Transcriptomic and physiological responses of Quercus acutissima and Quercus palustris to drought stress and rewatering.

Establishment of oak seedlings, which is an important factor in forest restoration, is affected by drought that hampers the survival, growth, and development of seedlings. Therefore, it is necessary to understand how seedlings respond to and recover from water-shortage stress. We subjected seedlings of two oak species, Quercus acutissima and Quercus palustris, to drought stress for one month and then rewatered them for six days to observe physiological and genetic expression changes. Phenotypically, the growth of Q. acutissima was reduced and severe wilting and recovery failure were observed in Q. palustris after an increase in plant temperature. The two species differed in several physiological parameters during drought stress and recovery. Although the photosynthesis-related indicators did not change in Q. acutissima, they were decreased in Q. palustris. Moreover, during drought, content of soluble sugars was significantly increased in both species, but it recovered to original levels only in Q. acutissima. Malondialdehyde content increased in both the species during drought, but it did not recover in Q. palustris after rewatering. Among the antioxidant enzymes, only superoxide dismutase activity increased in Q. acutissima during drought, whereas activities of ascorbate peroxidase, catalase, and glutathione reductase increased in Q. palustris. Abscisic acid levels were increased and then maintained in Q. acutissima, but recovered to previous levels after rewatering in Q. palustris. RNA samples from the control, drought, recovery day 1, and recovery day 6 treatment groups were compared using transcriptome analysis. Q. acutissima exhibited 832 and 1076 differentially expressed genes (DEGs) related to drought response and recovery, respectively, whereas Q. palustris exhibited 3947 and 1587 DEGs, respectively under these conditions. Gene ontology enrichment of DEGs revealed "response to water," "apoplast," and "Protein self-association" to be common to both the species. However, in the heatmap analysis of genes related to sucrose and starch synthesis, glycolysis, antioxidants, and hormones, the two species exhibited very different transcriptome responses. Nevertheless, the levels of most DEGs returned to their pre-drought levels after rewatering. These results provide a basic foundation for understanding the physiological and genetic expression responses of oak seedlings to drought stress and recovery.

Genome-Wide Characterization of Glyceraldehyde-3-Phosphate Dehydrogenase Genes and Their Expression Profile under Drought Stress in Quercus rubra.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is crucial in plant metabolism and responses to various abiotic stresses. In the glycolysis pathway, glyceraldehyde-3-phosphate (G3P) is oxidized to 1,3-bisphosphate glycerate (1,3-BPG) through the catalytic action of GAPDH. However, the GAPDH gene family in Quercus rubra has been minimally researched. In this study, we identified 13 GAPDH-encoding genes in Q. rubra through a bioinformatics analysis of genomic data. Evolutionary studies suggest that these QrGAPDH genes are closely related to those in Glycine max and Triticum aestivum. We conducted a comprehensive whole-genome study, which included predictions of subcellular localization, gene structure analysis, protein motif identification, chromosomal placement, and analysis of cis-acting regions. We also examined the expression of GAPDH proteins and genes in various tissues of Q. rubra and under drought stress. The results indicated diverse expression patterns across different tissues and differential expression under drought conditions. Notably, the expression of Qurub.02G290300.1, Qurub.10G209800.1, and Qrub.M241600.1 significantly increased in the leaf, stem, and root tissues under drought stress. This study provides a systematic analysis of QrGAPDH genes, suggesting their pivotal roles in the drought stress response of trees.

Genome-wide identification of the PFK gene family and their expression analysis in Quercus rubra.

The glycolytic pathway involves phosphofructokinase (PFK), a rate-limiting enzyme that catalyzes the phosphorylation of fructose-6-phosphate. In plants, the two PFK members are ATP-dependent phosphofructokinase (PFK) and pyrophosphate-fructose-6-phosphate phosphotransferase (PFP). However, the functions of the PFK family members in Quercus rubra are not well understood. The purpose of this study was to investigate the genome-wide distribution of the PFK family members and their roles in Q. rubra by performing a systematic study of the phylogenetic relationships, molecular characteristics, motifs, chromosomal and subcellular locations, and cis-elements of QrPFKs. We identified 14 QrPFK genes in the genome of Q. rubra, followed by examining their expression in different tissues, including the roots, stems, and leaves. The phylogenetic tree divided the 14 QrPFK genes into two groups: 11 belonging to PFK and three belonging to PFP. The expression profiles of all 14 proteins were relatively the same in leaves but differed between stems and roots. Four genes (Qurub.02G189400.1, Qurub.02G189400.2, Qurub.09G134300.1, and Qurub.09G134300.2) were expressed at very low levels in both stems and roots, while two (Qurub.05G235500.1 and Qurub.05G235500.1) were expressed at low levels and the others showed relatively high expression in all tissues.

Transcriptomic and Physiological Analysis Reveals Genes Associated with Drought Stress Responses in Populus alba × Populus glandulosa.

Drought stress affects plant productivity by altering plant responses at the morphological, physiological, and molecular levels. In this study, we identified physiological and genetic responses in Populus alba × Populus glandulosa hybrid clones 72-30 and 72-31 after 12 days of exposure to drought treatment. After 12 days of drought treatment, glucose, fructose, and sucrose levels were significantly increased in clone 72-30 under drought stress. The Fv/Fo and Fv/Fm values in both clones also decreased under drought stress. The changes in proline, malondialdehyde, and H2O2 levels were significant and more pronounced in clone 72-30 than in clone 72-31. The activities of antioxidant-related enzymes, such as catalase and ascorbate peroxidase, were significantly higher in the 72-31 clone. To identify drought-related genes, we conducted a transcriptomic analysis in P. alba × P. glandulosa leaves exposed to drought stress. We found 883 up-regulated and 305 down-regulated genes in the 72-30 clone and 279 and 303 in the 72-31 clone, respectively. These differentially expressed genes were mainly in synthetic pathways related to proline, abscisic acid, and antioxidants. Overall, clone 72-31 showed better drought tolerance than clone 72-30 under drought stress, and genetic changes also showed different patterns.

Developmental Transcriptome Analysis of Red-Spotted Apollo Butterfly, Parnassius bremeri.

Parnassius bremeri (P. bremeri), a member of the genus Snow Apollo in the swallowtail family (Papilionidae), is a high alpine butterfly that lives in Russia, Korea, and China. It is an endangered wildlife (Class I) in South Korea and is a globally endangered species. The lack of transcriptomic and genomic resources of P. bremeri significantly hinders the study of its population genetics and conservation. The detailed information of the developmental stage-specific gene expression patterns of P. bremeri is of great demand for its conservation. However, the molecular mechanism underlying the metamorphic development of P. bremeri is still unknown. In the present study, the differentially expressed genes (DEGs) across the metamorphic developmental stages were compared using high-throughput transcriptome sequencing. We identified a total of 72,161 DEGs from eight comparisons. GO enrichment analysis showed that a range of DEGs were responsible for cuticle development and the melanin biosynthetic pathway during larval development. Pathway analysis suggested that the signaling pathways, such as the Wnt signaling pathway, hedgehog signaling pathway and Notch signaling pathway, are regulated during the developmental stages of P. bremeri. Furthermore, sensory receptors were also activated, especially during the larval to adult transition stage. Collectively, the results of this study provide a preliminary foundation and understanding of the molecular mechanism in their transcriptomes for further research on the metamorphic development of P. bremeri.

Rosmarinus officinalis L. Leaf Extracts and Their Metabolites Inhibit the Aryl Hydrocarbon Receptor (AhR) Activation In Vitro and in Human Keratinocytes: Potential Impact on Inflammatory Skin Diseases and Skin Cancer.

Aryl hydrocarbon receptor (AhR) activation by environmental agents and microbial metabolites is potentially implicated in a series of skin diseases. Hence, it would be very important to identify natural compounds that could inhibit the AhR activation by ligands of microbial origin as 6-formylindolo[3,2-b]carbazole (FICZ), indirubin (IND) and pityriazepin (PZ) or the prototype ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five different dry Rosmarinus officinalis L. extracts (ROEs) were assayed for their activities as antagonists of AhR ligand binding with guinea pig cytosol in the presence of [3H]TCDD. The methanolic ROE was further assayed towards CYP1A1 mRNA induction using RT-PCR in human keratinocytes against TCDD, FICZ, PZ, and IND. The isolated metabolites, carnosic acid, carnosol, 7-O-methyl-epi-rosmanol, 4',7-O-dimethylapigenin, and betulinic acid, were assayed for their agonist and antagonist activity in the presence and absence of TCDD using the gel retardation assay (GRA). All assayed ROE extracts showed similar dose-dependent activities with almost complete inhibition of AhR activation by TCDD at 100 ppm. The methanol ROE at 10 ppm showed 99%, 50%, 90%, and 85% inhibition against TCDD, FICZ, IND, and PZ, respectively, in human keratinocytes. Most assayed metabolites exhibited dose-dependent antagonist activity. ROEs inhibit AhR activation by TCDD and by the Malassezia metabolites FICZ, PZ, and IND. Hence, ROE could be useful for the prevention or treatment of skin diseases mediated by activation of AhR.

Assessing the estrogenic activity of chemicals present in resin based dental composites and in leachates of commercially available composites using the ERα-CALUX bioassay.

OBJECTIVE: The biocompatibility of resin based dental composites has not yet been fully characterized even though certain monomers used in these composites are synthesized from Bisphenol A (BPA), a well-known estrogenic endocrine disruptor. As a result, they show structural relationship to BPA and can contain it as an impurity. Therefore, the estrogenic activity of 9 monomers, 2 photoinitiators, one photostabilizer and leachates of 4 commercially available composites was determined. METHODS: The ERα-CALUX bioassay was used to determine both agonistic and antagonistic estrogenic activities of the pure compounds (BPA, BisDMA, BisGMA, BisEMA(3), BisEMA(6), BisEMA(10), TEGDMA, TCD-DI-HEA, BADGE, UDMA, HMBP, DMPA, CQ) and the leachates of cured composite disks. The leachates of 4 commercially available composites (Solitaire 2, Ceram.x Spectra ST, G-ænial Posterior and Filtek Supreme XTE) in water and 0.1 M NaOH (pH = 13, 'worst-case scenario') were tested for estrogenic activity (pooled leachates from 10 cured composite disks). RESULTS: Agonistic estrogenic activity was found for the monomer BisDMA, the photostabilizer HMBP and photoinitiator DMPA. All leachates from the 4 tested composites showed significant agonistic estrogenic activity higher than the DMSO control, and the highest activity (potency and efficacy) was found for Solitaire 2, followed by Ceram.x Spectra ST. Furthermore, antagonistic estrogenic activity was found in the leachates from G-ænial Posterior. SIGNIFICANCE: These results show that significant estrogenic activity was found in all leachates of the cured composite disks, and that this estrogenicity is most likely due to a mixture effect of multiple estrogenic compounds (including BPA, HMBP and DMPA). This indicates that further research into the endocrine activity of all the compounds that are present in these composites (even at low quantities) and their possible mixture effect is warranted to guarantee their safe use.

Woodsmoke Extracts Cross-Link Proteins and Induce Cornified Envelope Formation without Stimulating Keratinocyte Terminal Differentiation.

Air pollution poses a serious risk to human health. To help understand the contribution of smoke from wood burning to the harmfulness of air pollution toward the skin, we studied the effects of liquid smoke, aqueous extracts of wood smoke condensate, a commercially available food flavor additive, in cultured keratinocytes. We report that liquid smoke can react with and cross-link keratinocyte cellular proteins, leading to abnormal cross-linked envelope formation. Instead of inducing genes ordinarily involved in terminal differentiation, liquid smoke induced expression of genes associated with stress responses. When transglutaminase activity was inhibited, liquid smoke still promoted protein cross-linking and envelope formation in keratinocytes. This phenomenon likely results from oxidative stress and protein adducts from aldehydes as either preloading the cells with N-acetylcysteine or reducing the aldehyde content of liquid smoke decreased its ability to promote protein cross-linking and envelope formation. Finally, liquid smoke-induced envelopes were found to have elevated protein content, suggesting oxidative cross-linking and formation of protein adducts might impair barrier function by inducing abnormal incorporation of cellular proteins into envelopes. Since the cross-linked protein envelope provides structural stability to the stratum corneum and serves as a scaffold for the organization of the corneocyte lipid envelope (hydrophobic barrier to the environment), these findings provide new insight into the mechanism by which pro-oxidative air pollutants can impair epidermal function.

Using Estrogenic Activity and Nontargeted Chemical Analysis to Identify Contaminants in Sewage Sludge.

Diverse organic compounds, many derived from consumer products, are found in sewage sludge worldwide. Understanding which of these poses the most significant environmental threat following land application can be investigated through a variety of predictive and cell-based toxicological techniques. Nontargeted analysis using high-resolution mass spectrometry with predictive estrogenic activity modeling was performed on sewage sludge samples from 12 wastewater treatment plants in California. Diisobutyl phthalate and dextrorphan were predicted to exhibit estrogenic activity and identified in >75% of sludge samples, signifying their universal presence and persistence. Additionally, the application of an estrogen-responsive cell bioassay revealed reductions in agonistic activity during mesophilic and thermophilic treatment but significant increases in antagonism during thermophilic treatment, which warrants further research. Ten nontarget features were identified (metoprolol, fenofibric acid, erythrohydrobupropion, oleic acid, mestranol, 4'-chlorobiphenyl-2,3-diol, medrysone, scillarenin, sudan I, and N,O-didesmethyltramadol) in treatment set samples and are considered to have influenced the in vitro estrogenic activity observed. The combination of predictive and in vitro estrogenicity with nontargeted analysis has led to confirmation of 12 estrogen-active contaminants in California sewage sludge and has highlighted the importance of evaluating both agonistic and antagonistic responses when evaluating the bioactivity of complex samples.

Bioanalytical screening of low levels of dioxins and dioxin-like PCBs in pig meat (pork) for checking compliance with EU maximum and action levels using highly sensitive "third generation" recombinant H4L7.5c2 rat hepatoma cells.

BACKGROUND: Low maximum and action levels set by the European Union for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in pig meat (pork) have led to a demand for reliable and cost-effective bioanalytical screening methods implemented upstream of gas chromatography/high-resolution mass spectrometry confirmatory technology, that can detect low levels of contamination in EU-regulated foods with quick turn-around times. RESULTS: Based on the Chemically Activated LUciferase gene eXpression (CALUX) bioassay, extraction and clean-up steps were optimized for recovery and reproducibility within working ranges significantly lower than in current bioassays. A highly sensitive "3rd generation" recombinant rat hepatoma cell line (H4L7.5c2) containing 20 dioxin responsive elements was exposed to pork sample extracts, and their PCDD/Fs and DL-PCBs levels were evaluated by measuring luciferase activity. The method was validated according to the provisions of Commission Regulation (EU) 2017/644 of 5 April 2017 with spiking experiments performed selectively for PCDD/Fs and DL-PCBs and individual calibration for PCDD/Fs, DL-PCBs and the calculated sum of PCDD/Fs and DL-PCBs. The resulting performance parameters met all legal specifications as confirmed by re-calibration using authentic samples. Cut-off concentrations for assessing compliance with low maximum levels and action levels set for PCDD/Fs and DL-PCBs within a range of 0.50-1.25 pg WHO-TEQ/g fat were derived, ensuring low rates of false-compliant results (ß-error < 1%) and keeping the rate of false-noncompliant results well under control (α-error < 12%). CONCLUSIONS: We present a fast and efficient bioanalytical routine method validated according to the European Union's legal requirements on the basis of authentic samples, allowing the analyst to reliably identify pork samples and any other EU-regulated foods of animal origin suspected to be noncompliant with a high level of performance and turn-around times of 52 h. This was facilitated in particular by a quick and efficient extraction step followed by selective clean-up, use of a highly sensitive "3rd generation" H4L7.5c2 recombinant rat hepatoma cell CALUX bioassay, and optimized assay performance with improved calibrator precision and reduced lack-of-fit errors. New restrictions are proposed for the calibrator bias and the unspecific background contribution to reportable results. The procedure can utilize comparably small sample amounts and allows an annual throughput of 840-1000 samples per lab technician. The described bioanalytical method contributes to the European Commission's objective of generating accurate and reproducible analytical results according to Commission Regulation (EU) 2017/644 across the European Union.

Identifying Toxicologically Significant Compounds in Urban Wildfire Ash Using In Vitro Bioassays and High-Resolution Mass Spectrometry.

Urban wildfires may generate numerous unidentified chemicals of toxicity concern. Ash samples were collected from burned residences and from an undeveloped upwind reference site, following the Tubbs fire in Sonoma County, California. The solvent extracts of ash samples were analyzed using GC- and LC-high-resolution mass spectrometry (HRMS) and using a suite of in vitro bioassays for their bioactivity toward nuclear receptors [aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and androgen receptor (AR)], their influence on the expression of genetic markers of stress and inflammation [interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2)], and xenobiotic metabolism [cytochrome P4501A1 (CYP1A1)]. Genetic markers (CYP1A1, IL-8, and COX-2) and AhR activity were significantly higher with wildfire samples than in solvent controls, whereas AR and ER activities generally were unaffected or reduced. The bioassay responses of samples from residential areas were not significantly different from the samples from the reference site despite differing chemical compositions. Suspect and nontarget screening was conducted to identify the chemicals responsible for elevated bioactivity using the multiple streams of HRMS data and open-source data analysis workflows. For the bioassay endpoint with the largest available database of pure compound results (AhR), nontarget features statistically related to whole sample bioassay response using Spearman's rank-order correlation coefficients or elastic net regression were significantly more likely (by 10 and 15 times, respectively) to be known AhR agonists than the overall population of compounds tentatively identified by nontarget analysis. The findings suggest that a combination of nontarget analysis, in vitro bioassays, and statistical analysis can identify bioactive compounds in complex mixtures.

Evaluation of the dioxin-like toxicity in soil samples from Thua Thien Hue province using the AhR-CALUX bioassay - An update of Agent Orange contamination in Vietnam.

In this study, an AhR-responsive reporter-gene cell-based bioassay CALUX was used to assess the biological potency of dioxins and dioxin-like PCBs (dl-PCBs) in top soil samples collected from a former airbase (A-So) and remote regions from urban and agricultural areas in Thua Thien Hue, Vietnam. In top soil collected from A-So airbase, Bioanalytical EQuivalent (BEQ) concentrations of up to 2700 pg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) per g dry weight (pg BEQ-TCDD g-1 dw) were assessed. Interestingly, while BEQ values for dl-PCBs were found to be up to 13 pg BEQ-TCDD g-1 dw, the dl-PCB activity was not detected in all the hotspot sample extracts. In contrasts, BEQ values for dioxin like compounds from remote regions were much lower and occasionally below the quantification limits of the method. The BEQ activities obtained in this study have a similar trend to the WHO-TEQ results for the samples collected in the A-So airbase. However, BEQ values were higher than those of TEQ, probably reflecting the presence of additional AhR ligands and/or possible non-additive interactions in the sample mixture. This study confirms that after more than 60 years, a strong residual pollution of PCDD/Fs remains on this former air base following the use and storage of Agent Orange during the Vietnam War, raising a health risk for populations exposed in this area because livestock animals graze there.

Transcriptome wide SSR discovery cross-taxa transferability and development of marker database for studying genetic diversity population structure of Lilium species.

Lily belongs to family liliaceae, which mainly propagates vegetatively. Therefore, sufficient number of polymorphic, informative, and functional molecular markers are essential for studying a wide range of genetic parameters in Lilium species. We attempted to develop, characterize and design SSR (simple sequence repeat) markers using online genetic resources for analyzing genetic diversity and population structure of Lilium species. We found di-nucleotide repeat motif were more frequent (4684) within 0.14 gb (giga bases) transcriptome than other repeats, of which was two times higher than tetra-repeat motifs. Frequency of di-(AG/CT), tri-(AGG/CTT), tetra-(AAAT), penta-(AGAGG), and hexa-(AGAGGG) repeats was 34.9%, 7.0%, 0.4%, 0.3%, and 0.2%, respectively. A total of 3607 non-redundant SSR primer pairs was designed based on the sequences of CDS, 5'-UTR and 3'-UTR region covering 34%, 14%, 23%, respectively. Among them, a sub set of primers (245 SSR) was validated using polymerase chain reaction (PCR) amplification, of which 167 primers gave expected PCR amplicon and 101 primers showed polymorphism. Each locus contained 2 to 12 alleles on average 0.82 PIC (polymorphic information content) value. A total of 87 lily accessions was subjected to genetic diversity analysis using polymorphic SSRs and found to separate into seven groups with 0.73 to 0.79 heterozygosity. Our data on large scale SSR based genetic diversity and population structure analysis may help to accelerate the breeding programs of lily through utilizing different genomes, understanding genetics and characterizing germplasm with efficient manner.

An aryl hydrocarbon receptor from the caecilian Gymnopis multiplicata suggests low dioxin affinity in the ancestor of all three amphibian orders.

The aryl hydrocarbon receptor (AHR) plays pleiotropic roles in the development and physiology of vertebrates in conjunction with xenobiotic and endogenous ligands. It is best known for mediating the toxic effects of dioxin-like pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While most vertebrates possess at least one AHR that binds TCDD tightly, amphibian AHRs bind TCDD with very low affinity. Previous analyses of AHRs from Xenopus laevis (a frog; order Anura) and Ambystoma mexicanum (a salamander; order Caudata) identified three amino acid residues in the ligand-binding domain (LBD) that underlie low-affinity binding. In X. laevis AHR1ß, these are A354, A370, and N325. Here we extend the analysis of amphibian AHRs to the caecilian Gymnopis multiplicata, representing the remaining extant amphibian order, Gymnophiona. G. multiplicata AHR groups with the monophyletic vertebrate AHR/AHR1 clade. The LBD includes all three signature residues of low TCDD affinity, and a structural homology model suggests that its architecture closely resembles those of other amphibians. In transactivation assays, the EC50 for reporter gene induction by TCDD was 17.17 nM, comparable to X. laevis AhR1ß (26.23 nM) and Ambystoma AHR (34.09 nM) and dramatically higher than mouse AhR (0.13 nM), a trend generally reflected in direct measures of TCDD binding. These shared properties distinguish amphibian AHRs from the high-affinity proteins typical of both vertebrate groups that diverged earlier (teleost fish) and those that appeared more recently (other tetrapods). These findings suggest the hypothesis that AHRs with low TCDD affinity represent a characteristic that evolved in a common ancestor of all three extant amphibian groups.

ddRAD-seq derived genome-wide SNPs, high density linkage map and QTLs for fruit quality traits in strawberry (Fragaria x ananassa).

Understanding the genetic determinants are essential for improving the fruit quality traits of strawberry. In this study, we focused on mapping the loci for fruit-length (FL), -diameter (FD), -weight (FW) and -soluble solid content (SSC) using the genome-wide single nucleotide polymorphisms (SNPs) identified via ddRAD-sequencing of the F1 population raised from Maehyang (♀) X Festival (♂). A total of 12,698 high quality SNPs were identified of which 1554 SNPs that showed significant Mendelian segregation (p < 0.05) were mapped to 53 linkage groups (LG) spanning a total of 2937.93 cM with an average marker density of 2.14 cM/locus. Six QTLs for FL and four QTLs for each of FD, FW and SSC were identified that explained 24-35%, 21-42%, 24-54% and 23-50% of overall phenotypic variations, respectively. The genes that lie within these QTL regions were extracted and discussed thoroughly. In addition, a high resolution melting marker (MF154) were designed based on the SNP A1723G of the UDP-glucose 4-epimerase GEPI48-like gene FAN_iscf00021287. The marker detected the high vs low sugar containing F1 plants and commercial cultivars with 81.39% and 86.95% detection accuracy, respectively. These SNPs, linkage map, QTLs and candidate genes will be helpful in understanding and improving the fruit quality traits of strawberry.

Characterization, identification and expression profiling of genome-wide R-genes in melon and their putative roles in bacterial fruit blotch resistance.

BACKGROUND: Bacterial fruit blotch (BFB), a disease caused by Acidovorax citrulli, results in significant economic losses in melon. The causal QTLs and genes for resistance to this disease have yet to be identified. Resistance (R)-genes play vital roles in resistance to plant diseases. Since the complete genome sequence of melon is available and genome-wide identification of R-genes has been performed for this important crop, comprehensive expression profiling may lead to the identification of putative candidate genes that function in the response to BFB. RESULTS: We identified melon accessions that are resistant and susceptible to BFB through repeated bioassays and characterized all 70 R-genes in melon, including their gene structures, chromosomal locations, domain organizations, motif distributions, and syntenic relationships. Several disease resistance-related domains were identified, including NBS, TIR, LRR, CC, RLK, and DUF domains, and the genes were categorized based on the domains of their encoded proteins. In addition, we profiled the expression patterns of the genes in melon accessions with contrasting levels of BFB resistance at 12 h, 1 d, 3 d, and 6 d after inoculation with A. citrulli. Six R-genes exhibited consistent expression patterns (MELO3C023441, MELO3C016529, MELO3C022157, MELO3C022146, MELO3C025518, and MELO3C004303), with higher expression levels in the resistant vs. susceptible accession. CONCLUSION: We identified six putative candidate R-genes against BFB in melon. Upon functional validation, these genes could be targeted for manipulation via breeding and biotechnological approaches to improve BFB resistance in melon in the future.

The Cellular and Molecular Determinants of Naphthoquinone-Dependent Activation of the Aryl Hydrocarbon Receptor.

1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ) are clinically promising biologically active chemicals that have been shown to stimulate the aryl hydrocarbon receptor (AhR) signaling pathway, but whether they are direct or indirect ligands or activate the AhR in a ligand-independent manner is unknown. Given the structural diversity of AhR ligands, multiple mechanisms of AhR activation of gene expression, and species differences in AhR ligand binding and response, we examined the ability of 1,2-NQ and 1,4-NQ to bind to and activate the mouse and human AhRs using a series of in vitro AhR-specific bioassays and in silico modeling techniques. Both NQs induced AhR-dependent gene expression in mouse and human hepatoma cells, but were more potent and efficacious in human cells. 1,2-NQ and 1,4-NQ stimulated AhR transformation and DNA binding in vitro and was inhibited by AhR antagonists. Ligand binding analysis confirmed the ability of 1,2-NQ and 1,4-NQ to competitively bind to the AhR ligand binding cavity and the molecular determinants for interactions were predicted by molecular modeling methods. NQs were shown to bind distinctly differently from that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and differences were also observed between species. Mutation of amino acid residues (F289, M334, and M342) involved in critical NQ:AhR binding interactions, decreased NQ- and AhR-dependent gene expression, consistent with a role for these residues in binding and activation of the AhR by NQs. These studies provide insights into the molecular mechanism of action of NQs and contribute to the development of emerging NQ-based therapeutics.

Development of a novel recombinant cell line for detection and characterization of Ah receptor nuclear translocation in intact cells.

The Ah receptor (AhR) is a ligand-dependent transcriptional factor that mediates the effects of structurally diverse chemicals. Ligand binding stimulates nuclear translocation of the AhR and leads to AhR DNA binding and increased gene expression. Studies of the molecular mechanisms by which ligands bind to and activate the AhR and AhR-dependent signal transduction require methods to easily examine each step of the AhR signaling pathway. While current assays can measure ligand and DNA binding in vitro and gene expression in cells, there is no simple method to monitor AhR nuclear translocation. We developed a stably transfected mouse hepatoma cell line (yAHAYc6) that expresses yellow fluorescent protein-tagged AhR (yAhR) for use in qualitative or semiquantitative assessment of nuclear/cytoplasmic distribution of yAhR in living cells by fluorescent microscopy. yAhR nuclear translocation was stimulated in a concentration- and time-dependent manner by AhR agonists and inhibited by antagonists. Inhibition of nuclear export channels by leptomycin B, resulted in increased nuclear accumulation of yAhR in the absence of added ligand, indicating endogenous nucleocytoplasmic shuttling of unliganded AhR and demonstrating the utility of these cells. This novel cell line can be used to detect and characterize AhR ligands and will facilitate mechanistic studies of AhR signaling.

Transitional States in Ligand-Dependent Transformation of the Aryl Hydrocarbon Receptor into Its DNA-Binding Form.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of an AhR lacking the entire PASB structurally diverse chemicals, including halogenated aromatic hydrocarbons. Ligand-dependent transformation of the AhR into its DNA binding form involves a ligand-dependent conformational change, heat shock protein 90 (hsp90), dissociation from the AhR complex and AhR dimerization with the AhR nuclear translocator (ARNT) protein. The mechanism of AhR transformation was examined using mutational approaches and stabilization of the AhR:hsp90 complex with sodium molybdate. Insertion of a single mutation (F281A) in the hsp90-binding region of the AhR resulted in its constitutive (ligand-independent) transformation/DNA binding in vitro. Mutations of AhR residues within the Arg-Cys-rich region (R212A, R217A, R219A) and Asp371 (D371A) impaired AhR transformation without a significant effect on ligand binding. Stabilization of AhR:hsp90 binding with sodium molybdate decreased transformation/DNA binding of the wild type AhR but had no effect on constitutively active AhR mutants. Interestingly, transformation of the AhR in the presence of molybdate allowed detection of an intermediate transformation ternary complex containing hsp90, AhR, and ARNT. These results are consistent with a stepwise transformation mechanism in which binding of ARNT to the liganded AhR:hsp90 complex results in a progressive displacement of hsp90 and conversion of the AhR into its high affinity DNA binding form. The available molecular insights into the signaling mechanism of other Per-ARNT-Sim (PAS) domains and structural information on hsp90 association with other client proteins are consistent with the proposed transformation mechanism of the AhR.

Development of Molecular Marker Linked with Bacterial Fruit Blotch Resistance in Melon (Cucumis melo L.).

Bacterial fruit blotch (BFB) causes losses in melon marketable yield. However, until now, there has been no information about the genetic loci responsible for resistance to the disease or their pattern of inheritance. We determined the inheritance pattern of BFB resistance from a segregating population of 491 F2 individuals raised by crossing BFB-resistant (PI 353814) and susceptible (PI 614596) parental accessions. All F1 plants were resistant to Acidovorax citrulli strain KACC18782, and F2 plants segregated with a 3:1 ratio for resistant and susceptible phenotypes, respectively, in a seedling bioassay experiment, indicating that BFB resistance is controlled by a monogenic dominant gene. In an investigation of 57 putative disease-resistance related genes across the melon genome, only the MELO3C022157 gene (encoding TIR-NBS-LRR domain), showing polymorphism between resistant and susceptible parents, revealed as a good candidate for further investigation. Cloning, sequencing and quantitative RT-PCR expression of the polymorphic gene MELO3C022157 located on chromosome 9 revealed multiple insertion/deletions (InDels) and single nucleotide polymorphisms (SNPs), of which the SNP A2035T in the second exon of the gene caused loss of the LRR domain and truncated protein in the susceptible accession. The InDel marker MB157-2, based on the large (504 bp) insertion in the first intron of the susceptible accession, was able to distinguish resistant and susceptible accessions among 491 F2 and 22 landraces/inbred accessions with 98.17% and 100% detection accuracy, respectively. This novel PCR-based, co-dominant InDel marker represents a practical tool for marker-assisted breeding aimed at developing BFB-resistant melon accessions.