#METABOLOMICS WORKBENCH lim_20241024_042947 DATATRACK_ID:5314 STUDY_ID:ST003535 ANALYSIS_ID:AN005806 PROJECT_ID:PR002175 VERSION 1 CREATED_ON 03-20-2025 #PROJECT PR:PROJECT_TITLE Arsenic-induced enhancement of diazotrophic recruitment and nitrogen fixation in PR:PROJECT_TITLE Pteris vittata rhizosphere PR:PROJECT_SUMMARY The enrichment of diazotrophs and resulting nitrogen accumulation promoted PR:PROJECT_SUMMARY hyperaccumulator growth and facilitated phytoremediation. Nonetheless, the PR:PROJECT_SUMMARY regulatory mechanism of hyperaccumulator biological nitrogen fixation has PR:PROJECT_SUMMARY remained elusive. Here, we report the mechanism by which arsenic regulates PR:PROJECT_SUMMARY biological nitrogen fixation in the arsenic-hyperaccumulator Pteris vittata. PR:PROJECT_SUMMARY Field investigations and greenhouse experiments, based on multi-omics PR:PROJECT_SUMMARY approaches, reveal that elevated arsenic stress induces an enrichment of key PR:PROJECT_SUMMARY diazotrophs, enhances plant nitrogen acquisition, and thus improves plant PR:PROJECT_SUMMARY growth. Metabolomic analysis and microfluidic experiments further demonstrate PR:PROJECT_SUMMARY that the upregulation of specific root metabolites plays a crucial role in PR:PROJECT_SUMMARY recruiting key diazotrophic bacteria. These findings highlight the pivotal role PR:PROJECT_SUMMARY of nitrogen-acquisition mechanisms in the arsenic hyperaccumulation of Pteris PR:PROJECT_SUMMARY vittata, and provide valuable insights into the plant stress resistance. PR:INSTITUTE Institute of Soil and Water Resources and Environmental Science, College of PR:INSTITUTE Environmental and Resource Sciences, Zhejiang University PR:LAST_NAME Lin PR:FIRST_NAME Jiahui PR:ADDRESS 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, China PR:EMAIL linjh.link@zju.edu.cn PR:PHONE 18968220088 PR:DOI http://dx.doi.org/10.21228/M8GJ7D #STUDY ST:STUDY_TITLE Arsenic-induced enhancement of diazotrophic recruitment and nitrogen fixation in ST:STUDY_TITLE Pteris vittata rhizosphere ST:STUDY_SUMMARY Heavy metal contamination poses an escalating global challenge to soil ST:STUDY_SUMMARY ecosystems. Hyperaccumulators play a crucial role in environmental remediation ST:STUDY_SUMMARY and resource recovery. The enrichment of diazotrophs and resulting nitrogen ST:STUDY_SUMMARY accumulation promoted hyperaccumulator growth and facilitated phytoremediation. ST:STUDY_SUMMARY Nonetheless, the regulatory mechanism of hyperaccumulator biological nitrogen ST:STUDY_SUMMARY fixation has remained elusive. Here, we report the mechanism by which arsenic ST:STUDY_SUMMARY regulates biological nitrogen fixation in the arsenic-hyperaccumulator Pteris ST:STUDY_SUMMARY vittata. Field investigations and greenhouse experiments, based on multi-omics ST:STUDY_SUMMARY approaches, reveal that elevated arsenic stress induces an enrichment of key ST:STUDY_SUMMARY diazotrophs, enhances plant nitrogen acquisition, and thus improves plant ST:STUDY_SUMMARY growth. Metabolomic analysis and microfluidic experiments further demonstrate ST:STUDY_SUMMARY that the upregulation of specific root metabolites plays a crucial role in ST:STUDY_SUMMARY recruiting key diazotrophic bacteria. These findings highlight the pivotal role ST:STUDY_SUMMARY of nitrogen-acquisition mechanisms in the arsenic hyperaccumulation of Pteris ST:STUDY_SUMMARY vittata, and provide valuable insights into the plant stress resistance. ST:INSTITUTE Zhejiang University ST:DEPARTMENT College of Environmental and Resource Sciences ST:LABORATORY Institute of Soil and Water Resources and Environmental Science ST:LAST_NAME Lin ST:FIRST_NAME Jiahui ST:ADDRESS 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, China ST:EMAIL linjh.link@zju.edu.cn ST:PHONE 18968220088 ST:SUBMIT_DATE 2024-10-24 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Pteris vittata SU:TAXONOMY_ID 13821 SU:SPECIES_GROUP Plants #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS G1 s36 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s36.mzXML SUBJECT_SAMPLE_FACTORS G2 s37 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s37.mzXML SUBJECT_SAMPLE_FACTORS G3 s38 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s38.mzXML SUBJECT_SAMPLE_FACTORS G4 s39 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s39.mzXML SUBJECT_SAMPLE_FACTORS G5 s40 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s40.mzXML SUBJECT_SAMPLE_FACTORS G6 s41 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s41.mzXML SUBJECT_SAMPLE_FACTORS G7 s42 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s42.mzXML SUBJECT_SAMPLE_FACTORS G8 s43 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s43.mzXML SUBJECT_SAMPLE_FACTORS G9 s44 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s44.mzXML SUBJECT_SAMPLE_FACTORS G10 s45 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s45.mzXML SUBJECT_SAMPLE_FACTORS G11 s46 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s46.mzXML SUBJECT_SAMPLE_FACTORS G12 s47 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s47.mzXML SUBJECT_SAMPLE_FACTORS G13 s48 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s48.mzXML SUBJECT_SAMPLE_FACTORS G14 s49 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s49.mzXML SUBJECT_SAMPLE_FACTORS G15 s50 Sample source:Bulk soil | Soil condition:Severe arsenic RAW_FILE_NAME=s50.mzXML SUBJECT_SAMPLE_FACTORS 3 s11 Sample source:Bulk soil | Soil condition:Slight arsenic RAW_FILE_NAME=s11.mzXML SUBJECT_SAMPLE_FACTORS 4 s12 Sample source:Bulk soil | Soil condition:Slight arsenic RAW_FILE_NAME=s12.mzXML SUBJECT_SAMPLE_FACTORS 7 s13 Sample source:Bulk soil | Soil condition:Slight arsenic RAW_FILE_NAME=s13.mzXML SUBJECT_SAMPLE_FACTORS 9 s14 Sample source:Bulk soil | Soil condition:Slight arsenic RAW_FILE_NAME=s14.mzXML SUBJECT_SAMPLE_FACTORS 11 s15 Sample source:Bulk soil | Soil condition:Slight arsenic RAW_FILE_NAME=s15.mzXML SUBJECT_SAMPLE_FACTORS 12 s16 Sample source:Bulk soil | Soil condition:Slight arsenic RAW_FILE_NAME=s16.mzXML SUBJECT_SAMPLE_FACTORS 13 s17 Sample source:Bulk soil | Soil condition:Slight arsenic RAW_FILE_NAME=s17.mzXML SUBJECT_SAMPLE_FACTORS 14 s18 Sample source:Bulk soil | Soil condition:Slight arsenic RAW_FILE_NAME=s18.mzXML SUBJECT_SAMPLE_FACTORS 15 s19 Sample source:Bulk soil | Soil condition:Slight arsenic RAW_FILE_NAME=s19.mzXML SUBJECT_SAMPLE_FACTORS 16 s20 Sample source:Bulk soil | Soil condition:Slight arsenic RAW_FILE_NAME=s20.mzXML SUBJECT_SAMPLE_FACTORS G1 s21 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s21.mzXML SUBJECT_SAMPLE_FACTORS G2 s22 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s22.mzXML SUBJECT_SAMPLE_FACTORS G3 s23 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s23.mzXML SUBJECT_SAMPLE_FACTORS G4 s24 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s24.mzXML SUBJECT_SAMPLE_FACTORS G5 s25 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s25.mzXML SUBJECT_SAMPLE_FACTORS G6 s26 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s26.mzXML SUBJECT_SAMPLE_FACTORS G7 s27 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s27.mzXML SUBJECT_SAMPLE_FACTORS G8 s28 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s28.mzXML SUBJECT_SAMPLE_FACTORS G9 s29 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s29.mzXML SUBJECT_SAMPLE_FACTORS G10 s30 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s30.mzXML SUBJECT_SAMPLE_FACTORS G11 s31 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s31.mzXML SUBJECT_SAMPLE_FACTORS G12 s32 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s32.mzXML SUBJECT_SAMPLE_FACTORS G13 s33 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s33.mzXML SUBJECT_SAMPLE_FACTORS G14 s34 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s34.mzXML SUBJECT_SAMPLE_FACTORS G15 s35 Sample source:Rhizosphere | Soil condition:Severe arsenic RAW_FILE_NAME=s35.mzXML SUBJECT_SAMPLE_FACTORS 3 s1 Sample source:Rhizosphere | Soil condition:Slight arsenic RAW_FILE_NAME=s1.mzXML SUBJECT_SAMPLE_FACTORS 16 s10 Sample source:Rhizosphere | Soil condition:Slight arsenic RAW_FILE_NAME=s10.mzXML SUBJECT_SAMPLE_FACTORS 4 s2 Sample source:Rhizosphere | Soil condition:Slight arsenic RAW_FILE_NAME=s2.mzXML SUBJECT_SAMPLE_FACTORS 7 s3 Sample source:Rhizosphere | Soil condition:Slight arsenic RAW_FILE_NAME=s3.mzXML SUBJECT_SAMPLE_FACTORS 9 s4 Sample source:Rhizosphere | Soil condition:Slight arsenic RAW_FILE_NAME=s4.mzXML SUBJECT_SAMPLE_FACTORS 11 s5 Sample source:Rhizosphere | Soil condition:Slight arsenic RAW_FILE_NAME=s5.mzXML SUBJECT_SAMPLE_FACTORS 12 s6 Sample source:Rhizosphere | Soil condition:Slight arsenic RAW_FILE_NAME=s6.mzXML SUBJECT_SAMPLE_FACTORS 13 s7 Sample source:Rhizosphere | Soil condition:Slight arsenic RAW_FILE_NAME=s7.mzXML SUBJECT_SAMPLE_FACTORS 14 s8 Sample source:Rhizosphere | Soil condition:Slight arsenic RAW_FILE_NAME=s8.mzXML SUBJECT_SAMPLE_FACTORS 15 s9 Sample source:Rhizosphere | Soil condition:Slight arsenic RAW_FILE_NAME=s9.mzXML SUBJECT_SAMPLE_FACTORS G1 rootG1 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG1.mzXML SUBJECT_SAMPLE_FACTORS G10 rootG10 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG10.mzXML SUBJECT_SAMPLE_FACTORS G11 rootG11 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG11.mzXML SUBJECT_SAMPLE_FACTORS G12 rootG12 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG12.mzXML SUBJECT_SAMPLE_FACTORS G13 rootG13 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG13.mzXML SUBJECT_SAMPLE_FACTORS G14 rootG14 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG14.mzXML SUBJECT_SAMPLE_FACTORS G15 rootG15 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG15.mzXML SUBJECT_SAMPLE_FACTORS G2 rootG2 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG2.mzXML SUBJECT_SAMPLE_FACTORS G3 rootG3 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG3.mzXML SUBJECT_SAMPLE_FACTORS G4 rootG4 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG4.mzXML SUBJECT_SAMPLE_FACTORS G5 rootG5 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG5.mzXML SUBJECT_SAMPLE_FACTORS G6 rootG6 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG6.mzXML SUBJECT_SAMPLE_FACTORS G7 rootG7 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG7.mzXML SUBJECT_SAMPLE_FACTORS G8 rootG8 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG8.mzXML SUBJECT_SAMPLE_FACTORS G9 rootG9 Sample source:Root | Soil condition:Severe arsenic RAW_FILE_NAME=rootG9.mzXML SUBJECT_SAMPLE_FACTORS 3 root1 Sample source:Root | Soil condition:Slight arsenic RAW_FILE_NAME=root1.mzXML SUBJECT_SAMPLE_FACTORS 16 root10 Sample source:Root | Soil condition:Slight arsenic RAW_FILE_NAME=root10.mzXML SUBJECT_SAMPLE_FACTORS 4 root2 Sample source:Root | Soil condition:Slight arsenic RAW_FILE_NAME=root2.mzXML SUBJECT_SAMPLE_FACTORS 7 root3 Sample source:Root | Soil condition:Slight arsenic RAW_FILE_NAME=root3.mzXML SUBJECT_SAMPLE_FACTORS 9 root4 Sample source:Root | Soil condition:Slight arsenic RAW_FILE_NAME=root4.mzXML SUBJECT_SAMPLE_FACTORS 11 root5 Sample source:Root | Soil condition:Slight arsenic RAW_FILE_NAME=root5.mzXML SUBJECT_SAMPLE_FACTORS 12 root6 Sample source:Root | Soil condition:Slight arsenic RAW_FILE_NAME=root6.mzXML SUBJECT_SAMPLE_FACTORS 13 root7 Sample source:Root | Soil condition:Slight arsenic RAW_FILE_NAME=root7.mzXML SUBJECT_SAMPLE_FACTORS 14 root8 Sample source:Root | Soil condition:Slight arsenic RAW_FILE_NAME=root8.mzXML SUBJECT_SAMPLE_FACTORS 15 root9 Sample source:Root | Soil condition:Slight arsenic RAW_FILE_NAME=root9.mzXML #COLLECTION CO:COLLECTION_SUMMARY Wild Pteris vittata plants with intact roots, rhizosphere soils, and adjacent CO:COLLECTION_SUMMARY bulk soils (0.3 meters from the root) were collected from low-As (<1000 ppm, CO:COLLECTION_SUMMARY n=10) and high-As (>1000 ppm, n=15) sites in Wenshan, Yunnan, China. In the CO:COLLECTION_SUMMARY lab, metabolites from the rhizospheric soils, roots, and bulk soils were CO:COLLECTION_SUMMARY extracted for metabolomic analyses following the protocol outlined below. To CO:COLLECTION_SUMMARY collect rhizosphere metabolites, roots were gently shaken to remove loosely CO:COLLECTION_SUMMARY adhering soil, followed by the careful collection of the closely adhering soil. CO:COLLECTION_SUMMARY The collected rhizosphere soil was then freeze-dried and pulverized in a grinder CO:COLLECTION_SUMMARY with glass beads (55 Hz, 2 min). Subsequently, 5 g of rhizospheric soil was CO:COLLECTION_SUMMARY transferred into a sterile centrifuge tube containing 25 mL of a pre-cooled (4 CO:COLLECTION_SUMMARY â) mixed solution of acetonitrile: methanol: H2O (2:2:1, v/v/v). The mixture CO:COLLECTION_SUMMARY underwent two rounds of vortexing for 30 s each on a BE-96 Vortex mixer, coupled CO:COLLECTION_SUMMARY with 30-min ultrasonication at 60 Hz on a KW-100TDV ultrasonic cleaner. CO:COLLECTION_SUMMARY Following centrifugation at 10,000 Ãâg for 10 min at 4 â using a H1850-R CO:COLLECTION_SUMMARY refrigerated centrifuge, the supernatant was filtered through a 0.2-μm sterile CO:COLLECTION_SUMMARY membrane filter (Millipore, Bedford, USA) to isolate the rhizosphere CO:COLLECTION_SUMMARY metabolites. The metabolites were then concentrated into a 2-mL centrifuge tube, CO:COLLECTION_SUMMARY dried into a powder, and stored at -80 â. This procedure was also applied for CO:COLLECTION_SUMMARY the collection of bulk soil metabolites. CO:COLLECTION_PROTOCOL_FILENAME P_vittata_Methods.pdf CO:SAMPLE_TYPE Root, Rhizosphere, Soil #TREATMENT TR:TREATMENT_SUMMARY No treatment. Wild Pteris vittata plants with intact roots, rhizosphere soils, TR:TREATMENT_SUMMARY and adjacent bulk soils (0.3 meters from the root) were collected from low-As TR:TREATMENT_SUMMARY (<1000 ppm, n=10) and high-As (>1000 ppm, n=15) sites in Wenshan, Yunnan, TR:TREATMENT_SUMMARY China. In the lab, metabolites from the rhizospheric soils, roots, and bulk TR:TREATMENT_SUMMARY soils were extracted for metabolomic analyses without any additional laboratory TR:TREATMENT_SUMMARY treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Equal amounts of preprocessed metabolome powder were re-dissolved by the precise SP:SAMPLEPREP_SUMMARY addition of 300 μL of an acetonitrile solution containing 4 ppm SP:SAMPLEPREP_SUMMARY 2-amino-3-(2-chlorophenyl)-propionic acid, prepared with 0.1% formic acid (1:9, SP:SAMPLEPREP_SUMMARY v/v; stored at 4 â). The supernatant was then filtered through 0.2-μm SP:SAMPLEPREP_SUMMARY membranes (Millipore, Bedford, USA) and transferred into vials for further SP:SAMPLEPREP_SUMMARY detection. SP:SAMPLEPREP_PROTOCOL_FILENAME P_vittata_Methods.pdf #CHROMATOGRAPHY CH:METHODS_FILENAME P_vittata_Methods.pdf CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:COLUMN_TEMPERATURE 40 CH:FLOW_GRADIENT 2:98 (A/B, v/v) at 0 min, 2:98 (A/B, v/v) at 1.0 min, 50:50 (A/B, v/v) at 9.0 CH:FLOW_GRADIENT min, 98:2 (A/B, v/v) at 12.0 min, 98:2 (A/B, v/v) at 13.5 min, 2:98 (A/B, v/v) CH:FLOW_GRADIENT at 14.0 min, 2:98 (A/B, v/v) at 17.0 min CH:FLOW_RATE 0.25 mL/min CH:SOLVENT_A 100% acetonitrile CH:SOLVENT_B 100% water; 5 mM ammonium formate CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE P_vittata_Methods.pdf #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS In untargeted metabolomics analysis, the raw MS data were converted to mzXML MS:MS_COMMENTS format by MSConvert in ProteoWizard software package (v3.0) and processed by MS:MS_COMMENTS XCMS70 (v3.2) for peak picking and alignment. The XCMS settings were as follows: MS:MS_COMMENTS method = âcentWaveâ, ppm = 15, peakwidth = c(5,30), mzwid = 0.015, mzdiff = MS:MS_COMMENTS 0.01, and bw = 2. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST003535_AN005806_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END