mount a robust flavonoid-based host defense upon caterpillar attack.

Current efforts to detect and evaluate crop resistance to insect pests are limited by traditional phenotyping methods, which are time-consuming and highly variable. Sugarcane aphid (SCA; Melanaphis sacchari) is a major pest of sorghum in North America that has emerged over the last decade and negatively impacts plant growth and development. The spectral reflectance data in visible, near infrared and shortwave infrared range (VIS-NIR-SWIR; 400-2500 nm) have been used to measure plant traits related to stress responses, nutrient dynamics, and physiological status. We examined the potential of spectral features (VIS-NIR-SWIR) to improve the current phenotyping methods in monitoring sorghum resistance mechanisms to SCA. We used eight sorghum lines that displayed varied levels of resistance to SCA and collected data from control and aphid-infested plants. Spectral feature data were collected using a leaf spectrometer, while plant physiological and chlorophyll fluorescence parameters were measured with LICOR and MultispeQ devices. The random forest classifier model differentiated the control and aphid-infested plants with a high accuracy of 87.4% with important spectral features in the VIS-NIR spectral range, particularly from 508 to 573 nm and 715 to 728 nm. The spectral indices exhibit significant difference in Greenness Index and Plant Senescence Reflectance Index in aphid-infested susceptible lines (BTx623, SC1345) compared with control plants. In addition, plant physiological parameters, such as stomatal conductance and chlorophyll fluorescence, showed significantly higher value for aphid-infested resistant line (Tx2783) compared with susceptible line (BTx623) in both treatments. Further, a partial least square regression model demonstrated medium predictive capability for plant physiological parameters related to fluorescence. In summary, spectral features at VIS-NIR range demonstrated promising results in differentiating aphid-infested sorghum plants. This is a proof-of-concept study on potential of spectral sensing to develop an effective monitoring and phenotyping plant resistance to aphids.

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.

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI’s unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

The aim of this work was to evaluate the use of stover from brown midrib (bmr) grain sorghum to produce polyhydroxyalkanoates (PHAs). These mutants produce lignin with novel compositions and/ or reduced lignin content that was hypothesized to result in higher PHA production than when using a wild-type sorghum stover. Five sorghum stover samples [wild-type (control) and four bmr mutants [bmr2, bmr6, and bmr12, and a double bmr mutant: bmr6 and bmr12 (stacked)] were evaluated as substrates for poly(3-hydroxybutyrate) (P(3HB)) production. When the main sugars in non-detoxified alkali-pretreated sorghum stover hydrolysates (SSHs) were metabolized by Paraburkholderia sacchari for P(3HB) production, the SSHs from bmr plants were demonstrated to be promising feedstocks with a maximum P(3HB) concentration of 7.2 g/L from the bmr6 SSH, while a maximum P(3HB) concentration of 4.2 g/L P(3HB) was produced using wild-type SSH. Moreover, the highest maximum Y[P(3HB)/sugars] were achieved using stacked SSH and bmr12 SSH (0.44 g/g and 0.42 g/g, respectively). Furthermore, Cuprivadus necator was utilized to evaluate P(3HB) synthesis from the alkaline pretreated liquor (APL) remaining after sorghum stover pretreatment and an oxidative enzyme-mediator-surfactant system (OEMS) was used to improve P(3HB) production from the APLs. A 28-fold increase in PHB production from the bmr2-APL with OEMS compared to the fermentation of the same APL with no OEMS was observed. This is the first published report to use grain sorghum stover to produce polyhydroxyalkanoates (PHAs). Also, a novel combination of PHA production from both sugars and lignin is reported.

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.

Summary Lignin, a complex heterogenous polymer present in virtually all plant cell walls, plays a critical role in protecting plants from various stresses. However, little is known about how lignin modifications in sorghum will impact plant defense against sugarcane aphids (SCA), a key pest of sorghum. We utilized the sorghum brown midrib (bmr) mutants, which are impaired in monolignol synthesis, to understand sorghum defense mechanisms against SCA. We found that loss of Bmr12 function and overexpression (OE) of Bmr12 provided enhanced resistance and susceptibility to SCA, respectively, as compared with wild‐type (WT; RTx430) plants. Monitoring of the aphid feeding behavior indicated that SCA spent more time in reaching the first sieve element phase on bmr12 plants compared with RTx430 and Bmr12‐OE plants. A combination of transcriptomic and metabolomic analyses revealed that bmr12 plants displayed altered auxin metabolism upon SCA infestation and specifically, elevated levels of auxin conjugate indole‐3‐acetic acid–aspartic acid (IAA–Asp) were observed in bmr12 plants compared with RTx430 and Bmr12‐OE plants. Furthermore, exogenous application of IAA–Asp restored resistance in Bmr12‐OE plants, and artificial diet aphid feeding trial bioassays revealed that IAA–Asp is associated with enhanced resistance to SCA. Our findings highlight the molecular underpinnings that contribute to sorghum bmr12‐mediated resistance to SCA.

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.

Flavonoids are potent antioxidants that play a role in defense against pathogens, UV-radiation, and the detoxification of reactive oxygen species. Dihydroflavonol 4-reductase (DFR) and flavanone 4-reductase (FNR) reduce dihydroflavonols and flavanones, respectively, using NAD(P)H to produce flavan-(3)-4-(di)ols in flavonoid biosynthesis. Anthocyanidin reductase (ANR) reduces anthocyanidins to flavan-3-ols. In addition to their sequences, the 3D structures of recombinant DFR, FNR and ANR from sorghum and switchgrass showed a high level of similarity. The catalytic mechanism, substrate-specificity and key residues of three reductases were deduced from crystal structures, site-directed mutagenesis, molecular docking, kinetics, and thermodynamic ana-lyses. Although DFR displayed its highest activity against dihydroflavonols, it also showed activity against flavanones and anthocyanidins. It was inhibited by the flavonol quercetin and high concentrations of dihydroflavonols/flavonones. SbFNR1 and SbFNR2 did not show any activity against dihydroflavonols. However, SbFNR1 displayed activity against flavanones and ANR activity against two anthocyanidins, cyanidin and pelargonidin. Therefore, SbFNR1 and SbFNR2 could be specific ANR isozymes without delphinidin activity. Sorghum has high concentrations of 3-deoxyanthocyanidins in vivo, supporting the observed high activity of SbDFR against flavonols. Mining of expression data indicated substantial induction of these three reductase genes in both switchgrass and sorghum in response to biotic stress. Key signature sequences for proper DFR/ANR classification are proposed and could form the basis for future metabolic engineering of flavonoid metabolism.

Sugarcane aphid (SCA; Melanaphis sacchari) is a devastating pest of sorghum (Sorghum bicolor) that colonizes sorghum plants at different growth stages. Leaf surface characteristics and sugars often influence aphid settling and feeding on host plants. However, how changes in cuticular waxes and sugar levels affect SCA establishment and feeding at different development stages of sorghum have not been explored. In this study, two- and six-week-old BTx623 plants, a reference line of sorghum, was used to evaluate plant-aphid interactions. Monitoring aphid feeding behavior using Electrical Penetration Graph (EPG) technique revealed that aphids spent more time in the sieve element phase of six-week-old plants compared to two-week-old plants. Significant differences were found in the time spent to reach the first sieve element and pathway phases between the two- and six-week-old plants. However, no-choice aphid bioassays displayed that SCA population numbers were higher in two-week-old plants compared to six-week-old plants. Differences in the abundance of wax and sugar contents were analyzed to determine how these plant components influenced aphid feeding and proliferation. Among the cuticular wax compounds analyzed, α-amyrin and isoarborinone increased after 10 days of aphid infestation only in six-week-old plants. Trehalose content was significantly increased by SCA feeding on two- and six-week-old plants. Furthermore, SCA feeding depressed sucrose content and increased levels of glucose and fructose in two-week-old but not in six-week-old plants. Overall, our study indicates that plant age is a determinant for SCA feeding, and subtle changes in triterpenoids and available sugars influence SCA establishment on sorghum plants.

In planta, H2O2 is produced as a by-product of enzymatic reactions and during defense responses. Ascorbate peroxidase (APX) is a key enzyme involved in scavenging cytotoxic H2O2. Here, we report the crystal structure of cytosolic APX from sorghum (Sorghum bicolor) (Sobic.001G410200). While the overall structure of SbAPX was similar to that of other APXs, SbAPX uniquely displayed four bound ascorbates rather than one. In addition to the ɣ-heme pocket identified in other APXs, ascorbates were bound at the δ-meso and two solvent-exposed pockets. Consistent with the presence of multiple binding sites, our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Bound ascorbate at two surface sites established an intricate proton network with ascorbate at the ɣ-heme edge and δ-meso sites. Based on crystal structures, steady-state kinetics, and site-directed mutagenesis results, both ascorbate molecules at the ɣ-heme edge and the one at the surface are expected to participate in the oxidation reaction. We provide evidence that the H2O2-dependent oxidation of ascorbate by APX produces a C2-hydrated bicyclic hemiketal form of dehydroascorbic acid at the ɣ-heme edge, indicating two successive electron transfers from a single-bound ascorbate. In addition, the δ-meso site was shared with several organic compounds, including p-coumaric acid and other phenylpropanoids, for the potential radicalization reaction. Site-directed mutagenesis of the critical residue at the ɣ-heme edge (R172A) only partially reduced polymerization activity. Thus, APX removes stress-generated H2O2 with ascorbates, and also uses this same H2O2 to potentially fortify cell walls via oxidative polymerization of phenylpropanoids in response to stress.

High-biomass-yielding southerly adapted switchgrasses (Panicum virgatum L.) frequently suffer from unpredictable winter hardiness at more northerly sites arising from damage to rhizomes that prevent effective spring regrowth. Previously, changes occurring over the growing season in rhizomes sampled from a cold-adapted tetraploid upland cultivar, Summer, demonstrated a role for abscisic acid (ABA), starch accumulation, and transcriptional reprogramming as drivers of dormancy onset and potential keys to rhizome health during winter dormancy. Here, rhizome metabolism of a high-yielding southerly adapted tetraploid switchgrass cultivar, Kanlow-which is a significant source of genetics for yield improvement-was studied over a growing season at a northern site. Metabolite levels and transcript abundances were combined to develop physiological profiles accompanying greening through the onset of dormancy in Kanlow rhizomes. Next, comparisons of the data to rhizome metabolism occurring in the adapted upland cultivar Summer were performed. These data revealed both similarities as well as numerous differences in rhizome metabolism that were indicative of physiological adaptations unique to each cultivar. Similarities included elevated ABA levels and accumulation of starch in rhizomes during dormancy onset. Notable differences were observed in the accumulation of specific metabolites, the expression of genes encoding transcription factors, and several enzymes linked to primary metabolism.

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.

MAIN CONCLUSION:Quantification of cuticular waxes coupled with insect bioassays and feeding behavior analysis demonstrate that long-chain C32 fatty alcohol impacts host plant selection by aphids. Cuticular waxes constitute the first point of contact between plants and their environment, and it also protect plants from external stresses. However, the role of waxes in Sorghum bicolor (sorghum) against sugarcane aphid (Melanaphis sacchari), a relatively new and devastating pest of sorghum in the U.S., is not fully understood. In this study, we monitored sugarcane aphid behavior on two genotypes of young sorghum plants with different wax chemistry: a wild-type plant (bloom) with lower C32 alcohol cuticular wax, and a mutant plant (bloomless) with 1.6 times the amount of wax compared to wild-type plants. No-choice aphid bioassays revealed that sugarcane aphid reproduction did not vary between wild-type and the bloomless plants. Electrical Penetration Graph (EPG) monitoring indicated that the sugarcane aphids spent comparable amount of time feeding from the sieve elements of the wild-type and bloomless plants. However, aphids spent more time feeding on the xylem sap of the bloomless plants compared to the wild-type plants. Furthermore, aphid choice assays revealed that the sugarcane aphids preferred to settle on bloomless compared to wild-type plants. Overall, our results suggest that cuticular waxes on young sorghum leaves play a critical role in influencing host plant selection by sugarcane aphids.

Waxy starches from cereal grains contain >90% amylopectin due to naturally occurring mutations that block amylose biosynthesis. Waxy starches have unique organoleptic characteristics (e.g. sticky rice) as well as desirable physicochemical properties for food processing. Using isogenic pairs of wild type sorghum lines and their waxy derivatives, we studied the effects of waxy starches in the whole grain context on the human gut microbiome. In vitro fermentations with human stool microbiomes show that beneficial taxonomic and metabolic signatures driven by grain from wild type parental lines are lost in fermentations of grain from the waxy derivatives and the beneficial signatures can be restored by addition of resistant starch. These undesirable effects are conserved in fermentations of waxy maize, wheat, rice and millet. We also demonstrate that humanized gnotobiotic mice fed low fiber diets supplemented with 20% grain from isogenic pairs of waxy vs. wild type parental sorghum have significant differences in microbiome composition and show increased weight gain. We conclude that the benefits of waxy starches on food functionality can have unintended tradeoff effects on the gut microbiome and host physiology that could be particularly relevant in human populations consuming large amounts of waxy grains.

Sorghum (Sorghum bicolor) is drought-tolerant and has diverse germplasm for food, feed, forage and bioenergy. However, stalk diseases reduce quality and yield of biomass and grain, especially under drought. Previously, brown midrib (bmr) mutations in monolignol biosynthesis were shown to reduce lignin content and alter composition but were not more susceptible to stalk diseases than wild-type lines. Recently characterized bmr mutations were shown to affect flavonoid biosynthesis (chalcone isomerase; bmr30-1) or 1-carbon metabolism (folylpolyglutamate synthase; bmr19-1107, bmr19-1168 and bmr19-1937). Two other mutations, bmr29-1 and bmr31-1, have not yet been characterized. The six mutations were incorporated into elite genetic backgrounds (RTx430, BTx623 and BWheatland) to develop near-isogenic lines containing each mutation. Using peduncle inoculations with Fusarium thapsinum and F. proliferatum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot) under well-watered conditions, most bmr lines were at least as resistant as the corresponding wild type, except for RTx430 bmr19-1937 that had significantly longer mean lesion lengths when inoculated with M. phaseolina. Based on significantly reduced lesion lengths following inoculations with F. proliferatum and F. thapsinum, respectively, bmr29-1 and bmr31-1 lines were screened using basal stalk inoculations under well-watered and water-deficit conditions. The bmr lines were at least as resistant as the corresponding wild-type lines. Wild-type BTx623 was highly susceptible to M. phaseolina under water deficit, but near-isogenic bmr29-1 and bmr31-1 lines had significantly shorter mean lesion lengths. Incorporation of these mutations can increase resistance to stalk pathogens in cultivar and hybrid development for feed, bioenergy and production of biomass-based green chemicals.