Exogenous auxin regulates multi-metabolic network and embryo development, controlling seed secondary dormancy and germination in Nicotiana tabacum L.

BACKGROUND: Auxin was recognized as a secondary dormancy phytohormone, controlling seed dormancy and germination. However, the exogenous auxin-controlled seed dormancy and germination remain unclear in physiological process and gene network. RESULTS: Tobacco seeds soaked in 1000 mg/l auxin solution showed markedly decreased germination compared with that in low concentration of auxin solutions and ddH2O. Using an electron microscope, observations were made on the seeds which did not unfold properly in comparison to those submerged in ddH2O. The radicle traits measured by WinRHIZO, were found to be also weaker than the other treatment groups. Quantified by ELISA, there was no significant difference found in ß-1,3glucanase activity and abscisic acid (ABA) content between the seeds imbibed in gradient concentration of auxin solution and those soaked in ddH2O. However, gibberellic acid (GA) and auxin contents were significantly higher at the time of exogenous auxin imbibition and were gradually reduced at germination. RNA sequencing (RNA-seq), revealed that the transcriptome of auxin-responsive dormancy seeds were more similar to that of the imbibed seeds when compared with primary dormancy seeds by principal component analysis. The results of gene differential expression analysis revealed that auxin-controlled seed secondary dormancy was associated with flavonol biosynthetic process, gibberellin metabolic process, adenylyl-sulfate reductase activity, thioredoxin activity, glutamate synthase (NADH) activity and chromatin regulation. In addition, auxin-responsive germination responded to ABA, auxin, jasmonic acid (JA) and salicylic acid (SA) mediated signaling pathway (red, far red and blue light), glutathione and methionine (Met) metabolism. CONCLUSIONS: In this study, exogenous auxin-mediated seed secondary dormancy is an environmental model that prevents seed germination in an unfavorable condition. Seeds of which could not imbibe normally, and radicles of which also could not develop normally and emerge. To complete the germination, seeds of which would stimulate more GA synthesis to antagonize the stimulation of exogenous auxin. Exogenous auxin regulates multi-metabolic networks controlling seed secondary dormancy and germination, of which the most important thing was that we found the auxin-responsive seed secondary dormancy refers to epigenetic regulation and germination to enhance Met pathway. Therefore, this study uncovers a previously unrecognized transcriptional regulatory networks and physiological development process of seed dormancy and germination with superfluous auxin signal activate.

Transportation of citrinin is regulated by the CtnC gene in the medicinal fungus Monascus purpureus. / 药用真菌紫色红曲霉通过CtnCåŸºå› è°ƒæŽ§æ¡”éœ‰ç´ çš„è½¬è¿.

Monascus is one of the most essential microbial resources in China, with thousands of years of history. Modern science has proved that Monascus can produce pigment, ergosterol, monacolin K, γ-aminobutyric acid, and other functionally active substances. Currently, Monascus is used to produce a variety of foods, health products, and pharmaceuticals, and its pigments are widely used as food additives. However, Monascus also makes a harmful polyketide component called citrinin in the fermentation process; citrinin has toxic effects on the kidneys such as teratogenicity, carcinogenicity, and mutagenicity (Gong et al., 2019). The presence of citrinin renders Monascus and its products potentially hazardous, which has led many countries to set limits and standards on citrinin content. For example, the citrinin limit is less than 0.04 mg/kg according to the Chinese document National Standard for Food Safety Food Additive Monascus (GB 1886.181-2016) (National Health and Family Planning Commission of the People's Republic of China, 2016), and the maximum level in food supplements based on rice fermented with Monascus purpureus is 100 µg/kg in the European Union (Commission of the European Union, 2019).

An oxidoreductase gene CtnD involved in citrinin biosynthesis in Monascus purpureus verified by CRISPR/Cas9 gene editing and overexpression.

Monascus produces a kind of mycotoxin, citrinin, whose synthetic pathway is still not entirely clear. The function of CtnD, a putative oxidoreductase located upstream of pksCT in the citrinin gene cluster, has not been reported. In this study, the CtnD overexpressed strain and the Cas9 constitutively expressed chassis strain were obtained by genetic transformation mediated by Agrobacterium tumefaciens. The pyrG and CtnD double gene-edited strains were then obtained by transforming the protoplasts of the Cas9 chassis strain with in vitro sgRNAs. The results showed that overexpression of CtnD resulted in significant increases in citrinin content of more than 31.7% and 67.7% in the mycelium and fermented broth, respectively. The edited CtnD caused citrinin levels to be reduced by more than 91% in the mycelium and 98% in the fermented broth, respectively. It was shown that CtnD is a key enzyme involved in citrinin biosynthesis. RNA-Seq and RT-qPCR showed that the overexpression of CtnD had no significant effect on the expression of CtnA, CtnB, CtnE, and CtnF but led to distinct changes in the expression of acyl-CoA thioesterase and two MFS transporters, which may play an unknown role in citrinin metabolism. This study is the first to report the important function of CtnD in M. purpureus through a combination of CRISPR/Cas9 editing and overexpression.

Study on species diversity of Akanthomyces (Cordycipitaceae, Hypocreales) in the Jinyun Mountains, Chongqing, China.

Akanthomyces species have only been reported from Guizhou and Qinghai Province, with few reports from other regions in China. In this research, the species diversity of Akanthomyces in the Jinyun Mountains, Chongqing was investigated. Fourteen infected spider specimens were collected and two new species (A.bashanensis and A.beibeiensis) and a known species (A.tiankengensis) were established and described according to a multi-locus phylogenetic analysis and the morphological characteristics. Our results reveal abundant Akanthomyces specimens and three species were found at Jinyun Mountain. Due to its being an important kind of entomopathogenic fungi, further attention needs to be paid to the diversity of other entomopathogenic fungi in Chongqing, China.

Two new species of Samsoniella (Cordycipitaceae, Hypocreales) from the Mayao River Valley, Guizhou, China.

Samsoniella species have been often found in the forest habitat and rarely found in special karst eco-environments, such as Tiankeng, valleys and caves. In this research, eleven cordyceps specimens were collected from Mayao River Valley. A known species (S.haniana) and two new species (S.duyunensis and S.vallis) were established and described according to a multilocus phylogenetic analysis and morphological characteristics. Our results provide insight that the richness of Samsoniella species in karst eco-environments and further attention should be paid to entomopathogenic fungi in such habitats.

Integrated transcriptome and metabolome analyses revealed regulatory mechanisms of flavonoid biosynthesis in Radix Ardisia.

Background: Radix Ardisia (Jab Bik Lik Jib) is a common Miao medicine and is widely distributed in the Guizhou region of southern China. The botanical origin of Radix Ardisia includes the dry root and rhizome of Ardisia Crenata Sims (ACS) or Ardisia Crispa (Thunb.) A.DC. (AC), which are closely related species morphologically. However, the secondary metabolites in their roots are different from one another, especially the flavonoids, and these differences have not been thoroughly explored at the molecular level. This project preliminarily identified regulatory molecular mechanisms in the biosynthetic pathways of the flavonoids between ACS and AC using a multi-omics association analysis. Methods: In this study, we determined the total levels of saponin, flavonoid, and phenolic in Radix Ardisia from different origins. Integrated transcriptome and metabolome analyses were used to identify the differentially expressed genes (DEGs) and differentially expressed metabolites (DEM). We also performed conjoint analyses on DEGs and DEMs to ascertain the degree pathways, and explore the regulation of flavonoid biosynthesis. Results: The total flavonoid and phenolic levels in ACS were significantly higher than in AC (P < 0.05). There were 17,685 DEGs between ACS vs. AC, 8,854 were upregulated and 8,831 were downregulated. Based on this, we continued to study the gene changes in the flavonoid biosynthesis pathway, and 100 DEGs involving flavonoid biosynthesis were differentially expressed in ACS and AC. We validated the accuracy of the RNA-seq data using qRT-PCR. Metabolomic analyses showed that 11 metabolites were involved in flavonoid biosynthesis including: Naringenin, Luteolin, Catechin, and Quercetin. A conjoint analysis of the genome-wide connection network revealed the differences in the types and levels of flavonoid compounds between ACS and AC. The correlation analysis showed that Naringenin, Luteolin, Catechin, and Quercetin were more likely to be key compounds in the flavonoid biosynthesis pathway also including 4CL, AOMT, CHS, CHI, DFR, F3'5'H, FLS, and LAR. Conclusions: This study provides useful information for revealing the regulation of flavonoid biosynthesis and the regulatory relationship between metabolites and genes in the flavonoid biosynthesis pathway in Radix Ardisia from different origins.

Multigene phylogeny, phylogenetic network, and morphological characterizations reveal four new arthropod-associated Simplicillium species and their evolutional relationship.

Simplicillium species are widely distributed and commonly found on various substrates. A minority of species are associated with arthropods. A spider-associated species Simplicillium araneae, and three insect-associated species, Simplicillium coleopterorum, Simplicillium guizhouense, and Simplicillium larvatum, are proposed as novel species based on a multi-locus phylogenetic analysis and morphological characteristics. These Simplicillium species completely fit the nutritional model of Hypocreales fungi and could be used as a model to study their evolutionary relationship. A phylogenetic network analysis based on ITS sequences suggests that a host jump was common among Simplicillium species, and S. araneae may have originally come from an insect host and then jumped to a spider host. However, the evolutionary relationship of S. coleopterorum, S. guizhouense, and S. larvatum was not clear in the phylogenetic network and more sequencing information should be added to the network. In addition, strain CBS 101267 was identified as Simplicillium subtropicum.

Phylogenetic, ecological and morphological characteristics reveal two new spider-associated genera in Clavicipitaceae.

Clavicipitaceous fungi are pathogenic to scale insects, white flies and other insect orders. However, a few species are spider-associated. Two new genera from China, Neoaraneomyces and Pseudometarhizium, are described based on phylogenetic, ecological and morphological characteristics. Two spider-associated species, Neoaraneomycesaraneicola, Pseudometarhiziumaraneogenum, and an insect-associated species Pseudometarhiziumlepidopterorum are included. The morphological characteristics of paecilomyces-like conidiogenous structures, present in many insect/spiders associated species make species-level identifications difficult. A phylogenetic analysis of the combined dataset (ITS, LSU, RPB2 and TEF), placed the two new genera in Clavicipitaceae. The new spider-associated species may be the result of convergent evolution to adapt to the ecological environment and may have undergone host jumping or altered their nutritional preferences.

Species Diversity of Cordyceps-Like Fungi in the Tiankeng Karst Region of China.

Tiankeng acts as a refugium for biodiversity amid a changing global climate, and a previous study has shown that some ancient (Alsophila spinulosa) and unique plants (cool-adapted plants) are present in Tiankeng. However, there are few reports on Ascomycota from the Tiankeng karst region. In this research, the species diversity of Cordyceps-like fungi in Monkey-Ear Tiankeng was investigated. Seven species in the genera Akanthomyces, Beauveria, Cordyceps, and Samsoniella were identified based on internal transcribed spacer sequences and morphological characteristics. Eight new species in the genera Akanthomyces, Cordyceps, and Samsoniella were established and described according to a multilocus phylogenetic analysis and morphological characteristics. Our results revealed that Cordyceps-like fungi were abundant in Monkey-Ear Tiankeng, providing new insights into the diversity of Ascomycota in this special eco-environment. IMPORTANCE Karst Tiankeng has a special eco-environment and acts as a refugium for biodiversity. However, there are few reports on Ascomycota from the Tiankeng karst region. In this research, seven known species and eight new species in the genera Akanthomyces, Beauveria, Cordyceps, and Samsoniella were reported. The results showed that Cordyceps-like fungi are abundant in Monkey-Ear Tiankeng. Interestingly, the month of the sampling was November, which is not an active period of growth and reproduction for Cordyceps-like fungi. These results revealed that unconventional time sampling should not be ignored, especially for a special eco-environment, and provided new insights into the diversity of Ascomycota in this special eco-environment.

Cryptic Diversity of Isaria-like Species in Guizhou, China.

Many Isaria-like species have recently been moved into more appropriate genera. However, more robust molecular phylogenetic analyses are still required for Isaria-like fungi to ensure accurate taxonomic identification. We analyzed these Isaria-like strains using multi-gene phylogenetics. Cryptic diversity was discovered in several Isaria farinosa strains, and two new species, Samsoniella pseudogunnii and S. pupicola, are proposed. Our results reveal that more attention needs to be paid to cryptic intraspecific diversity across different isolates and genotypes of the Isaria-like species, some of which will need to be transferred to Samsoniella. Interestingly, S. hepiali, with a very broad host distribution, has been widely used as a medicinal and edible cordycipitoid fungus.

Transient expression of organophosphorus hydrolase to enhance the degrading activity of tomato fruit on coumaphos.

We constructed an expression cassette of the organophosphorus pesticide degrading (opd) gene under the control of the E8 promoter. Then opd was transformed into tomato fruit using an agroinfiltration transient expression system. beta-Glucuronidase (GUS) staining, reverse transcription-polymerase chain reaction (RT-PCR), wavelength scanning, and fluorescent reaction were performed to examine the expression of the opd gene and the hydrolysis activity on coumaphos of organophosphorus hydrolase (OPH) in tomato fruit. The results show that the agroinfiltrated tomato fruit-expressed OPH had the maximum hydrolysis activity of about 11.59 U/mg total soluble protein. These results will allow us to focus on breeding transgenic plants that could not only enhance the degrading capability of fruit and but also hold no negative effects on pest control when spraying organophosphorus pesticides onto the seedlings in fields.

Analyzing 74,248 Samples Confirms the Association Between CLU rs11136000 Polymorphism and Alzheimer's Disease in Caucasian But Not Chinese population.

Clusterin (CLU) is considered one of the most important roles for pathogenesis of Alzheimer's Disease (AD). The early genome-wide association studies (GWAS) identified the CLU rs11136000 polymorphism is significantly associated with AD in Caucasian. However, the subsequent studies are unable to replicate these findings in different populations. Although two independent meta-analyses show evidence to support significant association in Asian and Caucasian populations by integrating the data from 18 and 25 related GWAS studies, respectively, many of the following 18 studies also reported the inconsistent results. Moreover, there are six missed and a misclassified GWAS studies in the two meta-analyses. Therefore, we suspected that the small-scale and incompletion or heterogeneity of the samples maybe lead to different results of these studies. In this study, large-scale samples from 50 related GWAS studies (28,464 AD cases and 45,784 controls) were selected afresh from seven authoritative sources to reevaluate the effect of rs11136000 polymorphism to AD risk. Similarly, we identified that the minor allele variant of rs11136000 significantly decrease AD risk in Caucasian ethnicity using the allele, dominant and recessive model. Different from the results of the previous studies, however, the results showed a negligible or no association in Asian and Chinese populations. Collectively, our analysis suggests that, for Asian and Chinese populations, the variant of rs11136000 may be irrelevant to AD risk. We believe that these findings can help to improve the understanding of the AD's pathogenesis.

Genetic Variant rs755622 Regulates Expression of the Multiple Sclerosis Severity Modifier D-Dopachrome Tautomerase in a Sex-Specific Way.

Multiple sclerosis (MS) is a sex-specific autoimmune disease involving central nervous system. Previous studies determined that macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (DDT) sex-specifically affect MS progression. Moreover, other studies reported that rs755622 polymorphism in promoter region of MIF gene is associated with risk of MS and affects the promoter activity to regulate MIF expression in a sex-specific way. Given that MIF and DDT share a part of promoter sequence, we surmise that rs755622 can also regulate DDT expression in a sex-specific way. However, this has not yet been studied. Here, we used five large-scale expression quantitative trait loci (eQTLs) and two RNA-seq datasets from brain and blood to assess the potential influence of rs755622 variant on expression of DDT in different genders by the linear regression and differential expression analysis. The results show that the minor allele frequency of rs755622 and expression of DDT are significantly increased in males for MS subjects and this minor allele variant can significantly upregulate DDT expression for males but not females, which suggests that the regulation of DDT expression level by rs755622 can affect MS progression in males. These findings further support and expand conclusions of previous studies and may help to better understand the mechanisms of MS.

Integrated analysis of tobacco miRNA and mRNA expression profiles under PVY infection provids insight into tobacco-PVY interactions.

Potato virus Y (PVY) is a globally and economically important pathogen of potato, tobacco, tomato and other staple crops and caused significant yield losses and reductions in quality.To explore the molecular PVY-host interactions, we analysed changes in the miRNA and mRNA profiles of tobacco in response to PVY infection. A total of 81 differentially expressed miRNAs belonging to 29 families and 8133 mRNAs were identified. The Gene Ontology (GO) enrichment analyses showed that genes encoding the DNA/RNA binding, catalytic activity and signalling molecules were all significantly enriched. Moreover, 88 miRNA-mRNA interaction pairs were identified through a combined analysis of the two datasets. We also found evidence showing that the virus-derived siRNAs (vsiRNAs) from the PVY genome target tobacco translationally controlled tumor protein (NtTCTP) mRNA and mediate plant resistance to PVY. Together, our findings revealed that both miRNA and mRNA expression patterns can be changed in response to PVY infection and novel vsiRNA-plant interactions that may regulate plant resistance to PVY. Both provide fresh insights into the virus-plant interactions.

NtPHYB1K326, a homologous gene of Arabidopsis PHYB, positively regulates the content of phenolic compounds in tobacco.

Polyphenols are important secondary metabolites and bioactive compounds in plants. Light is a vital abiotic factor that greatly impacts the content of polyphenols in plants. In spite of their importance the mechanism of polyphenol regulation still remains unknown in tobacco. A phytochrome B homolog, NtPHYB1K326, was isolated from Nicotiana tabacum cv. K326 to investigate the role of light receptors in the regulation of polyphenol metabolism in tobacco leaves. Furthermore, role of NtPHYB1K326 in polyphenol metabolism was analyzed by over-expression and RNAi-silencing approaches. Consistent and complemented results indicated involvement of NtPHYB1K326 in the regulation of polyphenol metabolism in tobacco leaves. Moreover, high levels of NtPHYB1K326 transcripts favor the accumulation of chlorogenic acid and its isomers, the key polyphenol component in tobacco leaves. Transcriptome analysis was also carried out for exploring the regulation mechanism of NtPHYB1K326 in the polyphenol metabolism. Compared with WT, 1665 and 1421 differentially-expressed genes were found in NtPHYB1K326-GFP and NtPHYB1K326-RNAi transgenic lines, respectively. Among these, about 30 genes were related to phenylpropanoid pathway, which is predominantly involved in synthesis of polyphenols. Further evidences from quantitative RT-PCR confirmed that NtPHYB1K326 may control phenylpropanoid pathway through regulating the transcription of PAL4 (phenylalanine ammonialyase 4), 4CL1 (4-coumarate:coenzyme A ligase 1) and COMT (caffeic acid 3-O-methyltransferase) genes.

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance.

DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits.

[Influence of temperature on hydrolysis and acidogenesis of kitchen wastes in two-phase anaerobic digestion].

The experiment evaluated the solubilization rate and VFA production rate of kitchen wastes with controlled-temperature at 25 degrees C, 37 degrees C, 40 degrees C and 50 degrees C, and subsequently investigated the lactic acid production and constituents of individual organic acids at various temperature levels. Results show that the increase of temperature when below 37 degrees C may improve both hydrolysis and acidogenesis rates and acidogenesis rate decreases while hydrolysis rate increases above 37 degrees C. The maximum VFA concentration of 34.4 g x L(-1) was achieved at 37 degrees C and a higher hydrolysis rate of 82% was obtained at 50 degrees C. Formic and acetic acid were predominant VFA, while propionic and butyric acid took a relatively little part. And lactic acid concentration remained relatively high. Accroding to the experiment an optimum temperature to optimize both hydrolysis and acidification is 37 degrees C.