Impaired Brown midrib12 function orchestrates sorghum resistance to aphids via an auxin conjugate indole-3-acetic acid-aspartic acid.

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.

Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein.

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.

Responses of Wheat (Triticum aestivum) Constitutively Expressing Four Different Monolignol Biosynthetic Genes to Fusarium Head Blight Caused by Fusarium graminearum.

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.

Phenylpropanoids Following Wounding and Infection of Sweet Sorghum Lines Differing in Responses to Stalk Pathogens.

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.

Deleterious mutations predicted in the sorghum (Sorghum bicolor) Maturity (Ma) and Dwarf (Dw) genes from whole-genome resequencing.

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.

Structural Similarities and Overlapping Activities among Dihydroflavonol 4-Reductase, Flavanone 4-Reductase, and Anthocyanidin Reductase Offer Metabolic Flexibility in the Flavonoid Pathway.

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.

Temporal transcriptomic profiling elucidates sorghum defense mechanisms against sugarcane aphids.

BACKGROUND: The sugarcane aphid (SCA; Melanaphis sacchari) has emerged as a key pest on sorghum in the United States that feeds from the phloem tissue, drains nutrients, and inflicts physical damage to plants. Previously, it has been shown that SCA reproduction was low and high on sorghum SC265 and SC1345 plants, respectively, compared to RTx430, an elite sorghum male parental line (reference line). In this study, we focused on identifying the defense-related genes that confer resistance to SCA at early and late time points in sorghum plants with varied levels of SCA resistance. RESULTS: We used RNA-sequencing approach to identify the global transcriptomic responses to aphid infestation on RTx430, SC265, and SC1345 plants at early time points 6, 24, and 48 h post infestation (hpi) and after extended period of SCA feeding for 7 days. Aphid feeding on the SCA-resistant line upregulated the expression of 3827 and 2076 genes at early and late time points, respectively, which was relatively higher compared to RTx430 and SC1345 plants. Co-expression network analysis revealed that aphid infestation modulates sorghum defenses by regulating genes corresponding to phenylpropanoid metabolic pathways, secondary metabolic process, oxidoreductase activity, phytohormones, sugar metabolism and cell wall-related genes. There were 187 genes that were highly expressed during the early time of aphid infestation in the SCA-resistant line, including genes encoding leucine-rich repeat (LRR) proteins, ethylene response factors, cell wall-related, pathogenesis-related proteins, and disease resistance-responsive dirigent-like proteins. At 7 days post infestation (dpi), 173 genes had elevated expression levels in the SCA-resistant line and were involved in sucrose metabolism, callose formation, phospholipid metabolism, and proteinase inhibitors. CONCLUSIONS: In summary, our results indicate that the SCA-resistant line is better adapted to activate early defense signaling mechanisms in response to SCA infestation because of the rapid activation of the defense mechanisms by regulating genes involved in monolignol biosynthesis pathway, oxidoreductase activity, biosynthesis of phytohormones, and cell wall composition. This study offers further insights to better understand sorghum defenses against aphid herbivory.

Effects of Altering Three Steps of Monolignol Biosynthesis on Sorghum Responses to Stalk Pathogens and Water Deficit.

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.

Divergent Metabolic Changes in Rhizomes of Lowland and Upland Switchgrass (Panicum virgatum) from Early Season through Dormancy Onset.

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.

Sugars and cuticular waxes impact sugarcane aphid (Melanaphis sacchari) colonization on different developmental stages of sorghum.

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.

Activity of Cytosolic Ascorbate Peroxidase (APX) from Panicum virgatum against Ascorbate and Phenylpropanoids.

APX is a key antioxidant enzyme in higher plants, scavenging H2O2 with ascorbate in several cellular compartments. Here, we report the crystal structures of cytosolic ascorbate peroxidase from switchgrass (Panicum virgatum L., Pvi), a strategic feedstock plant with several end uses. The overall structure of PviAPX was similar to the structures of other APX family members, with a bound ascorbate molecule at the ɣ-heme edge pocket as in other APXs. Our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Significantly, our study suggested that PviAPX can oxidize a broad range of phenylpropanoids with δ-meso site in a rather similar efficiency, which reflects its role in the fortification of cell walls in response to insect feeding. Based on detailed structural and kinetic analyses and molecular docking, as well as that of closely related APX enzymes, the critical residues in each substrate-binding site of PviAPX are proposed. Taken together, these observations shed new light on the function and catalysis of PviAPX, and potentially benefit efforts improve plant health and biomass quality in bioenergy and forage crops.

A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids.

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.

Sorghum cuticular waxes influence host plant selection by aphids.

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.

Dynamic regulation of phenylpropanoid pathway metabolites in modulating sorghum defense against fall armyworm.

Plants undergo dynamic metabolic changes at the cellular level upon insect infestation to better defend themselves. Phenylpropanoids, a hub of secondary plant metabolites, encompass a wide range of compounds that can contribute to insect resistance. Here, the role of sorghum (Sorghum bicolor) phenylpropanoids in providing defense against the chewing herbivore, fall armyworm (FAW), Spodoptera frugiperda, was explored. We screened a panel of nested association mapping (NAM) founder lines against FAW and identified SC1345 and Ajabsido as most resistant and susceptible lines to FAW, respectively, compared to reference parent, RTx430. Gene expression and metabolomic studies suggested that FAW feeding suppressed the expression level of genes involved in monolignol biosynthetic pathway and their associated phenolic intermediates at 10 days post infestation. Further, SC1345 genotype displayed elevated levels of flavonoid compounds after FAW feeding for 10 days, suggesting a diversion of precursors from lignin biosynthesis to the flavonoid pathway. Additionally, bioassays with sorghum lines having altered levels of flavonoids provided genetic evidence that flavonoids are crucial in providing resistance against FAW. Finally, the application of FAW regurgitant elevated the expression of genes associated with the flavonoid pathway in the FAW-resistant SC1345 genotype. Overall, our study indicates that a dynamic regulation of the phenylpropanoid pathway in sorghum plants imparts resistance against FAW.

Genetic analysis of seed traits in Sorghum bicolor that affect the human gut microbiome.

Prebiotic fibers, polyphenols and other molecular components of food crops significantly affect the composition and function of the human gut microbiome and human health. The abundance of these, frequently uncharacterized, microbiome-active components vary within individual crop species. Here, we employ high throughput in vitro fermentations of pre-digested grain using a human microbiome to identify segregating genetic loci in a food crop, sorghum, that alter the composition and function of human gut microbes. Evaluating grain produced by 294 sorghum recombinant inbreds identifies 10 loci in the sorghum genome associated with variation in the abundance of microbial taxa and/or microbial metabolites. Two loci co-localize with sorghum genes regulating the biosynthesis of condensed tannins. We validate that condensed tannins stimulate the growth of microbes associated with these two loci. Our work illustrates the potential for genetic analysis to systematically discover and characterize molecular components of food crops that influence the human gut microbiome.

Reprogramming of sorghum proteome in response to sugarcane aphid infestation.

Sugarcane aphid (SCA; Melanaphis sacchari Zehntner) is a key piercing-sucking pest of sorghum (Sorghum bicolor) that cause significant yield losses. While feeding on host plants, complex signaling networks are invoked from recognition of insect attack to induction of plant defenses. Consequently, these signaling networks lead to the production of insecticidal compounds or limited access of nutrients to insects. Previously, several studies were published on the transcriptomics analysis of sorghum in response to SCA infestation, but no information is available on the physiological changes of sorghum at the proteome level. We used the SCA resistant sorghum genotype SC265 for the global proteomics analysis after 1 and 7 days of SCA infestation using the TMT-plex technique. Peptides matching a total of 4211 proteins were identified and 158 proteins were differentially expressed at day 1 and 7. Overall, proteome profiling of SC265 after SCA infestation at days 1 and 7 revealed the suppression of plant defense-related proteins and upregulation of plant defense and signaling-related proteins, respectively. The plant defense responses based on proteome data were validated using electrical penetration graph (EPG) technique to observe changes in aphid feeding. Feeding behavior analyses revealed that SCA spent significantly longer time in phloem phase on SCA infested plants for day 1 and lesser time in day 7 SCA infested sorghum plants, compared to their respective control plants. Overall, our study provides insights into underlying mechanisms that contribute to sorghum resistance to SCA.

Dichotomous Role of Jasmonic Acid in Modulating Sorghum Defense Against Aphids.

The precursors and derivatives of jasmonic acid (JA) contribute to plant protective immunity to insect attack. However, the role of JA in sorghum (Sorghum bicolor) defense against sugarcane aphid (SCA) (Melanaphis sacchari), which is considered a major threat to sorghum production, remains elusive. Sorghum SC265, previously identified as a SCA-resistant genotype among the sorghum nested association mapping founder lines, transiently increased JA at early stages of aphid feeding and deterred aphid settling. Monitoring of aphid feeding behavior using electropenetrography, a technique to unveil feeding process of piercing-sucking insects, revealed that SC265 plants restricted SCA feeding from the phloem sap. However, exogenous application of JA attenuated the resistant phenotype and promoted improved aphid feeding and colonization on SC265 plants. This was further confirmed with sorghum JA-deficient plants, in which JA deficiency promoted aphid settling, however, it also reduced aphid feeding from the phloem sap and curtailed SCA population. Exogenous application of JA caused enhanced feeding and aphid proliferation on JA-deficient plants, suggesting that JA promotes aphid growth and development. SCA feeding on JA-deficient plants altered the sugar metabolism and enhanced the levels of fructose and trehalose compared with wild-type plants. Furthermore, aphid artificial diet containing fructose and trehalose curtailed aphid growth and reproduction. Our findings underscore a previously unknown dichotomous role of JA, which may have opposing effects by deterring aphid settling during the early stage and enhancing aphid proliferative capacity during later stages of aphid colonization on sorghum plants. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Functional and structural insight into the flexibility of cytochrome P450 reductases from Sorghum bicolor and its implications for lignin composition.

Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3'-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin. Therefore, to understand the structure and function of three CPR subunits from sorghum, recombinant subunits SbCPR2a, SbCPR2b, and SbCPR2c were subjected to X-ray crystallography and kinetic assays. Steady-state kinetic analyses demonstrated that all three CPR subunits supported the oxidation reactions catalyzed by SbC4H1 (CYP73A33) and SbC3'H (CYP98A1). Furthermore, comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance and suggested that the plant flavin mononucleotide-binding domain might be more flexible than mammalian homologs. In addition, the elucidated structure of SbCPR2b included the first observation of NADP+ in a native CPR. Overall, we conclude that the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage sorghums through protein engineering.