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.

Dynamic Reconfiguration of Switchgrass Proteomes in Response to Rust (Puccinia novopanici) Infection.

Switchgrass (Panicum virgatum L.) can be infected by the rust pathogen (Puccinia novopanici) and results in lowering biomass yields and quality. Label-free quantitative proteomics was conducted on leaf extracts harvested from non-infected and infected plants from a susceptible cultivar (Summer) at 7, 11, and 18 days after inoculation (DAI) to follow the progression of disease and evaluate any plant compensatory mechanisms to infection. Some pustules were evident at 7 DAI, and their numbers increased with time. However, fungal DNA loads did not appreciably change over the course of this experiment in the infected plants. In total, 3830 proteins were identified at 1% false discovery rate, with 3632 mapped to the switchgrass proteome and 198 proteins mapped to different Puccinia proteomes. Across all comparisons, 1825 differentially accumulated switchgrass proteins were identified and subjected to a STRING analysis using Arabidopsis (A. thaliana L.) orthologs to deduce switchgrass cellular pathways impacted by rust infection. Proteins associated with plastid functions and primary metabolism were diminished in infected Summer plants at all harvest dates, whereas proteins associated with immunity, chaperone functions, and phenylpropanoid biosynthesis were significantly enriched. At 18 DAI, 1105 and 151 proteins were significantly enriched or diminished, respectively. Many of the enriched proteins were associated with mitigation of cellular stress and defense.

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.

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.

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.

Co-Transcriptomic Analysis of the Maize-Western Corn Rootworm Interaction.

The Western corn rootworm (WCR; Diabrotica virgifera virgifera) is an economically important belowground pest of maize. Belowground feeding by WCR is damaging because it weakens the roots system, diminishes nutrient uptake, and creates entry points for fungal and bacterial pathogens and increases lodging, all of which can significantly suppress maize yields. Previously, it was demonstrated that belowground herbivory can trigger plant defense responses in the roots and the shoots, thereby impacting intraplant communication. Although several aspects of maize-WCR interactions have been reported, co-transcriptomic remodeling in the plant and insect are yet to be explored. We used a maize genotype, Mp708, that is resistant to a large guild of herbivore pests to study the underlying plant defense signaling network between below and aboveground tissues. We also evaluated WCR compensatory transcriptome responses. Using RNA-seq, we profiled the transcriptome of roots and leaves that interacted with WCR infestation up to 5 days post infestation (dpi). Our results suggest that Mp708 shoots and roots had elevated constitutive and WCR-feeding induced expression of genes related to jasmonic acid and ethylene pathways, respectively, before and after WCR feeding for 1 and 5 days. Similarly, extended feeding by WCR for 5 days in Mp708 roots suppressed many genes involved in the benzoxazinoid pathway, which is a major group of indole-derived secondary metabolites that provides resistance to several insect pests in maize. Furthermore, extended feeding by WCR on Mp708 roots revealed several genes that were downregulated in WCR, which include genes related to proteolysis, neuropeptide signaling pathway, defense response, drug catabolic process, and hormone metabolic process. These findings indicate a dynamic transcriptomic dialog between WCR and WCR-infested maize plants.

Effect of cultivar and temperature on the synergistic interaction between panicum mosaic virus and satellite panicum mosaic virus in switchgrass.

Panicum mosaic virus (PMV), the type member of the genus Panicovirus in the family Tombusviridae, naturally infects switchgrass (Panicum virgatum L.). PMV and its molecular partner, satellite panicum mosaic virus (SPMV), interact synergistically in coinfected millets to exacerbate the disease phenotype and increase the accumulation of PMV compared to plants infected with PMV alone. In this study, we examined the reaction of switchgrass cvs. Summer and Kanlow to PMV and PMV+SPMV infections at 24°C and 32°C. Switchgrass cv. Summer was susceptible to PMV at both temperatures. In contrast, cv. Kanlow was tolerant to PMV at 24°C, but not at 32°C, suggesting that Kanlow harbors temperature-sensitive resistance to PMV. At 24°C, PMV was readily detected in inoculated leaves, but not in upper uninoculated leaves of Kanlow, suggesting that resistance to PMV was likely mediated by abrogation of long-distance virus transport. Coinfection by PMV and SPMV at 24°C and 32°C in cv. Summer, but not in Kanlow, caused increased symptomatic systemic infection and mild disease synergism with slightly increased PMV accumulation compared to plants infected with PMV alone. These data suggest that the interaction between PMV and SPMV in switchgrass is cultivar-dependent, manifested in Summer but not in Kanlow. However, co-inoculation of cv. Kanlow with PMV+SPMV caused an enhanced asymptomatic infection, suggesting a role of SPMV in enhancement of symptomless infection in a tolerant cultivar. These data suggest that enhanced asymptomatic infections in a virus-tolerant switchgrass cultivar could serve as a source of virus spread and play an important role in panicum mosaic disease epidemiology under field conditions. Our data reveal that the cultivar, coinfection with SPMV, and temperature influence the severity of symptoms elicited by PMV in switchgrass.

Interplay of phytohormones facilitate sorghum tolerance to aphids.

KEY MESSAGE: Interactions among phytohormones are essential for providing tolerance of sorghum plants to aphids. Plant's encounter with insect herbivores trigger defense signaling networks that fine-tune plant resistance to insect pests. Although it is well established that phytohormones contribute to antixenotic- and antibiotic-mediated resistance to insect pests, their role in conditioning plant tolerance, the most durable and promising category of host plant resistance, is largely unknown. Here, we screened a panel of sorghum (Sorghum bicolor) inbred lines to identify and characterize sorghum tolerance to sugarcane aphids (SCA; Melanaphis sacchari Zehntner), a relatively new and devastating pest of sorghum in the United States. Our results suggest that the sorghum genotype SC35, the aphid-tolerant line identified among the sorghum genotypes, displayed minimal plant biomass loss and a robust photosynthetic machinery, despite supporting higher aphid population. Phytohormone analysis revealed significantly higher basal levels of 12-oxo-phytodienoic acid, a precursor in the jasmonic acid biosynthesis pathway, in the sorghum SCA-tolerant SC35 plants. Salicylic acid accumulation appeared as a generalized plant response to aphids in sorghum plants, however, SCA feeding-induced salicylic acid levels were unaltered in the sorghum tolerant genotype. Conversely, basal levels of abscisic acid and aphid feeding-induced cytokinins were accumulated in the SCA-tolerant sorghum genotype. Our findings imply that the aphid-tolerant sorghum genotype tightly controls the relationship among phytohormones, as well as provide significant insights into the underlying mechanisms that contribute to plant tolerance to sap-sucking aphids.

Transcriptomic and volatile signatures associated with maize defense against corn leaf aphid.

BACKGROUND: Maize (Zea mays L.) is a major cereal crop, with the United States accounting for over 40% of the worldwide production. Corn leaf aphid [CLA; Rhopalosiphum maidis (Fitch)] is an economically important pest of maize and several other monocot crops. In addition to feeding damage, CLA acts as a vector for viruses that cause devastating diseases in maize. We have shown previously that the maize inbred line Mp708, which was developed by classical plant breeding, provides heightened resistance to CLA. However, the transcriptomic variation conferring CLA resistance to Mp708 has not been investigated. RESULTS: In this study, we contrasted the defense responses of the resistant Mp708 genotype to those of the susceptible Tx601 genotype at the transcriptomic (mRNA-seq) and volatile blend levels. Our results suggest that there was a greater transcriptomic remodeling in Mp708 plants in response to CLA infestation compared to the Tx601 plants. These transcriptomic signatures indicated an activation of hormonal pathways, and regulation of sesquiterpenes and terpenoid synthases in a constitutive and inducible manner. Transcriptomic analysis also revealed that the resistant Mp708 genotype possessed distinct regulation of ethylene and jasmonic acid pathways before and after aphid infestation. Finally, our results also highlight the significance of constitutive production of volatile organic compounds (VOCs) in Mp708 and Tx601 plants that may contribute to maize direct and/or indirect defense responses. CONCLUSIONS: This study provided further insights to understand the role of defense signaling networks in Mp708's resistance to CLA.

Differential Defense Responses of Upland and Lowland Switchgrass Cultivars to a Cereal Aphid Pest.

Yellow sugarcane aphid (YSA) (Sipha flava, Forbes) is a damaging pest on many grasses. Switchgrass (Panicum virgatum L.), a perennial C4 grass, has been selected as a bioenergy feedstock because of its perceived resilience to abiotic and biotic stresses. Aphid infestation on switchgrass has the potential to reduce the yields and biomass quantity. Here, the global defense response of switchgrass cultivars Summer and Kanlow to YSA feeding was analyzed by RNA-seq and metabolite analysis at 5, 10, and 15 days after infestation. Genes upregulated by infestation were more common in both cultivars compared to downregulated genes. In total, a higher number of differentially expressed genes (DEGs) were found in the YSA susceptible cultivar (Summer), and fewer DEGs were observed in the YSA resistant cultivar (Kanlow). Interestingly, no downregulated genes were found in common between each time point or between the two switchgrass cultivars. Gene co-expression analysis revealed upregulated genes in Kanlow were associated with functions such as flavonoid, oxidation-response to chemical, or wax composition. Downregulated genes for the cultivar Summer were found in co-expression modules with gene functions related to plant defense mechanisms or cell wall composition. Global analysis of defense networks of the two cultivars uncovered differential mechanisms associated with resistance or susceptibility of switchgrass in response to YSA infestation. Several gene co-expression modules and transcription factors correlated with these differential defense responses. Overall, the YSA-resistant Kanlow plants have an enhanced defense even under aphid uninfested conditions.

Greenbug (Schizaphis graminum) herbivory significantly impacts protein and phosphorylation abundance in switchgrass (Panicum virgatum).

Switchgrass (Panicum virgatum L.) is an important crop for biofuel production but it also serves as host for greenbugs (Schizaphis graminum Rondani; GB). Although transcriptomic studies have been done to infer the molecular mechanisms of plant defense against GB, little is known about the effect of GB infestation on the switchgrass protein expression and phosphorylation regulation. The global response of the switchgrass cultivar Summer proteome and phosphoproteome was monitored by label-free proteomics shotgun in GB-infested and uninfested control plants at 10 days post infestation. Peptides matching a total of 3,594 proteins were identified and 429 were differentially expressed proteins in GB-infested plants relative to uninfested control plants. Among these, 291 and 138 were up and downregulated by GB infestation, respectively. Phosphoproteome analysis identified 310 differentially phosphorylated proteins (DP) from 350 phosphopeptides with a total of 399 phosphorylated sites. These phosphopeptides had more serine phosphorylated residues (79%), compared to threonine phosphorylated sites (21%). Overall, KEGG pathway analysis revealed that GB feeding led to the enriched accumulation of proteins important for biosynthesis of plant defense secondary metabolites and repressed the accumulation of proteins involved in photosynthesis. Interestingly, defense modulators such as terpene synthase, papain-like cysteine protease, serine carboxypeptidase, and lipoxygenase2 were upregulated at the proteome level, corroborating previously published transcriptomic data.

Aphid-Responsive Defense Networks in Hybrid Switchgrass.

Aphid herbivory elicits plant defense-related networks that are influenced by host genetics. Plants of the upland switchgrass (Panicum virgatum) cultivar Summer can be a suitable host for greenbug aphids (Schizaphis graminum; GB), and yellow sugarcane aphids (Sipha flava, YSA), whereas the lowland cultivar Kanlow exhibited multi-species resistance that curtails aphid reproduction. However, stabilized hybrids of Summer (♀) x Kanlow (♂) (SxK) with improved agronomics can be damaged by both aphids. Here, hormone and metabolite analyses, coupled with RNA-Seq analysis of plant transcriptomes, were utilized to delineate defense networks induced by aphid feeding in SxK switchgrass and pinpoint plant transcription factors (TFs), such as WRKYs that potentially regulate these responses. Abscisic acid (ABA) levels were significantly higher in GB infested plants at 5 and 10 days after infestation (DAI). ABA levels were highest at 15DAI in YSA infested plants. Jasmonic acid levels were significantly elevated under GB infestation, while salicylic acid levels were signifi40cantly elevated only at 15 DAI in YSA infested plants. Similarly, levels of several metabolites were altered in common or specifically to each aphid. YSA infestation induced a significant enrichment of flavonoids consistent with an upregulation of many genes associated with flavonoid biosynthesis at 15DAI. Gene co-expression modules that responded singly to either aphid or in common to both aphids were differentiated and linked to specific TFs. Together, these data provide important clues into the interplay of metabolism and transcriptional remodeling accompanying defense responses to aphid herbivory in hybrid switchgrass.

Enhanced metabolism and selection of pyrethroid-resistant western corn rootworms (Diabrotica virgifera virgifera LeConte).

Western corn rootworm (WCR) pyrethroid resistance has been previously reported in the United States (US) western Corn Belt, and cross-resistance and synergism studies suggested that both target site insensitivity and enhanced metabolism may be conferring WCR resistance to pyrethroids. The present study aimed to investigate the potential mechanisms of WCR pyrethroid resistance and to estimate the heritability of the resistance trait. Biochemical assays using model substrates and spectrophotometry revealed 2-4-fold higher activity of P450s and esterases in pyrethroid-resistant WCR populations, whereas the biological activity of glutathione S-transferase was similar between populations tested. No mutation in the voltage-gated sodium channel was detected in pyrethroid-resistant WCR individuals by sequencing PCR products containing the para-homologous L1014, T929, and M918 amino acid positions that are commonly associated with target site mutations in other pyrethroid-resistant insects. A pilot estimation of pyrethroid resistance heritability obtained during laboratory selection of a WCR population suggested a major genetic component of the resistance trait and predicted a 10-fold increase in WCR bifenthrin resistance within ~7 generations of insecticide lethal exposure. Results support earlier indirect evidence that enhanced metabolism may be contributing to WCR resistance to pyrethroids and illustrates the potential of WCR pyrethroid resistance evolution.

Overexpression of ferulate 5-hydroxylase increases syringyl units in Sorghum bicolor.

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.

Plant hemoglobins: a journey from unicellular green algae to vascular plants.

Globins (Glbs) are widely distributed in archaea, bacteria and eukaryotes. They can be classified into proteins with 2/2 or 3/3 α-helical folding around the heme cavity. Both types of Glbs occur in green algae, bryophytes and vascular plants. The Glbs of angiosperms have been more intensively studied, and several protein structures have been solved. They can be hexacoordinate or pentacoordinate, depending on whether a histidine is coordinating or not at the sixth position of the iron atom. The 3/3 Glbs of class 1 and the 2/2 Glbs (also called class 3 in plants) are present in all angiosperms, whereas the 3/3 Glbs of class 2 have been only found in early angiosperms and eudicots. The three Glb classes are expected to play different roles. Class 1 Glbs are involved in hypoxia responses and modulate NO concentration, which may explain their roles in plant morphogenesis, hormone signaling, cell fate determination, nutrient deficiency, nitrogen metabolism and plant-microorganism symbioses. Symbiotic Glbs derive from class 1 or class 2 Glbs and transport O2 in nodules. The physiological roles of class 2 and class 3 Glbs are poorly defined but could involve O2 and NO transport and/or metabolism, respectively. More research is warranted on these intriguing proteins to determine their non-redundant functions.

Transcriptome divergence during leaf development in two contrasting switchgrass (Panicum virgatum L.) cultivars.

The genetics and responses to biotic stressors of tetraploid switchgrass (Panicum virgatum L.) lowland cultivar 'Kanlow' and upland cultivar Summer are distinct and can be exploited for trait improvement. In general, there is a paucity of data on the basal differences in transcription across tissue developmental times for switchgrass cultivars. Here, the changes in basal and temporal expression of genes related to leaf functions were evaluated for greenhouse grown 'Kanlow', and 'Summer' plants. Three biological replicates of the 4th leaf pooled from 15 plants per replicate were harvested at regular intervals beginning from leaf emergence through senescence. Increases and decreases in leaf chlorophyll and N content were similar for both cultivars. Likewise, multidimensional scaling (MDS) analysis indicated both cultivar-independent and cultivar-specific gene expression. Cultivar-independent genes and gene-networks included those associated with leaf function, such as growth/senescence, carbon/nitrogen assimilation, photosynthesis, chlorophyll biosynthesis, and chlorophyll degradation. However, many genes encoding nucleotide-binding leucine rich repeat (NB-LRRs) proteins and wall-bound kinases associated with detecting and responding to environmental signals were differentially expressed. Several of these belonged to unique cultivar-specific gene co-expression networks. Analysis of genomic resequencing data provided several examples of NB-LRRs genes that were not expressed and/or apparently absent in the genomes of Summer plants. It is plausible that cultivar (ecotype)-specific genes and gene-networks could be one of the drivers for the documented differences in responses to leaf-borne pathogens between these two cultivars. Incorporating broad resistance to plant pathogens in elite switchgrass germplasm could improve sustainability of biomass production under low-input conditions.

Fall armyworm (Spodoptera frugiperda Smith) feeding elicits differential defense responses in upland and lowland switchgrass.

Switchgrass (Panicum virgatum L.) is a low input, high biomass perennial grass being developed for the bioenergy sector. Upland and lowland cultivars can differ in their responses to insect herbivory. Fall armyworm [FAW; Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae)] is a generalist pest of many plant species and can feed on switchgrass as well. Here, in two different trials, FAW larval mass were significantly reduced when fed on lowland cultivar Kanlow relative to larvae fed on upland cultivar Summer plants after 10 days. Hormone content of plants indicated elevated levels of the plant defense hormone jasmonic acid (JA) and its bioactive conjugate JA-Ile although significant differences were not observed. Conversely, the precursor to JA, 12-oxo-phytodienoic acid (OPDA) levels were significantly different between FAW fed Summer and Kanlow plants raising the possibility of differential signaling by OPDA in the two cultivars. Global transcriptome analysis revealed a stronger response in Kanlow plant relative to Summer plants. Among these changes were a preferential upregulation of several branches of terpenoid and phenylpropanoid biosynthesis in Kanlow plants suggesting that enhanced biosynthesis or accumulation of antifeedants could have negatively impacted FAW larval mass gain on Kanlow plants relative to Summer plants. A comparison of the switchgrass-FAW RNA-Seq dataset to those from maize-FAW and switchgrass-aphid interactions revealed that key components of plant responses to herbivory, including induction of JA biosynthesis, key transcription factors and JA-inducible genes were apparently conserved in switchgrass and maize. In addition, these data affirm earlier studies with FAW and aphids that the cultivar Kanlow can provide useful genetics for the breeding of switchgrass germplasm with improved insect resistance.

Wheat streak mosaic virus alters the transcriptome of its vector, wheat curl mite (Aceria tosichella Keifer), to enhance mite development and population expansion.

Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae) is an economically important wheat virus that is transmitted by the wheat curl mite (WCM; Aceria tosichella Keifer) in a persistent manner. Virus-vector coevolution may potentially influence vector gene expression to prolong viral association and thus increase virus transmission efficiency and spread. To understand the transcriptomic responses of WCM to WSMV, RNA sequencing was performed to assemble and analyse transcriptomes of WSMV viruliferous and aviruliferous mites. Among 7291 de novo-assembled unigenes, 1020 were differentially expressed between viruliferous and aviruliferous WCMs using edgeR at a false discovery rate ≤0.05. Differentially expressed unigenes were enriched for 108 gene ontology terms, with the majority of the unigenes showing downregulation in viruliferous mites in comparison to only a few unigenes that were upregulated. Protein family and metabolic pathway enrichment analyses revealed that most downregulated unigenes encoded enzymes and proteins linked to stress response, immunity and development. Mechanistically, these predicted changes in mite physiology induced by viral association could be suggestive of pathways needed for promoting virus-vector interactions. Overall, our data suggest that transcriptional changes in viruliferous mites facilitate prolonged viral association and alter WCM development to expedite population expansion, both of which could enhance viral transmission.