Microwave-Assisted Grafting of Coal onto Nitrogen-Doped Carbon Dots with a High Quantum Yield and Enhanced Photoluminescence Properties.

The fluorescent nitrogen-doped carbon dots (N-CDs) were synthesized via a facile one-pot solvothermal process using coal (Jin 15 Anthracite and Shaerhu lignite) as raw materials and dimethyl formamide (DMF) as the solvent, employing a microwave pyrolysis method. This approach demonstrates remarkable efficacy in the development of nitrogen-doped carbon dots (N-CDs) with a high quantum yield (QY). The N-CDs prepared have strong photoluminescence properties. Moreover, the obtained N-CDs emit blue PL and are easily dispersed in polymethyl methacrylate (PMMA), preserving the inherent advantages of N-CDs and the PMMA matrix. The JN-CDs exhibit a high quantum yield (QY) of 49.5% and a production yield of 25.7%, respectively. In contrast, the SN-CDs demonstrate a quantum yield of 40% and a production yield of 35.1%. It is worth noting that the production yield and quantum yield of coal-based carbon dots are inversely related indices. The lower metamorphic degree of subbituminous coal favors an enhanced product yield, while the higher metamorphic degree of anthracite promotes an improved quantum yield in the product, which may be attributed to the presence of amorphous carbon within it. Consequently, we propose and discuss potential mechanisms underlying N-CD formation.

Sequential thermal dissolution of two low-rank coals and characterization of their structures by high-performance liquid chromatography/time-of-flight mass spectrometry and gas chromatography/mass spectrometry.

RATIONALE: Gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography/time-of-flight mass spectrometry (HPLC/TOF-MS) were used to separate and reveal the molecular characteristics of organic matter in low-rank coals. METHODS: Six soluble portions (SPs) were obtained by sequential thermal dissolution (TD) of two low-rank coals in the order of cyclohexane, acetone and methanol solvents at 300°C. Organic matter with different molecular characteristics were enriched in eachTD extract, which was further separated and analyzed by GC/MS and HPLC/TOF-MS using an electrospray ionization source in positive mode to obtain a comprehensive understanding of the structural composition of coals. RESULTS: Low polarity compounds like alkanes and arenes have a better solubility in cyclohexane. Phorone has the highest relative abundance in the acetone SPs, and the main compounds detected in the methanol SPs are alcohols and phenols. According to the data from HPLC/TOF-MS, most of the oxygen atoms are in the form of carbonyl and alkoxy groups. The nitrogen-containing compounds in SPs are mainly saturated aliphatic amines and pyridines. The sulfur-containing compounds mainly exist in the form of thioalkanes and thiophenes. CONCLUSIONS: Non-destructive methods were used to obtain soluble matter from coals, and different chromatographic and mass spectrometric techniques were used to separate and analyze the organic matter in coals. Detailed molecular structural information was obtained for the efficient and clean utilization of low-rank coals.

Insight into molecular characteristics of a Chinese coal via separation, characterization, and data processing.

Dayan lignite was subjected to thermal dissolution sequentially with cyclohexane, acetone, and methanol. Each thermal dissolution extract was subjected to further separation/enrichment using column chromatography, which was sequentially eluted with petroleum ether, a mixture of ethyl acetate and petroleum ether (vol:vol = 1:1), and ethyl acetate. The three thermal dissolution extracts and nine enrichment subfractions were characterized by an Orbitrap mass spectrometry equipped with an atmospheric pressure chemical ionization ion source. The mass spectrometry data were also statistically analyzed by principal component analysis, which can reduce the dimensionality of data and classify multiple samples according to principal components. Identified compounds in the extracts and subfractions are classified into eight classes according to the heteroatom distribution. Hydrocarbon class is mainly presented in the petroleum ether fraction, and oxygen class, nitrogen class, and oxygen-nitrogen class are distributed in both petroleum ether/ethyl acetate and ethyl acetate subfractions. The combination of different analytical methods enhances the understanding of coal at the molecular level and provides important data for downstream refining processes.

Steroidal Glycosides from Allium tuberosum Seeds and Their Roles in Promoting Testosterone Production of Rat Leydig Cells.

A systematic phytochemical study on the components in the seeds of Allium tuberosum was performed, leading to the isolation of 27 steroidal glycosides (SGs 1-27). The structures of SGs were identified mainly by nuclear magnetic resonance and mass spectrometries as well as the necessary chemical evidence. In the SGs, 1-10 and 22-26 are new steroidal saponin analogues. An in vitro bioassay indicates that 1, 2, 7, 8, 10, 13-15, 20, 23, and 26 display promotional roles in testosterone production of rat Leydig cells with the EC50 values of 1.0 to 4.5 µM, respectively.

Insight into molecular information of Huolinguole lignite obtained by Fourier transform ion cyclotron resonance mass spectrometry and statistical methods.

RATIONALE: Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was applied to the characterization of organic compounds in coal extracts at the molecular level. Large volumes of data obtained by FT-ICR MS were processed via statistical methods to extract valuable information on the molecular structures and compositions of organic compounds in coal. METHODS: A low-rank coal was subjected to ultrasonic extraction sequentially with six solvents to separate and enrich species with different molecular characteristics. Complex mass spectra of the six extracts were obtained by a FT-ICR MS system equipped with two ionization sources. Two multivariate statistical methods, hierarchical clustering analysis (HCA) and principle component analysis (PCA), were introduced to mine useful information from the complex MS data and visually exhibit comprehensive molecular details in coal extracts. RESULTS: Similarities and differences between the 17 MS data sets from six coal extracts ionized by different ion sources were visually exhibited in plots via data processing using HCA and PCA. For HCA, all of the identified compounds were divided into seven classes (CH, O, N, S, ON, OS, and NS), and detailed differences in the relative abundance were revealed. In addition, PCA discriminated the differences in molecular composition for organic compounds from the six extracts. CONCLUSIONS: Multivariate statistical analysis is a promising methodology which can interpret the chemical composition of coals and coal derivatives at the molecular level, especially for the analysis of multiple complex samples presenting in a single plot.

Optimization of Ultrasonic-Microwave Assisted Extraction and Hepatoprotective Activities of Polysaccharides from Trametes orientalis.

Ultrasonic-microwave assisted extraction (UMAE) of Trametes orientalis polysaccharides was optimized by response surface methodology. Hepatoprotective effects of a purified T. orientalis polysaccharide (TOP-2) were evaluated by alcohol-induced liver injury model mice. The optimal UMAE parameters were indicated as below: ratio of water to raw material 28 mL/g, microwave power 114 W, extraction time 11 min. The polysaccharides yield was 7.52 ± 0.12%, which was well consistent with the predicted value of 7.54%. Pre-treatment with TOP-2 effectively increased the liver index and spleen index in alcohol-treated mice. The elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of mice after alcohol exposure were inhibited by TOP-2 administration. The liver tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) levels have decreased significantly as a result of alcohol exposure, while pre-treatment with TOP-2 could mitigate these consequences. Furthermore, pre-treatment with TOP-2 could efficiently boost the superoxidase dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities, and observably constrain the malondialdehyde (MDA) level. The findings suggest that TOP-2 might be useful for alleviating the alcohol-induced hepatotoxicity via its antioxidant and anti-inflammatory potential.

Application of mass spectrometry in the characterization of chemicals in coal-derived liquids.

Coal-derived liquids (CDLs) are primarily generated from pyrolysis, carbonization, gasification, direct liquefaction, low-temperature extraction, thermal dissolution, and mild oxidation. CDLs are important feedstocks for producing value-added chemicals and clean liquid fuels as well as high performance carbon materials. Accordingly, the compositional characterization of chemicals in CDLs at the molecular level with advanced analytical techniques is significant for the efficient utilization of CDLs. Although reviews on advancements have been rarely reported, great progress has been achieved in this area by using gas chromatography/mass spectrometry (GC/MS), two-dimensional GC-time of flight mass spectrometry (GC × GC-TOFMS), and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). This review focuses on characterizing hydrocarbon, oxygen-containing, nitrogen-containing, sulfur-containing, and halogen-containing chemicals in various CDLs with these three mass spectrometry techniques. Small molecular (< 500 u), volatile and semi-volatile, and less polar chemicals in CDLs have been identified with GC/MS and GC × GC-TOFMS. By equipped with two-dimensional GC, GC × GC-TOFMS can achieve a clearly chromatographic separation of complex chemicals in CDLs without prior fractionation, and thus can overcome the disadvantages of co-elution and serious peak overlap in GC/MS analysis, providing much more compositional information. With ultrahigh resolving power and mass accuracy, FT-ICR MS reveals a huge number of compositionally distinct compounds assigned to various chemical classes in CDLs. It shows excellent performance in resolving and characterizing higher-molecular, less volatile, and polar chemicals that cannot be detected by GC/MS and GC × GC-TOFMS. The application of GC × GC-TOFMS and FT-ICR MS to chemical characterization of CDLs is not as prevalent as that of petroleum and largely remains to be developed in many respects. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:543-579, 2017.

Evaluation of coal-related model compounds using tandem mass spectrometry.

RATIONALE: Gas chromatography/mass spectrometry (GC/MS) is a routine and basic instrumental method for the analysis of complex coal conversion products in the chemical industry. To further enhance the practical potential of GC/MS in chemical industry, a tandem MS method for the selection of ion pairs applied in monitoring coal conversions was established using GC/quadrupole time-of-flight MS (GC/Q-TOF MS). The corresponding fragmentation pathways were explored and suitable ion pairs were screened. METHODS: Fourteen coal-related model compounds (CRMCs) were analyzed using GC/Q-TOF MS with different collision-induced dissociation (CID) energies (5-20 eV). The fragmentation pathways can offer a better understanding of chemical bond breaking, hydrogen transfer, rearrangement reactions and elimination of neutral fragments for CRMCs during the CID process. RESULTS: The precursor ions of aromatic hydrocarbons without alkyl chains were difficult to fragment with a CID energy of 20 eV. But aromatic hydrocarbons with branched chains were prone to fragment via the loss of alkyl chains and further fragmented through ring-opening reactions. Compared with the Calk Car bond, the Car Car bond was difficult to fragment due to its high bond dissociation energy. The existence of heteroatoms facilitated fragmentation that was conducive to the screening of ion pairs. CONCLUSIONS: The CID technique of GC/Q-TOF MS will contribute to studies on the organic composition of coals and to building monitoring methods for coal conversions via fragmentation and ion pair selection.

Rapid analysis of carboxylic acids and esters with a direct analysis in real time ion source.

RATIONALE: Coal oxidation produces carboxylic acids (CAs), including aliphatic acids, benzoic acids, and benzenepolycarboxylic acids, which are important fine chemicals which could be used to understand the structural features of coals. However, detecting CAs usually presents great challenges due to extremely troublesome pretreatments. Therefore, it is essential to develop an analytical method for the rapid detection of CAs from coal oxidation. METHODS: A series of model compounds (MCs) of oxidation products and two practical samples were investigated by direct analysis in real time time-of-flight mass spectrometry (DART-TOFMS) under three different analytical conditions (ionizing gas temperature, organic solvent, and MC concentration). RESULTS: Ionizing methyl benzoate, dimethyl phthalate, and dimethyl adipate produces typical ions of methyl esters, including [M - OCH3 ]+ , [M + H]+ , and [M + NH4 ]+ . In contrast, the characteristic ions generated from CAs are polymer ions, such as [2 M + NH4 ]+ , [3 M + NH4 ]+ , [4 M + NH4 ]+ , and [5 M + NH4 ]+ , indicating the strong intermolecular hydrogen-bond interaction among CAs. CONCLUSIONS: Results suggest that DART-TOFMS could rapidly analyze CAs or esters in coal oxidation products according to their typical ions to further gain deep insights into the coal structure.

Analysis of soluble components in coals and interpretations for the complex mass spectra.

RATIONALE: The deduction of useful information from the mass spectra of a complex mixture like coals remains difficult, which limits the clean and efficient utilization of coals. It is necessary to explore the data interpretation methods for mass spectra and visualize the analytical data of coals for industrial utilization such as feedstock selection. METHODS: Coal sample and methanol were mixed and heated to 310 °C and kept at that temperature for 2 h. The solvent was under supercritical state at 310 °C and the solubility for the solid mixture increased. Soluble products from thermal dissolution of two Chinese coals were analyzed by high-performance liquid chromatography/atmospheric pressure chemical ionization orbitrap mass spectrometry. RESULTS: The iso-abundance plot for molecules in coals was upgraded to display the distributions of isomers which are indicated as concentric circles or triangles with the same carbon number and value of double-bond equivalent. The concentration ratio was introduced from economics to describe the content inequality of organic species within the same class of coal molecules. CONCLUSIONS: Interpretation methods for mass spectra visualize and simplify the understanding of complex components in coals for industrial utilization. Coals with a high concentration ratio for a specific class should take priority as a feedstock for chemicals and receive more attention. Copyright © 2016 John Wiley & Sons, Ltd.

Sequential ultrasonic extraction of a Chinese coal and characterization of nitrogen-containing compounds in the extracts using high-performance liquid chromatography with mass spectrometry.

Dongming lignite was sequentially extracted with petroleum ether, carbon disulfide, methanol, acetone, and isometric carbon disulfide/acetone mixed solvent at room temperature to afford extracts 1-5, respectively. High-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry was used to separate and characterize heteroatomic species in the extracts at molecular level. Molecular mass of compounds in the extracts is mainly distributed from 300 to 800 u, and the relative abundance of compounds with molecular mass over 800 u in the carbon disulfide extract is 135 times of that in the petroleum ether extract. The acetone extract has the highest relative abundance for organonitrogen compounds. Double bond equivalence numbers of detected species indicate that most of the organonitrogen compounds contain N-heterocyclic aromatic rings, including pyridine, quinoline and pyrrole. Some organonitrogen isomers in Dongming lignite were separated and identified by high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry, and the corresponding structural information was proposed.

Removal of hexavalent chromium from aqueous solution by calcined Zn/Al-LDHs.

In this study, Zn/Al-layered double hydroxides (Zn/Al-LDHs) were synthesized by a co-precipitation method and characterized with X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy. Then the hexavalent chromium Cr(VI) adsorption experiments on calcined Zn/Al-LDHs were carried out to analyze the effects of pH, temperature, adsorption time, initial Cr(VI) concentration and adsorbent dosage on the removal of Cr(VI) from aqueous solutions. The maximum adsorption capacity for Cr(VI) on calcined Zn/Al-LDHs under optimal conditions was found to be over 120 mg/g. The kinetic and isotherm of Cr(VI) adsorption on calcined Zn/Al-LDHs can be described with the pseudo-second-order kinetic model and Langmuir isotherm, respectively.

Investigation on Compositional Structures of the Organic Matter and Biomarker Distributions in Shengli Lignite.

The structural characteristics of the organic matter and biomarker distributions in Shengli lignite (SL) were comprehensively studied by combining a variety of modern analytical techniques and solvent extraction/thermal dissolution. Characterization of SL with Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, solid 13C nuclear magnetic resonance spectroscopy and thermogravimetry showed that organic matter in SL is rich in oxygen functional groups, such as C-O, >C=O, and -COOH, and hydrogen bonds. The hydrogen bonds mainly include -OH···π, self-associated -OH, -OH···ether O, tightly bound cyclic -OH, -OH···N, -COOH dimers, and -SH···N. The highest content of organic nitrogen and sulfur on SL surface are pyrrole nitrogen and aromatic sulfur, respectively. The proportions of aromatic and aliphatic carbons in SL are about 58% and 39%, respectively. The aromatic carbon is mainly composed of protonated aromatic and aromatic bridged carbons; methylene carbon has the highest content among the aliphatic carbons, with chains of average length of 1.43 carbon atoms. The average number of aromatic structural units in the carbon skeleton of SL is about 3, and each aromatic structural unit contains an average of 1-2 substituent groups. Thermogravimetric analysis clarified the distribution of the main types of covalent bonds in SL and their possible cracking temperatures during pyrolysis. The extracts and soluble portion of thermal dissolution from SL were analyzed by a gas chromatograph/mass spectrometer, and a series of biomarkers were identified, mainly concentrated in petroleum ether extract and cyclohexane thermal soluble portion. These included long-chain n-alkanes, isoprenoid alkanes, long-chain n-alkenes, terpenoids, n-alkan-2-ones, long-chain n-alkylbenzene, and long-chain n-alkyltoluene. The comprehensive characterization of the organic matter and the distribution of related biomarkers provided an important scientific basis for understanding the molecular structural characteristics and geochemical information on SL.

Characteristics of the Blockage from Air Nozzle Guide Duct in Circulating the Fluidized-Bed Coal Gasifier and Its Formation Mechanism.

Blockage is often generated in the air nozzle guide duct in a circulating fluidized-bed coal gasifier (CFBG), especially with Zhundong sub-bituminous coal (ZSBC) as the raw material. A typical example is found in one CFBG sample from Xinjiang Yihua Chemical Industry Co, Ltd. The serious blockage can be observed obviously. As so far, it is not clear for the characteristics and generation mechanism of the blockage. For analysis, the blockage can be classified into two parts, wall-layer blockage (WLB) and center-layer blockage (CLB). To inhibit its formation, it is of significance to analyze the composition, surface morphology, and formation mechanism of the two blockages. In our experiments, WLB and CLB were tested by XRF, XRD, FTIR, SEM-EDS, and SEM-mapping methods. Results showed that WLB presents high content of Fe, Cr, and Ni, and Fe mainly existed in the form of metal oxides. CLB is dominated by Si (43.04%), derived from silica and alkali and alkaline-earth metals silicates, and the migration of Fe, Cr, and Ni elements from the duct material was observed. Compared with WLB, from FTIR analysis, CLB contains more inorganic minerals, and the absorption peak of inorganic minerals is mainly attributed to asymmetric Si-O-Si. Many fine particles are attached to the surface of the WLB, while the surface of the CLB is smooth, and there is noticeable raised texture, which is presumed to be the result of particle melting and agglomerating as the bottom ash enters the duct in the gasification process. For the formation of the blockage, this paper speculates that it is mainly due to the difference in flow resistance near the air nozzle outlet, resulting in the formation of a flow dead zone at the bottom of the gasifier, which leads to large amounts of ash overcoming the outlet resistance and leaking into the air nozzle, and next, the ash corrodes in the tube, resulting in wall deposition and ultimately blocking the air guide duct. Two methods can be tried to avoid or inhibit the formation of blockage in the duct, including optimizing air nozzle with more wear-resistant and heat-resistant materials and adjusting the distance between air nozzles to avoid mutual interference from ash particles.

Separation and Analysis of Oxygen-Containing Compounds in a Shaanxi Middle/Low-Temperature Coal Tar.

A middle/low-temperature coal tar (M/LTCT) was obtained from a low-temperature carbonization plant in Shaanxi, China. The M/LTCT was separated into light components and coal tar pitch through extraction. A series of alkanes, aromatic hydrocarbons, oxygen-containing arenes (OCAs), and nitrogen-containing arenes were fractionated from light components by medium-pressure preparative chromatography with gradient elution using petroleum ether and ethyl acetate. They were analyzed using a gas chromatography-mass spectrometer (GC-MS) and a Fourier transform infrared spectrometer. The OCAs were analyzed by a Fourier transform Orbitrap MS (quadrupole exactive Orbitrap mass spectrometer), and the molecular distribution of the O 1-O 6 species was studied. OCAs are mainly oxygen-containing aromatic compounds, including aromatic phenols, furans, alkoxy aromatic hydrocarbons, aromatic ethers, aromatic aldehydes, aromatic ketones, and aromatic acids. The position of the oxygen atom on the aromatic ring and the condensation form of the aromatic ring are studied.

Production of Diethyl Maleate via Oxidative Depolymerization of Organosolv Lignin from Wheat Stalk over the Cooperative Acidic Ionic Liquid Pair.

Lignin, the second largest component of biomass, is considered as an important alternative source of fossil reserves for the production of fuels and chemicals. Here, we developed a novel method to oxidatively degrade organosolv lignin into value-added four-carbon esters, particularly diethyl maleate (DEM), with the cooperative catalyst consisting of 1-(3-sulfobutyl) triethylammonium hydrogen sulfate ([BSTEA]HSO4) and 1-butyl-3-methylimidazolium ferric chloride ([BMIM]Fe2Cl7). Under optimized conditions (1.00 MPa initial O2 pressure, 160 °C, 5 h), the lignin aromatic ring was effectively cleaved by oxidation to form DEM with a yield of 15.85% and a selectivity of 44.25% in the presence of the synergistic catalyst of [BMIM]Fe2Cl7-[BSMIM]HSO4 (1/3, mol/mol). The structure and composition analysis of lignin residues and liquid products confirmed that the aromatic units in lignin were effectively and selectively oxidized. Furthermore, the catalytic oxidation of lignin model compounds was explored for obtaining a possible reaction pathway of oxidative cleavage of lignin aromatic units to DEM. This study provides a promising alternative method for the production of traditional petroleum-based chemicals.

Producing Lignin and Ethyl Levulinate from Wheat Stalk Using 1-(3-Sulfobutyl) Triethylammonium Hydrogen Sulfate and USY Zeolite.

The facile, green, and efficient strategy for the separation of lignin from straw and subsequent production of value-added chemicals is crucial to the current utilization of straw. Herein, up to 23.72% of lignin was isolated from wheat stalk over cheap and green 1-(3-sulfobutyl) triethylammonium hydrogen sulfate ([BSTEA]HSO4) in aqueous ethanol (Vethanol: Vwater = 4:1). The acquired lignin was verified as a p-hydroxyphenyl-guaiacyl-syringyl type, which had a narrower molecular weight distribution, better thermal stability, and higher purity compared with those of the lignin obtained using 1-methyl-3-(4-sulfobutyl)-imidazolium hydrogen sulfate and 1-(3-sulfobutyl) pyridinium hydrogen sulfate. Moreover, a carbohydrate-rich liquid containing [BSTEA]HSO4 was obtained by water removal from the waste liquid after lignin separation and further converted to ethyl levulinate (EL) by a one-pot process in the presence of inexpensive and stable USY zeolite. The yield of EL reached 30.23% at 200 °C for 60 min over the presence of 40% [BSTEA]HSO4 and 60% USY zeolite. Under optimal conditions, the yields of lignin and EL can respectively reach 83.89 and 72.28% of those catalyzed by a fresh catalyst after five cycles. In short, the above-mentioned methods present a green, economic, and efficient route for the extraction of lignin and further treatment of the liquid waste generated during the extraction process.

Analysis of Pyrolysis Performance and Molecular Structure of Five Kinds of Low-Rank Coals in Xinjiang Based on the TG-DTG Method.

Taking five coal samples (FCSs) in Xinjiang as the research object, characterizations such as proximate analysis, ultimate analysis, Fourier transform infrared (FTIR), and thermogravimetry-differential thermal analysis (TG-DTG) were carried out. The Coats-Redfern model was used to simulate pyrolysis kinetics of FCSs under different reaction orders (ROs). The results showed that except for HSBC, the R 2 of the other four coal samples are all higher than 0.9, which showed a good correlation effect. FCSs present similar reaction activation energy in the same RO and temperature range. Results of FTIR showed that the hydroxyl groups of FCSs, in the range of 3100-3600 cm-1, were mainly self-associated hydroxyl hydrogen bonds and hydroxyl π bonds, and they occupied over 63%. Among them, the pyrolysis characteristic index (D) of XBC was 4.139 × 10-6, higher than those of other samples, and it showed good pyrolysis performance. Moreover, by reducing the temperature range appropriately, the fitting results showed a better correlation effect.

A zinc finger suppressor involved in stress resistance, cell wall integrity, conidiogenesis, and autophagy in the necrotrophic fungal pathogen Alternaria alternata.

The tangerine pathotype of Alternaria alternata can withstand high-level reactive oxygen species (ROS). By analyzing loss- and gain-of-function mutants, this study demonstrated that a Cys2His2 zinc finger-containing transcription regulator, A. alternata Stress Response Regulator 1 (AaSRR1), plays a negative role in resistance to peroxides and singlet-oxygen-generating compounds. AaSRR1 plays no role in cellular susceptibility or resistance to superoxide-producing compounds. AaSRR1 also negatively regulates conidiogenesis, maintenance of cell wall and membrane integrities, and chitin biosynthesis. Some wild-type hyphae displayed necrosis after exposure to 30 mM H2O2, whereas AaSRR1 deficient mutant (ΔAaSRR1) hyphae had visible granules and vacuoles. sGFP-AaATG8 proteolysis assays revealed that H2O2 and starvation could trigger autophagy formation in both wild type and ΔAaSRR1. Autophagy occurred at higher rates in ΔAaSRR1 than wild type under both conditions, particularly after H2O2 treatments, indicating that autophagy might contribute to ROS resistance. Upon exposure to H2O2 or under starvation, AaSRR1 was translocated into the nucleus, even though the expression of AaSRR1 was decreased. AaSRR1 is required for vegetative growth but is dispensable for fungal virulence as assayed on detached calamondin leaves. AaSRR1 suppressed the expression of the gene encoding a HOG1 mitogen-activated protein (MAP) kinase implicated in ROS resistance. Mutation of AaSRR1 increased catalase activity but decreased superoxide dismutase activity, leading to fewer ROS accumulation in the cytosol. Nevertheless, our results indicated that AaSRR1 is a transcription suppressor for ROS resistance. This study also revealed tradeoffs between stress responses and hyphal growth in A. alternata.

Pexophagy is critical for fungal development, stress response, and virulence in Alternaria alternata.

Alternaria alternata can resist high levels of reactive oxygen species (ROS). The protective roles of autophagy or autophagy-mediated degradation of peroxisomes (termed pexophagy) against oxidative stress remain unclear. The present study, using transmission electron microscopy and fluorescence microscopy coupled with a GFP-AaAtg8 proteolysis assay and an mCherry tagging assay with peroxisomal targeting tripeptides, demonstrated that hydrogen peroxide (H2 O2 ) and nitrogen depletion induced autophagy and pexophagy. Experimental evidence showed that H2 O2 triggered autophagy and the translocation of peroxisomes into the vacuoles. Mutational inactivation of the AaAtg8 gene in A. alternata led to autophagy impairment, resulting in the accumulation of peroxisomes, increased ROS sensitivity, and decreased virulence. Compared to the wild type, ΔAaAtg8 failed to detoxify ROS effectively, leading to ROS accumulation. Deleting AaAtg8 down-regulated the expression of genes encoding an NADPH oxidase and a Yap1 transcription factor, both involved in ROS resistance. Deleting AaAtg8 affected the development of conidia and appressorium-like structures. Deleting AaAtg8 also compromised the integrity of the cell wall. Reintroduction of a functional copy of AaAtg8 in the mutant completely restored all defective phenotypes. Although ΔAaAtg8 produced wild-type toxin levels in axenic culture, the mutant induced a lower level of H2 O2 and smaller necrotic lesions on citrus leaves. In addition to H2 O2 , nitrogen starvation triggered peroxisome turnover. We concluded that ΔAaAtg8 failed to degrade peroxisomes effectively, leading to the accumulation of peroxisomes and the reduction of the stress response. Autophagy-mediated peroxisome turnover could increase cell adaptability and survival under oxidative stress and starvation conditions.