Summary of Study ST002330

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001494. The data can be accessed directly via it's Project DOI: 10.21228/M8MD91 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002330
Study TitleEarly-stage responses to Plasmodiophora brassicae at the metabolome levels in clubroot resistant and susceptible oilseed Brassica napus
Study TypeTimecourse experiment
Study SummaryA total of 36 samples comprised two types of genotypes [CR (5 individuals pooled in each biological replicate) and CS (5 individuals pooled in each biological replicate)], two treatments (inoculated and uninoculated) and three biological replicates generated from three independent experiments and collected at 1-, 4-, and 7–DPI were used to extract primary and secondary metabolites and analyse the differences among the treatments.
Institute
Trent University
DepartmentBiology
LaboratoryEmery Lab
Last NameKisiala
First NameAnna
Address1600 West Bank Drive, Trent University
Emailannakisiala@trentu.ca
Phone7057481011
Submit Date2022-10-17
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2022-11-16
Release Version1
Anna Kisiala Anna Kisiala
https://dx.doi.org/10.21228/M8MD91
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001494
Project DOI:doi: 10.21228/M8MD91
Project Title:Early-stage responses to Plasmodiophora brassicae at the metabolome levels in clubroot resistant and susceptible oilseed Brassica napus
Project Type:MS quantitative analysis
Project Summary:Clubroot, a devastating soil-borne root disease, in Brassicaceae is caused by Plasmodiophora brassicae Woronin, an obligate biotrophic protist. Plant growth and development, as well as seed yield of Brassica crops, are severely affected due to this disease. Several reports described the molecular responses of B. napus to P. brassicae; however, information on the early stages of pathogenesis is limited. In this study, we have used metabolomics approach to characterize P. brassicae pathogenesis at 1-, 4-, and 7- days post-inoculation (DPI) in clubroot resistant (CR) and susceptible (CS) doubled-haploid (DH) lines. Several metabolites related to organic acids (e.g., citrate, pyruvate), amino acids (e.g., proline, aspartate), sugars, and mannitol, were differentially accumulated in roots in response to pathogen infection when the CR and CS genotypes were compared. Our results suggest important roles for these metabolites in mediating resistance to clubroot disease. To our knowledge, this is the first report of an integrated metabolome analysis aimed at characterizing the molecular basis of resistance to clubroot in canola.
Institute:Trent University
Department:Biology
Laboratory:Emery Lab
Last Name:Kisiala
First Name:Anna
Address:1600 West Bank Drive, Trent University, Peterborough, ON, K9L 0G2, Canada
Email:annakisiala@trentu.ca
Phone:7057481011
Funding Source:Alberta Agriculture and Forestry
Contributors:Dinesh Adhikary, Anna Kisiala, Ananya Sarkar, Urmila Basu, Habibur Rahman, Neil Emery, Nat N. V. Kav

Subject:

Subject ID:SU002417
Subject Type:Plant
Subject Species:Brassica napus
Taxonomy ID:3708

Factors:

Subject type: Plant; Subject species: Brassica napus (Factor headings shown in green)

mb_sample_id local_sample_id Developmental stage (DPI) Genotype Treatment
SA229641C18-RN3-negday 1 Resistant Control
SA229642HILIC-RN3-posday 1 Resistant Control
SA229643C18-RN1-posday 1 Resistant Control
SA229644HILIC-RN2-negday 1 Resistant Control
SA229645HILIC-RN3-negday 1 Resistant Control
SA229646C18-RN3-posday 1 Resistant Control
SA229647HILIC-RN2-posday 1 Resistant Control
SA229648C18-RN1-negday 1 Resistant Control
SA229649HILIC-RN1-negday 1 Resistant Control
SA229650HILIC-RN1-posday 1 Resistant Control
SA229651C18-RN2-posday 1 Resistant Control
SA229652C18-RN2-negday 1 Resistant Control
SA229653C18-RN6-negday 1 Resistant Treated with Plasmodiophora brassicae
SA229654HILIC-RN5-negday 1 Resistant Treated with Plasmodiophora brassicae
SA229655HILIC-RN6-negday 1 Resistant Treated with Plasmodiophora brassicae
SA229656HILIC-RN5-posday 1 Resistant Treated with Plasmodiophora brassicae
SA229657HILIC-RN6-posday 1 Resistant Treated with Plasmodiophora brassicae
SA229658C18-RN6-posday 1 Resistant Treated with Plasmodiophora brassicae
SA229659HILIC-RN4-posday 1 Resistant Treated with Plasmodiophora brassicae
SA229660C18-RN5-negday 1 Resistant Treated with Plasmodiophora brassicae
SA229661HILIC-RN4-negday 1 Resistant Treated with Plasmodiophora brassicae
SA229662C18-RN4-negday 1 Resistant Treated with Plasmodiophora brassicae
SA229663C18-RN5-posday 1 Resistant Treated with Plasmodiophora brassicae
SA229664C18-RN4-posday 1 Resistant Treated with Plasmodiophora brassicae
SA229665C18-RN9-posday 1 Susceptible Control
SA229666HILIC-RN8-negday 1 Susceptible Control
SA229667HILIC-RN9-posday 1 Susceptible Control
SA229668HILIC-RN9-negday 1 Susceptible Control
SA229669C18-RN9-negday 1 Susceptible Control
SA229670HILIC-RN8-posday 1 Susceptible Control
SA229671C18-RN7-negday 1 Susceptible Control
SA229672C18-RN8-negday 1 Susceptible Control
SA229673HILIC-RN7-posday 1 Susceptible Control
SA229674C18-RN7-posday 1 Susceptible Control
SA229675HILIC-RN7-negday 1 Susceptible Control
SA229676C18-RN8-posday 1 Susceptible Control
SA229677C18-RN12-negday 1 Susceptible Treated with Plasmodiophora brassicae
SA229678C18-RN12-posday 1 Susceptible Treated with Plasmodiophora brassicae
SA229679HILIC-RN12-negday 1 Susceptible Treated with Plasmodiophora brassicae
SA229680HILIC-RN11-negday 1 Susceptible Treated with Plasmodiophora brassicae
SA229681HILIC-RN12-posday 1 Susceptible Treated with Plasmodiophora brassicae
SA229682HILIC-RN10-posday 1 Susceptible Treated with Plasmodiophora brassicae
SA229683C18-RN10-posday 1 Susceptible Treated with Plasmodiophora brassicae
SA229684HILIC-RN11-posday 1 Susceptible Treated with Plasmodiophora brassicae
SA229685HILIC-RN10-negday 1 Susceptible Treated with Plasmodiophora brassicae
SA229686C18-RN10-negday 1 Susceptible Treated with Plasmodiophora brassicae
SA229687C18-RN11-posday 1 Susceptible Treated with Plasmodiophora brassicae
SA229688C18-RN11-negday 1 Susceptible Treated with Plasmodiophora brassicae
SA229689C18-RN15-posday 4 Resistant Control
SA229690C18-RN15-negday 4 Resistant Control
SA229691HILIC-RN15-negday 4 Resistant Control
SA229692HILIC-RN14-negday 4 Resistant Control
SA229693HILIC-RN15-posday 4 Resistant Control
SA229694HILIC-RN14-posday 4 Resistant Control
SA229695HILIC-RN13-posday 4 Resistant Control
SA229696C18-RN13-posday 4 Resistant Control
SA229697HILIC-RN13-negday 4 Resistant Control
SA229698C18-RN13-negday 4 Resistant Control
SA229699C18-RN14-posday 4 Resistant Control
SA229700C18-RN14-negday 4 Resistant Control
SA229701C18-RN18-posday 4 Resistant Treated with Plasmodiophora brassicae
SA229702C18-RN18-negday 4 Resistant Treated with Plasmodiophora brassicae
SA229703HILIC-RN18-negday 4 Resistant Treated with Plasmodiophora brassicae
SA229704HILIC-RN17-negday 4 Resistant Treated with Plasmodiophora brassicae
SA229705HILIC-RN18-posday 4 Resistant Treated with Plasmodiophora brassicae
SA229706C18-RN16-posday 4 Resistant Treated with Plasmodiophora brassicae
SA229707HILIC-RN16-posday 4 Resistant Treated with Plasmodiophora brassicae
SA229708HILIC-RN17-posday 4 Resistant Treated with Plasmodiophora brassicae
SA229709HILIC-RN16-negday 4 Resistant Treated with Plasmodiophora brassicae
SA229710C18-RN16-negday 4 Resistant Treated with Plasmodiophora brassicae
SA229711C18-RN17-posday 4 Resistant Treated with Plasmodiophora brassicae
SA229712C18-RN17-negday 4 Resistant Treated with Plasmodiophora brassicae
SA229713C18-RN21-negday 4 Susceptible Control
SA229714C18-RN21-posday 4 Susceptible Control
SA229715HILIC-RN21-negday 4 Susceptible Control
SA229716HILIC-RN20-negday 4 Susceptible Control
SA229717HILIC-RN21-posday 4 Susceptible Control
SA229718C18-RN19-posday 4 Susceptible Control
SA229719HILIC-RN19-posday 4 Susceptible Control
SA229720HILIC-RN20-posday 4 Susceptible Control
SA229721HILIC-RN19-negday 4 Susceptible Control
SA229722C18-RN19-negday 4 Susceptible Control
SA229723C18-RN20-posday 4 Susceptible Control
SA229724C18-RN20-negday 4 Susceptible Control
SA229725C18-RN24-posday 4 Susceptible Treated with Plasmodiophora brassicae
SA229726C18-RN24-negday 4 Susceptible Treated with Plasmodiophora brassicae
SA229727HILIC-RN24-negday 4 Susceptible Treated with Plasmodiophora brassicae
SA229728HILIC-RN23-negday 4 Susceptible Treated with Plasmodiophora brassicae
SA229729HILIC-RN24-posday 4 Susceptible Treated with Plasmodiophora brassicae
SA229730HILIC-RN23-posday 4 Susceptible Treated with Plasmodiophora brassicae
SA229731HILIC-RN22-posday 4 Susceptible Treated with Plasmodiophora brassicae
SA229732C18-RN22-posday 4 Susceptible Treated with Plasmodiophora brassicae
SA229733HILIC-RN22-negday 4 Susceptible Treated with Plasmodiophora brassicae
SA229734C18-RN22-negday 4 Susceptible Treated with Plasmodiophora brassicae
SA229735C18-RN23-posday 4 Susceptible Treated with Plasmodiophora brassicae
SA229736C18-RN23-negday 4 Susceptible Treated with Plasmodiophora brassicae
SA229737C18-RN27-posday 7 Resistant Control
SA229738C18-RN27-negday 7 Resistant Control
SA229739HILIC-RN27-negday 7 Resistant Control
SA229740HILIC-RN26-negday 7 Resistant Control
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Collection:

Collection ID:CO002410
Collection Summary:A total of 36 samples comprised two types of genotypes [CR (5 individuals pooled in each biological replicate) and CS (5 individuals pooled in each biological replicate)], two treatments (inoculated and uninoculated) and three biological replicates generated from three independent experiments and collected at 1-, 4-, and 7–DPI were used. Immediately after collection, all root samples were flash-frozen in liquid nitrogen and stored at -80 °C.
Collection Protocol Filename:Collection_protocol-canola
Sample Type:Plant
Collection Location:Alberta, Canada
Storage Conditions:-80℃
Collection Vials:2 mL round-bottom Eppendorf tubes
Storage Vials:2 mL round-bottom Eppendorf tubes

Treatment:

Treatment ID:TR002429
Treatment Summary:A single spore-derived isolate of P. brassicae pathotype 3H (P3H) was used in this study. To assess the disease progression post-inoculation, histology, and scanning electron microscopy (SEM) was conducted on P. brassicae inoculated and uninoculated root tissues. Samples were collected by Adhikary as follows: seedlings were randomly selected, gently uprooted from the soil, the roots were washed thoroughly and collected in triplicates at after 24 hours of inoculation [hereinafter indicated as 1-day post-inoculation (DPI)], 4-, and 7-DPI.

Sample Preparation:

Sampleprep ID:SP002423
Sampleprep Summary:Approximately 100 mg of fresh weight of root tissue samples (n=3) were aliquoted from finely homogenized frozen root tissues, placed into 2 mL safe-lock centrifuge tubes with two zirconium oxide beads, flash frozen in liquid nitrogen and stored at − 80 °C until further processing. To accommodate for different extraction procedures, two sample sets were prepared from the same bulk to independently analyze the content of primary metabolites (free amino acids, sugars and sugar phosphates, organic acids), and secondary metabolites (glucosinolates) using High Performance Liquid Chromatography – High-Resolution Accurate Mass – Full Scan Mass Spectrometry (HPLC-(HRAM)-MS). Samples for extraction of primary and secondary metabolites were spiked with 10ng of two labeled aromatic CKs (13C5-oT and 2H7-BAR) and extracted with ice-cold methanol (methanol-water [8:2, v/v]) following procedures of Chen et al 3 with modifications. Each filtered extract was split in half and each 500 µL transferred to a new 2 mL tube. Divided extracts were evaporated to dryness at ambient temperature in a speed vac concentrator. Sample residues designed for the analysis of primary metabolites were redissolved in 500 µl of 90% acetonitrile (acetonitrile: water, v/v) and samples for the analysis of glucosinolates were redissolved in 500 µL of 5% acetonitrile with 0.1% formic acid (acetonitrile: formic acid: water, v/v/v). Additionally, sample mixtures composed of 10uL of each sample extract were prepared separately for primary and secondary metabolite analysis and used to generate MS/MS data for compound identification. All samples were filtered using 0.2 µm PVDF spin filter with 2 mL receiver tubes (InnoSep Spin, Canadian Life Sciences, Peterborough, Canada) and transferred to insert-equipped 2 mL HPLC vials. A volume of 25 μL of each sample was injected into a Dionex UltiMate 3000 HPLC coupled to a QExactive Orbitrap mass spectrometer.
Sampleprep Protocol Filename:Protocol_methods-canola
Extraction Method:80% MeOH
Extract Cleanup:0.2 µm PVDF spin filter with 2 mL receiver tubes
Extract Storage:-20℃
Sample Resuspension:Primary metabolites - 90% ACN, glucosinolates - 5% ACN
Sample Spiking:two labeled aromatic CKs (13C5-oT and 2H7-BAR)

Combined analysis:

Analysis ID AN003801 AN003802 AN003803
Analysis type MS MS MS
Chromatography type HILIC HILIC Reversed phase
Chromatography system Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS Thermo Dionex Ultimate 3000 RS
Column Agilent InfinityLab Poroshell 120 HILIC-Z (100 x 2.1mm,2.7um) Agilent InfinityLab Poroshell 120 HILIC-Z (100 x 2.1mm,2.7um) Phenomonex Kinetex C18 (50 x 2.1mm,2.6um)
MS Type ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE NEGATIVE
Units normalized relative level normalized relative level normalized relative level

Chromatography:

Chromatography ID:CH002812
Chromatography Summary:Primary metabolites were resolved with an InfinityLab Poroshell 120 HILIC-Z column (2.1 × 100 mm, 2.7 µm; Agilent, Santa Clara, CA, US), using a flow rate of 0.2 mL min-1 with a mobile phase of 10 mM ammonium bicarbonate in water (A) and 10 mM ammonium bicarbonate in 95% acetonitrile (B). The following gradient was used to elute the analytes: mobile phase 100% B decreased to 90% over 2.5 min and to 50% over the next 5 min and returned to 100% over 0.5 min for 12 min of column re-equilibration.
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:Agilent InfinityLab Poroshell 120 HILIC-Z (100 x 2.1mm,2.7um)
Column Temperature:RT
Flow Rate:0.2 ml/min
Injection Temperature:4
Solvent A:100% water; 10 mM ammonium bicarbonate
Solvent B:95% acetonitrile/5% water; 10 mM ammonium bicarbonate
Chromatography Type:HILIC
  
Chromatography ID:CH002813
Chromatography Summary:Glucosinolates were resolved with a Kinetex C18 column (2.1 × 50 mm, 2.6 μm) using a flow rate of 0.3 mL min −1 with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The following gradient was used to elute the analytes: mobile phase B was held at 0% for 1.25 min before increasing to 50% over 2.75 min and to 100% over the next 0.5 min. Solvent B was then held at 100% for 2 min before returning to 0% over 0.5 min for 4 min of column re-equilibration.
Instrument Name:Thermo Dionex Ultimate 3000 RS
Column Name:Phenomonex Kinetex C18 (50 x 2.1mm,2.6um)
Column Temperature:RT
Flow Rate:0.3 ml/min
Injection Temperature:4
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS003543
Analysis ID:AN003801
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The Orbitrap QExactive was operated with a heated electrospray ionization (HESI) probe in positive mode. Temperatures of the HESI-II auxiliary gas heater and capillary were 450 and 300 °C, respectively, and the spray voltage was 3.9 kV. Sheath, auxiliary, and sweep gases were operated at 30, 8, and 0 (arbitrary units), respectively, and the S-lens RF level was 60. Each sample was analyzed using a mass range of m/z 70−900, and data were acquired at 70,000 resolution, automatic gain control (AGC) target of 1e6, and maximum injection time (IT) of 100 ms. Additionally, the top 10 data-dependent acquisition experiments were performed for the mixed sample from each group (HILIC and C18) to obtain compound MS/MS spectra. Processing of all full scan and ddMS2 data was conducted using the Xcalibur 4.1 software. Metabolites were identified by accurate mass, comparison of retention times to authentic standards or by accurate mass and comparison of fragmentation patterns to MS/MS databases (METLIN, PubChem). Metabolite relative concentration was normalized based on the average recoveries of 13C5-oT and 2H7-BAR as the internal standards
Ion Mode:POSITIVE
  
MS ID:MS003544
Analysis ID:AN003802
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The Orbitrap QExactive was operated with a heated electrospray ionization (HESI) probe in negative mode. Temperatures of the HESI-II auxiliary gas heater and capillary were 450 and 300 °C, respectively, and the spray voltage was 3.9 kV. Sheath, auxiliary, and sweep gases were operated at 30, 8, and 0 (arbitrary units), respectively, and the S-lens RF level was 60. Each sample was analyzed using a mass range of m/z 70−900, and data were acquired at 70,000 resolution, automatic gain control (AGC) target of 1e6, and maximum injection time (IT) of 100 ms. Additionally, the top 10 data-dependent acquisition experiments were performed for the mixed sample from each group (HILIC and C18) to obtain compound MS/MS spectra. Processing of all full scan and ddMS2 data was conducted using the Xcalibur 4.1 software. Metabolites were identified by accurate mass, comparison of retention times to authentic standards or by accurate mass and comparison of fragmentation patterns to MS/MS databases (METLIN, PubChem). Metabolite relative concentration was normalized based on the average recoveries of 13C5-oT and 2H7-BAR as the internal standards
Ion Mode:NEGATIVE
  
MS ID:MS003545
Analysis ID:AN003803
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The Orbitrap QExactive was operated with a heated electrospray ionization (HESI) probe in negative mode. Temperatures of the HESI-II auxiliary gas heater and capillary were 450 and 300 °C, respectively, and the spray voltage was 3.9 kV. Sheath, auxiliary, and sweep gases were operated at 30, 8, and 0 (arbitrary units), respectively, and the S-lens RF level was 60. Each sample was analyzed using a mass range of m/z 70−900, and data were acquired at 70,000 resolution, automatic gain control (AGC) target of 1e6, and maximum injection time (IT) of 100 ms. Additionally, the top 10 data-dependent acquisition experiments were performed for the mixed sample from each group (HILIC and C18) to obtain compound MS/MS spectra. Processing of all full scan and ddMS2 data was conducted using the Xcalibur 4.1 software. Metabolites were identified by accurate mass, comparison of retention times to authentic standards or by accurate mass and comparison of fragmentation patterns to MS/MS databases (METLIN, PubChem). Metabolite relative concentration was normalized based on the average recoveries of 13C5-oT and 2H7-BAR as the internal standards
Ion Mode:NEGATIVE
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