Occurrence of moisture in deep gas-bearing shale matrix and its impacts on methane adsorption/desorption capability under favorable reservoir conditions.

To comprehend impacts of moisture on exploring and producing shale gas, the rules of pseudo-in situ moisture occurrence in deep shales were revealed through low-pressure N2 adsorption and desorption, and CO2 adsorption measurements. The influences of pseudo-in situ moisture on CH4 adsorption/desorption in the shales were explored at 353.15 K and pressures up to 30 MPa by using the volumetric method. Results showed that the pseudo-in situ moisture content of the shales ranges between 0.57% and 0.94%, which positively correlates with clay mineral content but negatively correlates with organic matter and quartz. The clay minerals contribute more to moisture occurrence mainly via adsorption effect. The pores with the diameters of 1.10-4.10 nm of the shales serve as dominant space for accommodating moisture. Moreover, the pseudo-in situ moisture reduces saturated adsorption capacity and isosteric adsorption heat of CH4 on the shales, suggesting the weakened adsorption affinity toward CH4-shale system. Typically, the minor pseudo-in situ moisture could significantly weaken CH4 adsorption capability of the shales with low clay mineral content through blocking pore throats of organic matter-hosted pores. However, the abundant pseudo-in situ moisture only slightly reduces CH4 adsorption capability of the shales with high clay mineral content due to continuous distribution of organic matter-hosted pores. The aforementioned different roles are dominated by the difference in occurrence characteristics of organic matter-hosted pores and clay mineral-hosted pores between the shales with low clay mineral content and the shales with high clay mineral content. Furthermore, the pseudo-in situ moisture strengthens CH4 adsorption/desorption hysteresis on the shales associated with moisture uptake-induced clay mineral swelling, thereby raising difficulty for CH4 desorption from the shales.

Conserved and plant-specific histone acetyltransferase complexes cooperate to regulate gene transcription and plant development.

Although a conserved SAGA complex containing the histone acetyltransferase GCN5 is known to mediate histone acetylation and transcriptional activation in eukaryotes, how to maintain different levels of histone acetylation and transcription at the whole-genome level remains to be determined. Here we identify and characterize a plant-specific GCN5-containing complex, which we term PAGA, in Arabidopsis thaliana and Oryza sativa. In Arabidopsis, the PAGA complex consists of two conserved subunits (GCN5 and ADA2A) and four plant-specific subunits (SPC, ING1, SDRL and EAF6). We find that PAGA and SAGA can independently mediate moderate and high levels of histone acetylation, respectively, thereby promoting transcriptional activation. Moreover, PAGA and SAGA can also repress gene transcription via the antagonistic effect between PAGA and SAGA. Unlike SAGA, which regulates multiple biological processes, PAGA is specifically involved in plant height and branch growth by regulating the transcription of hormone biosynthesis and response related genes. These results reveal how PAGA and SAGA cooperate to regulate histone acetylation, transcription and development. Given that the PAGA mutants show semi-dwarf and increased branching phenotypes without reduction in seed yield, the PAGA mutations could potentially be used for crop improvement.

Comprehensive characterization of three classes of Arabidopsis SWI/SNF chromatin remodelling complexes.

Although SWI/SNF chromatin remodelling complexes are known to regulate diverse biological functions in plants, the classification, compositions and functional mechanisms of the complexes remain to be determined. Here we comprehensively characterized SWI/SNF complexes by affinity purification and mass spectrometry in Arabidopsis thaliana, and found three classes of SWI/SNF complexes, which we termed BAS, SAS and MAS (BRM-, SYD- and MINU1/2-associated SWI/SNF complexes). By investigating multiple developmental phenotypes of SWI/SNF mutants, we found that three classes of SWI/SNF complexes have both overlapping and specific functions in regulating development. To investigate how the three classes of SWI/SNF complexes differentially regulate development, we mapped different SWI/SNF components on chromatin at the whole-genome level and determined their effects on chromatin accessibility. While all three classes of SWI/SNF complexes regulate chromatin accessibility at proximal promoter regions, SAS is a major SWI/SNF complex that is responsible for mediating chromatin accessibility at distal promoter regions and intergenic regions. Histone modifications are related to both the association of SWI/SNF complexes with chromatin and the SWI/SNF-dependent chromatin accessibility. Three classes of SWI/SNF-dependent accessibility may enable different sets of transcription factors to access chromatin. These findings lay a foundation for further investigation of the function of three classes of SWI/SNF complexes in plants.

Arabidopsis Trithorax histone methyltransferases are redundant in regulating development and DNA methylation.

Although the Trithorax histone methyltransferases ATX1-5 are known to regulate development and stress responses by catalyzing histone H3K4 methylation in Arabidopsis thaliana, it is unknown whether and how these histone methyltransferases affect DNA methylation. Here, we found that the redundant ATX1-5 proteins are not only required for plant development and viability but also for the regulation of DNA methylation. The expression and H3K4me3 levels of both RNA-directed DNA methylation (RdDM) genes (NRPE1, DCL3, IDN2, and IDP2) and active DNA demethylation genes (ROS1, DML2, and DML3) were downregulated in the atx1/2/4/5 mutant. Consistent with the facts that the active DNA demethylation pathway mediates DNA demethylation mainly at CG and CHG sites, and that the RdDM pathway mediates DNA methylation mainly at CHH sites, whole-genome DNA methylation analyses showed that hyper-CG and CHG DMRs in atx1/2/4/5 significantly overlapped with those in the DNA demethylation pathway mutant ros1 dml2 dml3 (rdd), and that hypo-CHH DMRs in atx1/2/4/5 significantly overlapped with those in the RdDM mutant nrpe1, suggesting that the ATX paralogues function redundantly to regulate DNA methylation by promoting H3K4me3 levels and expression levels of both RdDM genes and active DNA demethylation genes. Given that the ATX proteins function as catalytic subunits of COMPASS histone methyltransferase complexes, we also demonstrated that the COMPASS complex components function as a whole to regulate DNA methylation. This study reveals a previously uncharacterized mechanism underlying the regulation of DNA methylation.

Characterization of dissolved black carbon and its binding behaviors to ceftazidime and diclofenac pharmaceuticals: Employing the molecular weight fractionation.

As the ubiquitous component of the aquatic environment, dissolved organic matter (DOM) readily bind with residual pharmaceutical contaminants (PCs) and influence their environmental behaviors. However, the binding mechanisms between dissolved black carbon (DBC), a vital part of the natural DOM pool, and PCs were poorly researched. In this study, the bulk DBC was divided into four fractions in molecular weight (MW) via an ultrafiltration system, and the properties of DBC and their binding interaction with two kinds of typical PCs (ceftazidime (CAZ) and diclofenac (DCF)) were explored concretely. The results showed that low MW component was the main contributor to bulk DBC, and the aromaticity increased with the increase of MW. The categories of chemical structures and fluorescent substances in different MW DBC were similar. Multispectral techniques showed that the oxygen-enriched compounds in DBC had the higher affinity to CAZ/DCF. The -NH-, -COOH, -NH2 groups in CAZ molecules appeared to form the hydrogen bond with DBC. Fluorescence quenching experiments were analyzed, and the binding mechanisms were specifically expounded from the thermodynamic perspective. The fluorophore of fulvic acid-like compounds (FA) were quenched by both static and dynamic quenching mechanisms, while only static quenching occurred for humic acid-like compounds (HA). For bulk DBC, the hydrogen bond and van der Waals force were the major forces in the HA-CAZ system, while the hydrophobic force made the primary contribution to the HA-DCF system, which might be ascribed to the higher hydrophobic nature of DCF. Notably, with the increase of HA MW, the main binding mode of HA-CAZ/DCF changed from hydrophobic force to hydrogen bond and van der Waals force gradually, which also directly proved that various noncovalent interactions co-driven the binding processes. Our findings are beneficial to better assess the fate of DBC and PCs and the corresponding complexes in the aquatic environment.

Characterization of an autonomous pathway complex that promotes flowering in Arabidopsis.

Although previous studies have identified several autonomous pathway components that are required for the promotion of flowering, little is known about how these components cooperate. Here, we identified an autonomous pathway complex (AuPC) containing both known components (FLD, LD and SDG26) and previously unknown components (EFL2, EFL4 and APRF1). Loss-of-function mutations of all of these components result in increased FLC expression and delayed flowering. The delayed-flowering phenotype is independent of photoperiod and can be overcome by vernalization, confirming that the complex specifically functions in the autonomous pathway. Chromatin immunoprecipitation combined with sequencing indicated that, in the AuPC mutants, the histone modifications (H3Ac, H3K4me3 and H3K36me3) associated with transcriptional activation are increased, and the histone modification (H3K27me3) associated with transcriptional repression is reduced, suggesting that the AuPC suppresses FLC expression at least partially by regulating these histone modifications. Moreover, we found that the AuPC component SDG26 associates with FLC chromatin via a previously uncharacterized DNA-binding domain and regulates FLC expression and flowering time independently of its histone methyltransferase activity. Together, these results provide a framework for understanding the molecular mechanism by which the autonomous pathway regulates flowering time.

Antimicrobial Effects and Active Compounds of the Root of Aucklandia Lappa Decne (Radix Aucklandiae).

The development of new biological fungicides using plant metabolites has become an important direction for pesticide development, and previous studies found that Radix Aucklandiae had a certain inhibitory effect on plant pathogens. In this study, we systematically studied the antimicrobial activity of extracts of Radix Aucklandiae, and the active compounds were isolated, purified and structurally identified. Ethanol extracts of Radix Aucklandiae had different inhibitory effects on seven common plant-pathogenic fungi, with EC50 (concentration for 50% of maximal effect) values ranging from 114.18 mg/L to 414.08 mg/L. The extract at concentration of 1,000 mg/L had a significant control effect on strawberry grey mould and wheat powdery mildew of more than 90%. Three active compounds were isolated and purified from the extract, which were identified as alantolactone, dehydrocostus lactone and costunolide. All three compounds showed significant inhibitory effects on Botrytis cinerea, and the MIC (minimal inhibitory concentration) values were 15.63 mg/L, 3.91 mg/L and 15.63 mg/L. Dehydrocostus lactone also showed obvious inhibitory effect on Fusarium graminearum with an MIC value of 62.25 mg/L. The extract of Radix Aucklandiae has high antimicrobial activity against some common plant-pathogenic fungi, and the work lays a foundation for the development of extracts of Radix Aucklandiae as botanical fungicides.

The binding characteristics of sediment-derived dissolved organic matter with ceftazidime: a microstructural and spectroscopic correlation study.

This research focused on the characterization of sediment-derived dissolved organic matter (SDOM) extracted from sediment of Yellow River and the binding behaviors of ceftazidime (CAZ) with the presence of SDOM. The morphology, surface composition and structure of SDOM and the complexation between SDOM and CAZ in terms of component features, binding capacity and sequence were studied by multiple approaches. Results showed that SDOM was in situ autochthonous-dominated with a low weight-average molecular weight and aromaticity (the value of SR was 2.523). The multiple morphological characteristics, high surface oxygen contents (53.49%) and more aliphatic (H/C = 1.91) of SDOM were further confirmed. Studies on SDOM-CAZ interaction suggested that the functional groups and chemical compositions of SDOM were susceptible to CAZ. In more detail, the aromatic protons and aliphatic protons of CAZ impacted significantly, and the binding between CAZ and SDOM might relate to noncovalent. The protein-like fractions were considered to be the primary participant with 49% fractions lost and the aromatics and amides as mainly active site interaction with CAZ. These findings have significant implications on the environmental fate of cephalosporin antibiotics and that of sediment-derived DOM.

The RNA recognition motif-containing protein UBA2c prevents early flowering by promoting transcription of the flowering repressor FLM in Arabidopsis.

FLOWERING LOCUS M (FLM) is a well-known MADS-box transcription factor that is required for preventing early flowering under low temperatures in Arabidopsis thaliana. Alternative splicing of FLM is involved in the regulation of temperature-responsive flowering. However, how the basic transcript level of FLM is regulated is largely unknown. Here, we conducted forward genetic screening and identified a previously uncharacterized flowering repressor gene, UBA2c. Genetic analyses indicated that UBA2c represses flowering at least by promoting FLM transcription. We further demonstrated that UBA2c directly binds to FLM chromatin and facilitates FLM transcription by inhibiting histone H3K27 trimethylation, a histone marker related to transcriptional repression. UBA2c encodes a protein containing two putative RNA recognition motifs (RRMs) and one prion-like domain (PrLD). We found that UBA2c forms speckles in the nucleus and that both the RRMs and PrLD are required not only for forming the nuclear speckles but also for the biological function of UBA2c. These results identify a previously unknown flowering repressor and provide insights into the regulation of flowering time.

Study on the fungicidal mechanism of glabridin against Fusarium graminearum.

Glabridin is a natural plant-derived compound that has been widely used in medicine and cosmetic applications. However, the fungicidal mechanism of glabridin against phytopathogens remains unclear. In this study, we determined the biological activity and physiological effects of glabridin against F. graminearum. Then the differentially expressed proteins of F. graminearum were screened. The EC50 values of glabridin in inhibiting the mycelial growth and conidial germination of F. graminearum were 110.70 mg/L and 40.47 mg/L respectively. Glabridin-induced cell membrane damage was indicated by morphological observations, DiBAC4(3) and PI staining, and measurements of relative conductivity, ergosterol content and respiratory rates. These assays revealed that the integrity of the membrane was destroyed, the content of ergosterol decreased, and the respiratory rate was inhibited. A proteomics analysis showed that 186 proteins were up-regulated and 195 proteins were down-regulated. Mechanically sensitive ion channel proteins related to transmembrane transport and ergosterol biosynthesis ERG4/ERG24, related to ergosterol synthesis were blocked. It is speculated that glabridin acts on ergosterol synthesis-related proteins to destroy the integrity of the cell membrane, resulting in abnormal transmembrane transport and an increased membrane potential. Finally, the morphology of mycelia was seriously deformed, growth and development were inhibited. As a result death was even induced.

COMPASS functions as a module of the INO80 chromatin remodeling complex to mediate histone H3K4 methylation in Arabidopsis.

In the INO80 chromatin remodeling complex, all of the accessory subunits are assembled on the following three domains of INO80: N-terminal domain (NTD), HSA domain, and ATPase domain. Although the ATPase and HSA domains and their interacting accessory subunits are known to be responsible for chromatin remodeling, it is largely unknown how the accessory subunits that interact with the INO80 NTD regulate chromatin status. Here, we identify both conserved and nonconserved accessory subunits that interact with the three domains in the INO80 complex in Arabidopsis thaliana. While the accessory subunits that interact with all the three INO80 domains can mediate transcriptional repression, the INO80 NTD and the accessory subunits interact with it can contribute to transcriptional activation even when the ATPase domain is absent, suggesting that INO80 has an ATPase-independent role. A subclass of the COMPASS histone H3K4 methyltransferase complexes interact with the INO80 NTD in the INO80 complex and function together with the other accessory subunits that interact with the INO80 NTD, thereby facilitating H3K4 trimethylation and transcriptional activation. This study suggests that the opposite effects of the INO80 complex on transcription are required for the balance between vegetative growth and flowering under diverse environmental conditions.

Arabidopsis RPD3-like histone deacetylases form multiple complexes involved in stress response.

The Arabidopsis thaliana RPD3-type histone deacetylases have been known to form conserved SIN3-type histone deacetylase complexes, but whether they form other types of complexes is unknown. Here, we perform affinity purification followed by mass spectrometry and demonstrate that the Arabidopsis RPD3-type histone deacetylases HDA6 and HDA19 interact with several previously uncharacterized proteins, thereby forming three types of plant-specific histone deacetylase complexes, which we named SANT, ESANT, and ARID. RNA-seq indicates that the newly identified components function together with HDA6 and HDA19 and coregulate the expression of a number of genes. HDA6 and HDA19 were previously thought to repress gene transcription by histone deacetylation. We find that the histone deacetylase complexes can repress gene expression via both histone deacetylation-dependent and -independent mechanisms. In the mutants of histone deacetylase complexes, the expression of a number of stress-induced genes is up-regulated, and several mutants of the histone deacetylase complexes show severe retardation in growth. Considering that growth retardation is thought to be a trade-off for an increase in stress tolerance, we infer that the histone deacetylase complexes identified in this study prevent overexpression of stress-induced genes and thereby ensure normal growth of plants under nonstress conditions.

Studies on the control effect of Bacillus subtilis on wheat powdery mildew.

BACKGROUND: Wheat powdery mildew is a worldwide fungal disease and one of the main diseases harming wheat production. Bacillus subtilis is a vital biocontrol bacteria with broad-spectrum antimicrobial activity. In this study, we systematically studied the control effect of B. subtilis on wheat powdery mildew. RESULTS: The control efficiency of 4 × 105 CFU ml-1 B. subtilis on wheat leaves was 71.75% in vitro and 70.31% in a pot experiment. Application of 4 × 105 CFU ml-1 B. subtilis significantly inhibited spore germination (spore germination rate of 22.23%) and increased appressorium deformity (appressorium deformity rate of 69.33%). This was significantly different from the results in the sterile water treatment. Through transcriptome sequencing analysis, we found that differentially expressed genes were mainly enriched in the biosynthesis and metabolism of amino acids (including phenylalanine), carbon metabolism, the pentose phosphate pathway and other pathways. In particular, the plant hormone signal pathway gene nonexpressor of pathogenesis-related genes 1 (NPR1) was significantly upregulated. CONCLUSION: B. subtilis at concentrations of 4 × 105 CFU ml-1 had a significant control effect on wheat powdery mildew and can inhibit germination of the conidial germ tubes and the normal development of appressorium. B. subtilis may induce disease resistance in wheat to control wheat powdery mildew, and this effect is related to the salicylic acid-dependent signal pathway. © 2021 Society of Chemical Industry.

FVE promotes RNA-directed DNA methylation by facilitating the association of RNA polymerase V with chromatin.

Association of RNA polymerase V (Pol V) with chromatin is a critical step for RNA- directed DNA methylation (RdDM) in plants. Although the methylated DNA-binding proteins SUVH2 and SUVH9 and the chromatin remodeler-containing complex DRD1-DMS3-RDM1 are known to be required for the association of Pol V with chromatin, the molecular mechanisms underlying the association of Pol V with different chromatin environments remain largely unknown. Here we found that SUVH9 interacts with FVE, a homolog of the mammalian retinoblastoma-associated protein, which has been previously identified as a shared subunit of the histone deacetylase complex and the polycomb-type histone H3K27 trimethyltransferase complex. We demonstrated that FVE facilitates the association of Pol V with chromatin and thus contributes to DNA methylation at a substantial subset of RdDM target loci. Compared with FVE-independent RdDM target loci, FVE-dependent RdDM target loci are more abundant in gene-rich chromosome arms than in pericentromeric heterochromatin regions. This study contributes to our understanding of how the association of Pol V with chromatin is regulated in different chromatin environments.

Three functionally redundant plant-specific paralogs are core subunits of the SAGA histone acetyltransferase complex in Arabidopsis.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an evolutionarily conserved histone acetyltransferase complex that has a critical role in histone acetylation, gene expression, and various developmental processes in eukaryotes. However, little is known about the composition and function of the SAGA complex in plants. In this study, we found that the SAGA complex in Arabidopsis thaliana contains not only conserved subunits but also four plant-specific subunits: three functionally redundant paralogs, SCS1, SCS2A, and SCS2B (SCS1/2A/2B), and a TAF-like subunit, TAFL. Mutations in SCS1/2A/2B lead to defective phenotypes similar to those caused by mutations in the genes encoding conserved SAGA subunits HAG1 and ADA2B, including delayed juvenile-to-adult phase transition, late flowering, and increased trichome density. Furthermore, we demonstrated that SCS1/2A/2B are required for the function of the SAGA complex in histone acetylation, thereby promoting the transcription of development-related genes. These results together suggest that SCS1/2A/2B are core subunits of the SAGA complex in Arabidopsis. Compared with SAGA complexes in other eukaryotes, the SAGA complexes in plants have evolved unique features that are necessary for normal growth and development.

Visible-LED-light-driven photocatalytic degradation of ofloxacin and ciprofloxacin by magnetic biochar modified flower-like Bi2WO6: The synergistic effects, mechanism insights and degradation pathways.

Bi2WO6 possesses good stability but poor photocatalytic activity under visible light. Herein, the coupling of Bi2WO6, Fe3O4 and biochar (Bi2WO6/Fe3O4/BC) was investigated to enhance the photocatalytic performance of Bi2WO6 through facile hydrothermal method, which almost completely degraded ofloxacin (OFL) and ciprofloxacin (CIP) within 30 min under energy-saving visible LED irradiation. The superior photocatalytic activity of Bi2WO6/Fe3O4/BC was ascribed to the stronger visible light adsorption capacity and the lower recombination of electron-hole pairs. O2- played a major role during the photocatalytic reaction. The characterization results suggested that the introduction of biochar avoided the agglomeration of Bi2WO6 microspheres and Fe3O4 nanoparticles, at the same time, the biochar participated in OFL and CIP photodegradation by consuming different oxygen-containing functional groups. In order to further evaluate the application potential of Bi2WO6/Fe3O4/BC, the effects of environment factors and the application in different actual water were carefully investigated. Various transformation products and the possible degradation pathways of OFL and CIP were analyzed based on high resolution mass spectrometry (HRMS) results, moreover, the toxicity evaluation results of Escherichia coli indicated these intermediates products were less toxic compared OFL and CIP. Overall, Bi2WO6/Fe3O4/BC can provide a potential way for the application of photocatalytic technology in ambient wastewater purification.

Depression-like behavior is associated with lower Per2 mRNA expression in the lateral habenula of rats.

Circadian rhythm dysfunction is primary symptom of depression and is closely related to depression onset. The role of the lateral habenula (LHb) of the thalamus in the pathogenesis of depression has been a research topic of great interest. The neuronal activity of this structure has circadian characteristics, which are related to the regulation of circadian rhythms. However, in depression model of rats, the role of clock genes in the LHb has not been assessed. To address this gap, we used a clomipramine (CLI) injection-induced depression model in rats to assess the daily expression of rhythmic genes in the LHb and depression-like behavior in rats at multiple time points. In determining the role of the Per2 gene in the development of depression-like behavior in the LHb, we found that the expression of this clock gene differed in a circadian manner. Per2 expression was also significantly decreased in CLI-treated rats in late afternoon (17:00) and in the middle of the night (1:00). Furthermore, silencing Per2 in the LHb of normal rats induced depression-like behavior at night, suggesting that Per2 may play an important role in the pathogenesis of depression. Collectively, these results indicate that decreased Per2 expression in the LHb may be related to increased depression-like behavior at night in depression model of rats.

The CBP/p300 histone acetyltransferases function as plant-specific MEDIATOR subunits in Arabidopsis.

In eukaryotes, MEDIATOR is a conserved multi-subunit complex that links transcription factors and RNA polymerase II and that thereby facilitates transcriptional initiation. Although the composition of MEDIATOR has been well studied in yeast and mammals, relatively little is known about the composition of MEDIATOR in plants. By affinity purification followed by mass spectrometry, we identified 28 conserved MEDIATOR subunits in Arabidopsis thaliana, including putative MEDIATOR subunits that were not previously validated. Our results indicated that MED34, MED35, MED36, and MED37 are not Arabidopsis MEDIATOR subunits, as previously proposed. Our results also revealed that two homologous CBP/p300 histone acetyltransferases, HAC1 and HAC5 (HAC1/5) are in fact plant-specific MEDIATOR subunits. The MEDIATOR subunits MED8 and MED25 (MED8/25) are partially responsible for the association of MEDIATOR with HAC1/5, MED8/25 and HAC1/5 co-regulate gene expression and thereby affect flowering time and floral development. Our in vitro observations indicated that MED8 and HAC1 form liquid-like droplets by phase separation, and our in vivo observations indicated that these droplets co-localize in the nuclear bodies at a subset of nuclei. The formation of liquid-like droplets is required for MED8 to interact with RNA polymerase II. In summary, we have identified all of the components of Arabidopsis MEDIATOR and revealed the mechanism underlying the link of histone acetylation and transcriptional regulation.

Insight into metal binding properties of biochar-derived DOM using EEM-PARAFAC and differential absorption spectra combined with two-dimensional correlation spectroscopy.

A large amount of biochar-derived dissolved organic matter (BDOM) will be released into the environment with biochars application into repairing soil/water, which may alter the fate and transport of contaminants. In this study, four DOM samples were extracted from cauliflower root biochar (CRBC), reed straw biochar (RSBC), corn stalks biochar (CSBC), and potato stalk biochar (PSBC). Excitation-emission matrix combined with parallel factor (EEM-PARAFAC) analysis, differential absorbance spectra (DAS), and two-dimensional correlation spectroscopy (2D-COS) analysis were applied to explore the complexation property of BDOM with metals. DAS showed sites heterogeneity within the DOM pool for metals complexing. Humic-like and fulvic-like substances were main fluorescent components identified by EEM-PARAFAC. 2D-COS analysis revealed that polysaccharides and aliphatic firstly responded to Pb(II) binding with CRBC-derived DOM and three other biochar-derived DOM, respectively. While aliphatic groups, aromatic N=O, and polysaccharides gave the fastest response to Cu(II) binding with CRBC, RSBC, and the other two biochar-derived DOM, respectively.