Integrated transcriptomics and metabolomics reveal the mechanism of intestinal damage upon acute patulin exposure in mice.

Mycotoxin contamination has become a major food safety issue and greatly threatens human and animal health. Patulin (PAT), a common mycotoxin in the environment, is exposed through the food chain and damages the gastrointestinal tract. However, its mechanism of enterotoxicity at the genetic and metabolic levels remains to be elucidated. Herein, the intestinal histopathological and biochemical indices, transcriptome, and metabolome of C57BL/6 J mice exposed to different doses of PAT were successively assessed, as well as the toxicokinetics of PAT in vivo. The results showed that acute PAT exposure induced damaged villi and crypts, reduced mucus secretion, decreased SOD and GSH-Px activities, and enhanced MPO activity in the small intestine and mild damage in the colon. At the transcriptional level, the genes affected by PAT were dose-dependently altered in the small intestine and fluctuated in the colon. PAT primarily affected inflammation-related signaling pathways and oxidative phosphorylation in the small intestine and immune responses in the colon. At the metabolic level, amino acids decreased, and extensive lipids accumulated in the small intestine and colon. Seven metabolic pathways were jointly affected by PAT in two intestinal sites. Moreover, changes in PAT products and GST activity were detected in the small intestinal tissue but not in the colonic tissue, explaining the different damage degrees of the two sites. Finally, the integrated results collectively explained the toxicological mechanism of PAT, which damaged the small intestine directly and the colon indirectly. These results paint a clear panorama of intestinal changes after PAT exposure and provide valuable information on the exposure risk and toxic mechanism of PAT.

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat.

Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication-related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement.

Mycoflora assessment, growth and toxigenic features of patulin-producers in kiwifruit in China.

BACKGROUND: Fungal development in agricultural products may cause mycotoxin contamination, which is a significant threat to food safety. Patulin (PAT) and PAT-producer contamination has been established as a worldwide problem. The present study aimed to investigate the mycoflora and PAT-producers present in kiwifruits and environmental samples collected from orchards and processing plants in Shaanxi Province, China. RESULTS: Variations in mycoflora were observed in different samples, with penicillia and aspergilli as the predominant genera. Approximately 42.86% of dropped fruits were contaminated with PAT-producers, which harbored the 6-methylsalicylic acid synthase and the isoepoxydon dehydrogenase genes that are involved in PAT biosynthesis. The growth of Penicillium expansum, Penicillium griseofulvum and Penicillium paneum in kiwi puree agar (KPA) medium and kiwi juice well fitted the modified Gompertz and Baranyi and Roberts models (R2 ≥ 0.95). A significant positive correlation between colony diameter and PAT content in KPA medium of P. expansum and P. griseofulvum was observed (P < 0.05). CONCLUSIONS: The present study analyzed the mycofloral composition and the potential risk for PAT and PAT-producer contamination in kiwifruit, which may be utilized in the establishment of proper management practices in the kiwifruit industry. © 2017 Society of Chemical Industry.

Genome-wide identification, phylogeny and expressional profiles of mitogen activated protein kinase kinase kinase (MAPKKK) gene family in bread wheat (Triticum aestivum L.).

BACKGROUND: Mitogen-activated protein kinase kinase kinases (MAPKKKs) are the important components of MAPK cascades, which play the crucial role in plant growth and development as well as in response to diverse stresses. Although this family has been systematically studied in many plant species, little is known about MAPKKK genes in wheat (Triticum aestivum L.), especially those involved in the regulatory network of stress processes. RESULTS: In this study, we identified 155 wheat MAPKKK genes through a genome-wide search method based on the latest available wheat genome information, of which 29 belonged to MEKK, 11 to ZIK and 115 to Raf subfamily, respectively. Then, chromosome localization, gene structure and conserved protein motifs and phylogenetic relationship as well as regulatory network of these TaMAPKKKs were systematically investigated and results supported the prediction. Furthermore, a total of 11 homologous groups between A, B and D sub-genome and 24 duplication pairs among them were detected, which contributed to the expansion of wheat MAPKKK gene family. Finally, the expression profiles of these MAPKKKs during development and under different abiotic stresses were investigated using the RNA-seq data. Additionally, 10 tissue-specific and 4 salt-responsive TaMAPKKK genes were selected to validate their expression level through qRT-PCR analysis. CONCLUSIONS: This study for the first time reported the genome organization, evolutionary features and expression profiles of the wheat MAPKKK gene family, which laid the foundation for further functional analysis of wheat MAPKKK genes, and contributed to better understanding the roles and regulatory mechanism of MAPKKKs in wheat.

Genome-wide identification, phylogeny and expression analysis of AP2/ERF transcription factors family in Brachypodium distachyon.

BACKGROUND: The AP2/ERF transcription factor is one of the most important gene families in plants, which plays the vital role in regulating plant growth and development as well as in response to diverse stresses. Although AP2/ERFs have been thoroughly characterized in many plant species, little is known about this family in the model plant Brachypodium distachyon, especially those involved in the regulatory network of stress processes. RESULTS: In this study, a comprehensive genome-wide search was performed to identify AP2/ERF gene family in Brachypodium and a total of 141 BdAP2/ERFs were obtained. Phylogenetic analysis classified them into four subfamilies, of which 112 belonged to ERF, four to RAV and 24 to AP2 as well as one to soloist subfamily respectively, which was in accordance with the number of AP2 domains and gene structure analysis. Chromosomal localization, gene structure, conserved protein motif and cis-regulatory elements as well as gene duplication events analysis were further performed to systematically investigate the evolutionary features of these BdAP2/ERF genes. Furthermore, the regulatory network between BdAP2/ERF and other genes were constructed using the orthology-based method, and 39 BdAP2/ERFs were found to be involved in the regulatory network and 517 network branches were identified. The expression profiles of BdAP2/ERF during development and under diverse stresses were investigated using the available RNA-seq and microarray data and ten tissue-specific and several stress-responsive BdAP2/ERF genes were identified. Finally, 11 AP2/ERF genes were selected to validate their expressions in different tissues and under different stress treatments using RT-PCR method and results verified that these AP2/ERFs were involved in various developmental and physiological processes. CONCLUSIONS: This study for the first time reported the characteristics of the BdAP2/ERF family, which will provide the invaluable information for further evolutionary and functional studies of AP2/ERF in Brachypodium, and also contribute to better understanding the molecular basis for development and stresses tolerance in this model species and beyond.

Characterization of microRNAs and their targets in wild barley (Hordeum vulgare subsp. spontaneum) using deep sequencing.

MicroRNAs (miRNA) are a class of small, endogenous RNAs that play a negative regulatory role in various developmental and metabolic processes of plants. Wild barley (Hordeum vulgare subsp. spontaneum), as the progenitor of cultivated barley (Hordeum vulgare subsp. vulgare), has served as a valuable germplasm resource for barley genetic improvement. To survey miRNAs in wild barley, we sequenced the small RNA library prepared from wild barley using the Illumina deep sequencing technology. A total of 70 known miRNAs and 18 putative novel miRNAs were identified. Sequence analysis revealed that all of the miRNAs identified in wild barley contained the highly conserved hairpin sequences found in barley cultivars. MiRNA target predictions showed that 12 out of 52 miRNA families were predicted to target transcription factors, including 8 highly conserved miRNA families in plants and 4 wheat-barley conserved miRNA families. In addition to transcription factors, other predicted target genes were involved in diverse physiological and metabolic processes and stress defense. Our study for the first time reported the large-scale investigation of small RNAs in wild barley, which will provide essential information for understanding the regulatory role of miRNAs in wild barley and also shed light on future practical utilization of miRNAs for barley improvement.

Antifungal mechanism of cinnamaldehyde and citral combination against Penicillium expansum based on FT-IR fingerprint, plasma membrane, oxidative stress and volatile profile.

Cinnamaldehyde (Cin) and citral (Cit) have been studied as antimicrobial agents and natural preservatives, but their action modes are controversial, and the knowledge of their antifungal mechanism against P. expansum is still incomplete. The present study was conducted to evaluate the antifungal mechanism of the combination of Cin and Cit (Cin/Cit) against P. expansum by observing the cellular ultrastructure, fourier transform infrared spectroscopy (FT-IR) fingerprints, plasma membrane, oxidative stress and volatile profile. Cin/Cit caused membrane invaginations, organelles and cytoplasm destruction, as shown by transmission electron microscopy (TEM) observations. The FT-IR spectra and followed principle component analysis (PCA) presented the significant differences in chemical compounds, particularly phospholipid, protein and fatty acids of cells exposed to Cin/Cit. Compared to controls, Cin/Cit induced a decrease of ergosterol by 39.40%, an increase of unsaturated fatty acid, and protein release level (3.5 times). Besides, membrane damage was further verified through the reduction of the membrane integrity by using a flow cytometer. Moreover, the increase of malondialdehyde (MDA) (40.09%) and reactive oxygen species (ROS) accumulation indicated an induction of oxidative stress in cells by Cin/Cit. To resist the unfavorable stress caused by Cin/Cit, P. expansum metabolized Cin or Cit to the predominant detoxification compounds, cinnamic alcohol, nerol, and geraniol. The alterations in volatile profile demonstrated the influences on specific metabolisms in P. expansum caused by Cin/Cit.

Effect of Cinnamaldehyde and Citral Combination on Transcriptional Profile, Growth, Oxidative Damage and Patulin Biosynthesis of Penicillium expansum.

Penicillium expansum, as a main postharvest pathogen of fruits, can secrete patulin (PAT), causing fruit decay and health problems. In this study, the antifungal test, SEM (scanning electron microscope) observation, transcriptional profile, PAT biosynthesis, and physiological characters of P. expansum exposed to cinnamaldehyde and citral combination (Cin/Cit) were evaluated. Cin/Cit could inhibit the mycelial growth and spore germination of P. expansum in a dose-dependent manner. Besides, Cin/Cit caused spores and mycelia wrinkled and depressed by SEM observation. Gene expression profiles of P. expansum were conducted by RNA sequencing (RNA-seq) in the presence or absence of Cin/Cit treatment. A total of 1713 differentially expressed genes (DEGs) were obtained, including 793 down-regulated and 920 up-regulated genes. Most of the DEGs participated in the biosynthesis of secondary metabolites, amino acid metabolism, and oxidation-reduction process, etc. Cin/Cit induced the dysfunction of the mitochondrial membrane, causing the potential influence on energy metabolism and reactive oxidative species production. The changes of superoxide dismutase (SOD) and catalase (CAT) activities combing with the increase of hydrogen peroxide content indicated the oxidative stress on P. expansum induced by Cin/Cit, which corresponded well with the transcriptional results. Moreover, both the RNA-seq data and the qRT-PCR showed the remarkable down-regulation of genes included in the PAT biosynthetic pathway under the Cin/Cit treatment. These findings provided more useful information about the antifungal mechanism of Cin/Cit against P. expansum at molecular and gene levels and suggested that Cin/Cit is a potential candidate to control P. expansum.

Genome-Wide Identification and Characterization of Salinity Stress-Responsive miRNAs in Wild Emmer Wheat (Triticum turgidum ssp. dicoccoides).

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs which regulate diverse molecular and biochemical processes at a post-transcriptional level in plants. As the ancestor of domesticated wheat, wild emmer wheat (Triticum turgidum ssp. dicoccoides) has great genetic potential for wheat improvement. However, little is known about miRNAs and their functions on salinity stress in wild emmer. To obtain more information on miRNAs in wild emmer, we systematically investigated and characterized the salinity-responsive miRNAs using deep sequencing technology. A total of 88 conserved and 124 novel miRNAs were identified, of which 50 were proven to be salinity-responsive miRNAs, with 32 significantly up-regulated and 18 down-regulated. miR172b and miR1120a, as well as mi393a, were the most significantly differently expressed. Targets of these miRNAs were computationally predicted, then Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the targets of salinity-responsive miRNAs were enriched in transcription factors and stress-related proteins. Finally, we investigated the expression profiles of seven miRNAs ranging between salt-tolerant and sensitive genotypes, and found that they played critical roles in salinity tolerance in wild emmer. Our results systematically identified the salinity-responsive miRNAs in wild emmer, not only enriching the miRNA resource but also laying the foundation for further study on the biological functions and evolution of miRNAs in wild wheat and beyond.

Genome-wide characterization of microsatellites in Triticeae species: abundance, distribution and evolution.

Microsatellites are an important constituent of plant genome and distributed across entire genome. In this study, genome-wide analysis of microsatellites in 8 Triticeae species and 9 model plants revealed that microsatellite characteristics were similar among the Triticeae species. Furthermore, genome-wide microsatellite markers were designed in wheat and then used to analyze the evolutionary relationship of wheat and other Triticeae species. Results displayed that Aegilops tauschii was found to be the closest species to Triticum aestivum, followed by Triticum urartu, Triticum turgidum and Aegilops speltoides, while Triticum monococcum, Aegilops sharonensis and Hordeum vulgare showed a relatively lower PCR amplification effectivity. Additionally, a significantly higher PCR amplification effectivity was found in chromosomes at the same subgenome than its homoeologous when these markers were subjected to search against different chromosomes in wheat. After a rigorous screening process, a total of 20,666 markers showed high amplification and polymorphic potential in wheat and its relatives, which were integrated with the public available wheat markers and then anchored to the genome of wheat (CS). This study not only provided the useful resource for SSR markers development in Triticeae species, but also shed light on the evolution of polyploid wheat from the perspective of microsatellites.

Genome-Wide Identification, Evolution, and Co-expression Network Analysis of Mitogen-Activated Protein Kinase Kinase Kinases in Brachypodium distachyon.

Mitogen-activated protein kinase (MAPK) cascades are the conserved and universal signal transduction modules in all eukaryotes, which play the vital roles in plant growth, development, and in response to multiple stresses. In this study, we used bioinformatics methods to identify 86 MAPKKK protein encoded by 73 MAPKKK genes in Brachypodium. Phylogenetic analysis of MAPKKK family from Arabidopsis, rice, and Brachypodium has classified them into three subfamilies, of which 28 belonged to MEKK, 52 to Raf, and 6 to ZIK subfamily, respectively. Conserved protein motif, exon-intron organization, and splicing intron phase in kinase domains supported the evolutionary relationships inferred from the phylogenetic analysis. And gene duplication analysis suggested the chromosomal segment duplication happened before the divergence of the rice and Brachypodium, while all of three tandem duplicated gene pairs happened after their divergence. We further demonstrated that the MAPKKKs have evolved under strong purifying selection, implying the conservation of them. The splicing transcripts expression analysis showed that the splicesome translating longest protein tended to be adopted. Furthermore, the expression analysis of BdMAPKKKs in different organs and development stages as well as heat, virus and drought stresses revealed that the MAPKKK genes were involved in various signaling pathways. And the circadian analysis suggested there were 41 MAPKKK genes in Brachypodium showing cycled expression in at least one condition, of which seven MAPKKK genes expressed in all conditions and the promoter analysis indicated these genes possessed many cis-acting regulatory elements involved in circadian and light response. Finally, the co-expression network of MAPK, MAPKK, and MAPKKK in Brachypodium was constructed using 144 microarray and RNA-seq datasets, and ten potential MAPK cascades pathway were predicted. To conclude, our study provided the important information for evolutionary and functional characterization of MAPKKK family in Brachypodium, which will facilitate the functional analysis of BdMAPKKK genes, and also will facilitate better understanding the MAPK signal pathway in Brachypodium and beyond.

Global Identification of MicroRNAs and Their Targets in Barley under Salinity Stress.

Salinity is a major limiting factor for agricultural production worldwide. A better understanding of the mechanisms of salinity stress response will aid efforts to improve plant salt tolerance. In this study, a combination of small RNA and mRNA degradome sequencing was used to identify salinity responsive-miRNAs and their targets in barley. A total of 152 miRNAs belonging to 126 families were identified, of which 44 were found to be salinity responsive with 30 up-regulated and 25 down-regulated respectively. The majority of the salinity-responsive miRNAs were up-regulated at the 8h time point, while down-regulated at the 3h and 27h time points. The targets of these miRNAs were further detected by degradome sequencing coupled with bioinformatics prediction. Finally, qRT-PCR was used to validate the identified miRNA and their targets. Our study systematically investigated the expression profile of miRNA and their targets in barley during salinity stress phase, which can contribute to understanding how miRNAs respond to salinity stress in barley and other cereal crops.

Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants.

Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simplicity BAC libraries are most preferred over other approaches for cloning large genomic DNA fragments for large-insert libraries. Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction. It requires the isolation of purified nuclei, embedding them into LMP agarose plugs, restriction digestion of the plugs, and quite often size selection using PFGE and electro-elution of insert DNA. The construction of BAC libraries is complex and challenging for most molecular laboratories. To facilitate the construction of BAC libraries, we present a step-by-step protocol for isolation of HMW DNA and construction of plant BAC libraries.