Gene identification, expression analysis, and molecular docking of SAT and OASTL in the metabolic pathway of selenium in Cardamine hupingshanensis.

KEY MESSAGE: Identification of selenium stress-responsive expression and molecular docking of serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) in Cardamine hupingshanensis. A complex coupled with serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) is the key enzyme that catalyzes selenocysteine (Sec) synthesis in plants. The functions of SAT and OASTL genes were identified in some plants, but it is still unclear whether SAT and OASTL are involved in the selenium metabolic pathway in Cardamine hupingshanensis. In this study, genome-wide identification and comparative analysis of ChSATs and ChOASTLs were performed. The eight ChSAT genes were divided into three branches, and the thirteen ChOASTL genes were divided into four branches by phylogenetic analysis and sequence alignment, indicating the evolutionary conservation of the gene structure and its association with other plant species. qRT-PCR analysis showed that the ChSAT and ChOASTL genes were differentially expressed in different tissues under various selenium levels, suggesting their important roles in Sec synthesis. The ChSAT1;2 and ChOASTLA1;2 were silenced by the VIGS system to investigate their involvement in selenium metabolites in C. hupingshanensis. The findings contribute to understanding the gene functions of ChSATs and ChOASTLs in the selenium stress and provide a reference for further exploration of the selenium metabolic pathway in plants.

Molecularly imprinted resin modified with ionic liquid for dispersive filter extraction and determination of perfluoroalkyl acids in eggs.

Perfluoroalkyl acids (PFAAs) are emerging pollutants that endangers food safety. Developing methods for the selective determination of trace PFAAs in complex samples remains challenging. Herein, an ionic liquid modified porous imprinted phenolic resin-dispersive filter extraction-liquid chromatography-tandem mass spectrometry (IL-PIPR-DFE-LC-MS/MS) method was developed for the determination of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in eggs. The new IL-PIPR adsorbent was prepared at room temperature, which avoids the disorder and instability of the template at high temperatures. The imprinting factor of IL-PIPR for PFOA and PFOS exceeded 7.3. DFE, combined with IL-PIPR (15 mg), was used to extract PFOA and PFOS from eggs within 15 min. The established method exhibits low limits of detection (0.01-0.02 ng/g) and high recoveries (84.7%-104.7%), which surpass those of previously reported methods. This work offers a new approach to explore advanced imprinted adsorbents for PFAAs, efficient sample pretreatment technique, and analytical method for pollutants in foods.

Whole genome identification, molecular docking and expression analysis of enzymes involved in the selenomethionine cycle in Cardamine hupingshanensis.

BACKGROUND: The selenomethionine cycle (SeMTC) is a crucial pathway for the metabolism of selenium. The basic bioinformatics and functions of four enzymes involved in the cycle including S-adenosyl-methionine synthase (MAT), SAM-dependent methyltransferase (MTase), S-adenosyl-homocysteine hydrolase (SAHH) and methionine synthase (MTR), have been extensively reported in many eukaryotes. The identification and functional analyses of SeMTC genes/proteins in Cardamine hupingshanensis and their response to selenium stress have not yet been reported. RESULTS: In this study, 45 genes involved in SeMTC were identified in the C. hupingshanensis genome. Phylogenetic analysis showed that seven genes from ChMAT were clustered into four branches, twenty-seven genes from ChCOMT were clustered into two branches, four genes from ChSAHH were clustered into two branches, and seven genes from ChMTR were clustered into three branches. These genes were resided on 16 chromosomes. Gene structure and homologous protein modeling analysis illustrated that proteins in the same family are relatively conserved and have similar functions. Molecular docking showed that the affinity of SeMTC enzymes for selenium metabolites was higher than that for sulfur metabolites. The key active site residues identified for ChMAT were Ala269 and Lys273, while Leu221/231 and Gly207/249 were determined as the crucial residues for ChCOMT. For ChSAHH, the essential active site residues were found to be Asn87, Asp139 and Thr206/207/208/325. Ile204, Ser111/329/377, Asp70/206/254, and His329/332/380 were identified as the critical active site residues for ChMTR. In addition, the results of the expression levels of four enzymes under selenium stress revealed that ChMAT3-1 genes were upregulated approximately 18-fold, ChCOMT9-1 was upregulated approximately 38.7-fold, ChSAHH1-2 was upregulated approximately 11.6-fold, and ChMTR3-2 genes were upregulated approximately 28-fold. These verified that SeMTC enzymes were involved in response to selenium stress to varying degrees. CONCLUSIONS: The results of this research are instrumental for further functional investigation of SeMTC in C. hupingshanensis. This also lays a solid foundation for deeper investigations into the physiological and biochemical mechanisms underlying selenium metabolism in plants.

Development of a three-dimensional porous ionic liquid-chitosan-graphene oxide aerogel for efficient extraction and detection of polyhalogenated carbazoles in sediment samples.

The three-dimensional porous ionic liquid-chitosan-graphene oxide aerogel (IL-CS-GOA) monolithic adsorbent with a through-hole structure was prepared using natural chitosan (CS) as the skeletal framework, graphene oxide (GO) as the support to provide mechanical strength, and ionic liquid (IL) as the porogen and modifier. The resulting IL-CS-GOA demonstrated a fluffy and porous structure with various pore sizes and excellent regeneration capability (over six cycles). Its specific surface area exceeded that of CS-GOA and IL-GOA by more than 7 times, enhancing its polyhalogenated carbazoles (PHCZs) adsorption capacity. Within 5 min, IL-CS-GOA (1.0 mg) exhibited adsorption amounts of 539 ng mg-1 for 3-bromocarbazole (3-BCZ), 716 ng mg-1 for 2,7-dibromocarbazole (2,7-BCZ), and 798 ng mg-1 for 1,3,6,8-tetrabromocarbazole (1,3,6,8-BCZ), showcasing its rapid mass transfer and high adsorption capabilities. IL-CS-GOA was utilized as the adsorbent for glass dropper extraction (GDE) in conjunction with gas chromatography-mass spectrometry (GC-MS/MS), to develop a highly efficient and accurate method for determining PHCZs in sediments. Under optimal conditions, the established method exhibited a wide linear range (0.4-250 ng g-1, r ≥ 0.9990), low detection limits (0.04-0.24 ng g-1), and satisfactory recoveries (80.5 %-93.8 %), enabling the accurate and rapid detection of PHCZs in sediment samples. This study presents a novel approach for creating three-dimensional porous aerogels, introduces a new form of sample pretreatment using GDE with a monolithic adsorbent, and offers a new method for the determination of PHCZs in environmental matrices.

Ant nest-like hierarchical porous imprinted resin-dispersive solid-phase extraction for selective extraction and determination of polychlorinated biphenyls in milk.

Polychlorinated biphenyls (PCBs) are persistent toxic, organic chemicals that tend to accumulate in the food chain. This study reports the rapid and selective extraction and determination of PCBs (PCB81, 153, 105, 126, and 157) in milk samples by a dispersive solid-phase extraction (DSPE) coupled with gas chromatography-tandem mass spectrometry (GC-MS/MS). An ionic liquid-molecularly imprinted porous resin (IL-MIPPR) as a DSPE adsorbent was synthesized from m-aminophenol, formaldehyde, and 2,2'-benzidinedisulfonic acid as the monomer, crosslinker, and virtual template, respectively. The IL-MIPPR had a fast mass transfer (1.0 min) and good selectivity (imprinting factors of 1.8-3.0). The IL-MIPPR - DSPE - GC-MS/MS method exhibited good linearity (R2 ≥ 0.9995), the limit of detections (LODs) < 0.6 pg/g, and the recoveries ranged from 82.8 % to 106 % with relative standard deviations ≤ 6.6 %. This method is thus better than previously reported methods in terms of the LOD, the adsorbent dosage, and the extraction time.

Novel molecularly imprinted phenolic resin-dispersive filter extraction for rapid determination of perfluorooctanoic acid and perfluorooctane sulfonate in milk.

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are emerging pollutants that threaten food safety. Herein, a rapid, accurate, and selective method for determination of PFOA and PFOS in milk was established by using new molecularly imprinted phenolic resin (MIP-PR) as the adsorbent of dispersive filter extraction (DFE) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MIP-PR was synthesized at room-temperature using m-aminophenol, glutaraldehyde, and perfluorononanoic acid as the monomers, cross-linkers, and virtual templates, respectively, and exhibited rapid mass transfer (30 s), high selectivity (imprinted factors > 3.7), and large adsorption capacity (>54.6 mg/g). Compared with reported methods, the developed MIP-PR-DFE method is fast, selective, inexpensive, and shows good purification and enrichment efficiency. The proposed MIP-PR-DFE-LC-MS/MS exhibited low limits of detection (0.006-0.022 ng/mL), high recoveries (94.7-109 %), and good precision (RSDs ≤ 9.5 %). This study provides a new idea for the development of imprinted resin adsorbents for perfluorinated compound, and a new method for sample pretreatment for monitoring of PFOA and PFOS in food.

Gene Identification, expression analysis and molecular docking of ATP sulfurylase in the selenization pathway of Cardamine hupingshanensis.

BACKGROUND: ATP sulfurylase (ATPS) is a crucial enzyme for the selenate assimilation pathway in plants. RESULTS: In this study, genome-wide and comparative analyses of ATPS in Cardamine hupingshanensis, including sequence and structural analyses, were performed. The expression of ChATPS gene family members in C. hupingshanensis under selenium (Se) stress was also investigated, and our results suggest that ChATPS1-2 play key roles in the response to Se stress. Nine ATPS genes were found from C. hupingshanensis, which share highly conserved sequences with ATPS from Arabidopsis thaliana. In addition, we performed molecular docking of ATP sulfurylase in complex with compounds ATP, selenate, selenite, sulfate, and sulfite. ChAPS3-1 was found to have stronger binding energies with all compounds tested. Among these complexes, amino acid residues Arg, Gly, Ser, Glu, and Asn were commonly present. CONCLUSION: Our study reveals the molecular mechanism of C. hupingshanensis ATP sulfurylase interacting with selenate, which is essential for understanding selenium assimilation. This information will guide further studies on the function of the ChATPS gene family in the selenium stress response and lay the foundation for the selenium metabolic pathway in higher plants.

Green synthesis of phloroglucinol-urotropine porous polymer: Ingenious miniaturized solid phase extraction for efficient purification and determination of polycyclic aromatic hydrocarbons in lotus roots.

Polycyclic aromatic hydrocarbons (PAHs) posed a serious threat to food safety and human health due to long-term emission. In this work, a new method was established using phloroglucinol-urotropine porous polymer (PU-PP) in a pipette tip for solid phase extraction (PT-SPE) for the first time and used prior to determination of four PAHs (phenanthrene, anthracene, fluoranthene, and pyrene) in lotus roots. Synthesis of the PU-PP adsorbent was green compared with alternatives; urotropine was used as a cross-linker and ethanol-water as the solvent. PU-PP-based PT-SPE had the advantages of low solvent consumption, good purification, practicability, stability, and low-cost. The proposed pre-purification method offered low limits of detection (0.09-0.28 ng/g) and good recoveries (84.6-114.3 %, RSDs ≤ 5.6 %) for determination of the four PAHs, which were detected at trace concentrations in samples. This new method provides an alternative for monitoring trace pollutants in aquatic plant ingredients.

Ionic Liquid Modified Porous Polymer as a Dispersive Filter Extraction Adsorbent for Simple, Sensitive, and Efficient Determination of Chlorotriazine Herbicides in Irrigation Water.

Triazine herbicides (TRZHs) are widely used in agricultural production, but their improper use can threaten the environment and organisms. Herein, rapid extraction of four chlorotriazine herbicides (Cl-TRZHs) in irrigation water was achieved using an ionic liquid modified porous m-aminophenol formaldehyde resin (IL-MAPFR) as a dispersive filter extraction (DFE) adsorbent. The IL-MAPFR shows excellent adsorption performance for four Cl-TRZHs (simazine, cyanazine, atrazine, and terbuthylazine), with a large specific surface area (158.1 m2 g-1) and fast mass transfer (2 min). The adsorption process conforms to the pseudo-second-order kinetics and Freundlich isotherm models. The four Cl-TRZHs were concentrated 12-16-fold after the IL-MAPFR-DFE method. Coupled with high-performance liquid chromatography, an accurate and sensitive determination method for four Cl-TRZHs in irrigation water was established, with low detection limit (0.11-0.20 ng mL-1), high recoveries (91.5-110%), and excellent precision (relative standard deviations (RSDs) ≤ 8.4%). This method provides technical support for agricultural irrigation water quality monitoring and has great application potential in water safety, especially pesticide residues.

Increased Drought Resistance 1 Mutation Increases Drought Tolerance of Upland Rice by Altering Physiological and Morphological Traits and Limiting ROS Levels.

To discover new mutants conferring enhanced tolerance to drought stress, we screened a mutagenized upland rice (Oryza sativa) population (cv. IAPAR9) and identified a mutant, named idr1-1 (increased drought resistance 1-1), with obviously increased drought tolerance under upland field conditions. The idr1-1 mutant possessed a significantly enhanced ability to tolerate high-drought stresses. Map-based cloning revealed that the gene LOC_Os05g26890, residing in the mapping region of IDR1 locus, carried a single-base deletion in the idr1-1 mutant. IDR1 encodes the Gα subunit of the heterotrimeric G protein (also known as RGA1), and this protein was localized in nucleus and to plasma membrane or cell periphery. Further investigations indicated that the significantly increased drought tolerance in idr1-1 mutants stemmed from a range of physiological and morphological changes, including greater leaf potentials, increased proline contents, heightened leaf thickness and upregulation of antioxidant-synthesizing and drought-induced genes, under drought-stressed conditions. Especially, reactive oxygen species (ROS) production might be remarkably impaired, while ROS-scavenging ability appeared to be markedly enhanced due to significantly elevated expression of ROS-scavenging enzyme genes in idr1-1 mutants under drought-stressed conditions. In addition, idr1-1 mutants showed reduced expression of OsBRD1. Altogether, these results suggest that mutation of IDR1 leads to alterations in multiple layers of regulations, which ultimately leads to changes in the physiological and morphological traits and limiting of ROS levels, and thereby confers obviously increased drought tolerance to the idr1-1 mutant.

One pot green synthesis of m-aminophenol-urea-glyoxal resin as pipette tip solid-phase extraction adsorbent for simultaneous determination of four plant hormones in watermelon juice.

Plant hormones (PHs) are a type of pesticide that can potentially affect human health. Therefore, their quantitative detection is particularly important. In this study, a green and economic method for the simultaneous extraction and determination of four PHs, namely thidiazuron, forchlorfenuron, 1-naphthylacetic acid, and 2-naphthoxyacetic acid, in watermelon juice was developed by using m-aminophenol-urea-glyoxal resin as the adsorbent for pipette tip solid phase extraction (PT-SPE) coupled with liquid chromatography. The resin was synthesized via a simple (one pot hydrothermal synthesis) and green (ethanol as the solvent and glyoxal as crosslinking agent) process. The synthesized resin possesses multiple functional groups (hydroxyl, amino, and imino, among others), high adsorption capacity, larger specific surface area than the urea-glyoxal resin and m-aminophenol-glyoxal resin, and can be regenerated easily. The PT-SPE device is simple, cheap, and easy to obtain, and the adsorbent dosage is only 5.0 mg. The proposed method has a wide linear detection range, high recovery, good precision, and high sensitivity, and satisfies the measurement requirements for detecting trace levels of PHs in fruits and vegetables.

Imidazolium ionic-liquid-modified phenolic resin for solid-phase extraction of thidiazuron and forchlorfenuron from cucumbers.

An imidazolium ionic-liquid-modified phenolic resin (ILPR) was synthesized using 3-aminophenol as a functional monomer, glyoxylic acid as a green cross-linker, and polyethylene glycol 6000 as a porogen. The obtained ILPR showed better extraction of benzoylurea plant hormones thidiazuron and forchlorfenuron than the unmodified phenolic resin because the imidazolium IL provides more interaction modes with the analytes. ILPR, as a tailored adsorbent for solid-phase extraction, was coupled with high-performance liquid chromatography (ILPR‒SPE‒HPLC) for the simultaneous determination of thidiazuron and forchlorfenuron in cucumbers. Good linearity of the ILPR‒SPE‒HPLC method was obtained, ranging from 0.0100 to 5.00 µg g-1 with a correlation coefficient (r) ≥ 0.9999. The recoveries of spiked samples ranged from 91.4% to 100.7% with a relative standard deviation of ≤ 6.0%.

Involvement of MEM1 in DNA demethylation in Arabidopsis.

KEY MESSAGE: MEM1 participates in ROS1-mediated DNA demethylation pathway, and acts functionally as ROS3 to counteract the effects of RdDM pathway.mem1mutation leads to large numbers of hyper-DMRs inArabidopsisgenome. In higher plants, DNA methylation performs important functions in silencing transcribed genes and transposable elements (TEs). Active DNA demethylation mediated by REPRESSOR OF SILENCING 1 (ROS1) is able to antagonize the action of DNA methylation caused by RNA-directed DNA methylation (RdDM) pathway, which plays critical roles in keeping DNA methylation at a proper level. In this study, a new mutant named mem1 (for methylation elevated mutant 1) was isolated from a genetic screen of T-DNA insertional mutant population for lines with elevated DNA methylation at a particular locus through Chop-PCR method. MEM1 possesses a Zf-C3HC domain, and is localized in nucleus as well as highly expressed in cotyledons. Whole-genome bisulfite sequencing data showed that knockout mutation of MEM1 leads to 4519 CG, 1793 CHG and 12739 CHH hyper-DMRs (for differentially methylated regions). Further analysis indicated that there are 2751, 2216 and 2042 overlapped CG hyper-DMRs between mem1-1and three mutants, i.e. ros1-4, rdd and ros3-2, respectively; 797, 2514, and 6766 overlapped CHH hyper-DMRs were observed between mem1-1 and three such mutants, respectively; mem1 nrpd1-3 and mem1 rdm1 double mutants showed nearly complete or partial loss of hypermethylation at 4 tested loci, suggesting that MEM1 performs similar functions as DNA glycosylase/lyases in counteracting excessive DNA methylation, and MEM1 plays important roles as REPRESSOR OF SILENCING 3 (ROS3) in erasing CHH methylation caused by the RdDM pathway. Together, these data demonstrate the involvement of MEM1 in ROS1-mediated DNA demethylation pathway and functional connections between MEM1 and ROS3.

Arabidopsis subtilase SASP is involved in the regulation of ABA signaling and drought tolerance by interacting with OPEN STOMATA 1.

Arabidopsis Senescence-Associated Subtilisin Protease (SASP) has previously been reported to participate in leaf senescence and in the development of inflorescences and siliques. Here, we describe a new role of SASP in the regulation of abscisic acid (ABA) signaling. SASP encodes a subtilase and its expression was considerably induced by darkness, ABA, and ethylene treatments. sasp knockout mutants displayed obvious developmental phenotypes such as early flowering and smaller leaves. In particular, the sasp mutants exhibited enhanced ABA sensitivity during seed germination and seedling growth, heightened ABA-mediated leaf senescence, and increased production of reactive oxygen species (ROS). Importantly, the sasp mutants also showed remarkably increased tolerance to drought, with expression of six ABA signaling-related genes being either up- or down-regulated following ABA treatment. Interaction assays demonstrated that SASP physically interacts with OPEN STOMATA 1 (OST1) at the cell periphery. Co-expression of SASP and OST1 led to degradation of OST1, whereas this degradation was impaired in sasp-1 protoplasts. ROS attenuation assays demonstrated that in sasp-1 mutant guard cells the attenuation rate markedly decreased. Taken together, the results demonstrate that SASP plays an important role in regulating ABA signaling and drought tolerance through interaction with OST1.

Analysis of the DNA methylation patterns and transcriptional regulation of the NB-LRR-encoding gene family in Arabidopsis thaliana.

KEY MESSAGE: The relationships between transcription and methylation were revealed in Arabidopsis thaliana NB-LRR-encoding genes in wild type (Col-0) and different mutants. Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins constitute a large family that plays predominant roles in disease resistance. However, the regulation of NB-LRR-encoding genes at the transcriptional level is still poorly understood. Recently, DNA cytosine methylation in eukaryotes has been described as serving an important function in regulating gene expression. Here, we analysed the DNA methylation patterns of NB-LRR-encoding genes in Arabidopsis thaliana in samples from a wild type (Col-0) and ago4, met1, cmt3, drm1/2, and ddm1 mutants. Our results revealed that the vast majority of the NB-LRR-encoding genes in Col-0 were methylated, and the DNA methylation occurred predominantly in the CG sequence context. Moreover, DNA methylation was widely distributed in both the promoters and the bodies of most NB-LRR-encoding genes. Our results also showed that the loss of AGO4, MET1, CMT3, DRM1/2 or DDM1 functions generally led to decreased cytosine methylation in the NB-LRR-encoding genes. Analysis of the available transcriptome data from the wild type and the met1, cmt3, drm1/2 and ddm1 mutants revealed that differences in the transcription levels between the wild type and mutants were statistically significant for 63 of the NB-LRR-encoding genes. Of these genes, 38 were significantly upregulated, and the other 25 were significantly downregulated. Some NB-LRR-encoding genes with differential expression levels, which were revealed by the mRNA-Seq data, were confirmed to be significantly upregulated or downregulated in the mutants compared to the wild type by using quantitative RT-PCR. These data suggest that some Arabidopsis NB-LRR-encoding genes are likely to be regulated by altered DNA methylation patterns.