Summary of study ST001056

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 PR000709. The data can be accessed directly via it's Project DOI: 10.21228/M81M4X This work is supported by NIH grant, U2C- DK119886.

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Study IDST001056
Study TitleEvaluation of the short-term effects of the allelochemical umbelliferone on Triticum durum L. metabolism through GC-MS based untargeted metabolomics
Study SummaryAllelopathy is a plant defense mechanism by which they protect themselves from competitive species using specialized biochemicals in the form of secretion or volatiles released to the environment. Though, umbelliferone is a well-known allelochemical, its mechanism of action in a short-term treatment is far from established. We used ≈ 10–days old wheat seedlings treated with104 µM umbelliferone over a time course experiment covering 6 times points, i.e., 0h, 6h, 12h, 24h, 48h, and 96h and compared the metabolomic changes to control (mock-treated) plants. Using gas chromatography mass-spectrometry (GC-MS) based metabolomics efforts, we collectively obtained quantitative data on 177 metabolites that were derivatized (either derivatized singly or multiple times) or not representing 139 non-redundant (unique) metabolites. Out of these 139 metabolites, 118 were associated with a unique HMDB identifier, while 113 were associated with a KEGG identifier. Relative quantification of these metabolites across the time-course of umbelliferone treatment, revealed 22 compounds (sugars, fatty acids, secondary metabolites, organic acids, and amino acids) that changed significantly (repeated measures ANOVA, P-value < 0.05) with time. Using multivariate partial least squares discriminant analysis (PLS-DA) we showed the grouping of samples based on time-course across the control and umbelliferone treated plants, whereas the metabolite-metabolite Pearson correlation revealed tightly formed clusters of umbelliferone-derived metabolites, fatty acids, amino acids, and carbohydrates. Also, time-course of umbelliferone treatment revealed, that phospho-L-serine, maltose, and dehydroquinic acid were the top three metabolites showing highest importance in discrimination among the time-points. The above indicate a system-wide changes induced by umbelliferone, through dysregulation of primary as well as specialized metabolism.
Institute
Università Mediterranea di Reggio Calabria
DepartmentDipartimento AGRARIA
Last NameAraniti
First NameFabrizio
AddressDepartment AGRARIA, University Mediterranea of Reggio Calabria, Località Feo di Vito, SNC I-89124, Reggio Calabria RC, Italy
Emailfabrizio.araniti@unirc.it
Phone+39-09651694283
Submit Date2018-09-12
Num Groups3
PublicationsIn process
Raw Data AvailableYes
Analysis Type DetailGC-MS
Release Date2020-01-13
Release Version1
Fabrizio Araniti Fabrizio Araniti
https://dx.doi.org/10.21228/M81M4X
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000709
Project DOI:doi: 10.21228/M81M4X
Project Title:Short-term effects of the phytotoxic allelochemicals umbelliferone on Triticum durum L. metabolism
Project Summary:Short-term effects of the phytotoxic allelochemicals umbelliferone on Triticum durum L. metabolism
Institute:Università Mediterranea di Reggio Calabria
Department:Dipartimento AGRARIA
Last Name:Misra
First Name:Biswapriya
Address:Medical Center Boulevard NRC Building, G#43, Medical Center Boulevard, Winston Salem, NC, 27157, USA
Email:bbmisraccb@gmail.com
Phone:3522156040
Funding Source:NA
Project Comments:NA
Publications:In process
Contributors:Biswapriya B. Misra, Araniti F, Abenavoli MR

Subject:

Subject ID:SU001363
Subject Type:Plant
Subject Species:Triticum durum
Taxonomy ID:4567
Genotype Strain:cv. Opera
Age Or Age Range:10 days
Gender:Not applicable

Factors:

Subject type: Plant; Subject species: Triticum durum (Factor headings shown in green)

mb_sample_id local_sample_id Time
SA093741RT-
SA093742m/z-
SA093743T40h
SA093744T10h
SA093745T20h
SA093746T30h
SA09374712HKR412h
SA09374812HKR512h
SA09374912HKR212h
SA09375012HKR112h
SA09375112HUKR412h
SA09375212HUKR512h
SA09375312HUKR312h
SA09375412HUKR112h
SA09375512HKR312h
SA09375612HUKR212h
SA09375724HKR224h
SA09375824HKR424h
SA09375924HKR324h
SA09376024HKR124h
SA09376124HUKR324h
SA09376224HUKR124h
SA09376324HUKR224h
SA09376424HKR524h
SA09376524HUKR424h
SA09376624HUKR524h
SA09376748HUKR348h
SA09376848HUKR248h
SA09376948HUKR148h
SA09377048HKR548h
SA09377148HUKR448h
SA09377248HUKR548h
SA09377348HKR248h
SA09377448HKR348h
SA09377548HKR448h
SA09377648HKR148h
SA0937776HUKR16h
SA0937786HUKR26h
SA0937796HUKR36h
SA0937806HUKR56h
SA0937816HKR56h
SA0937826HUKR46h
SA0937836HKR46h
SA0937846HKR16h
SA0937856HKR36h
SA0937866HKR26h
SA09378796HKR496h
SA09378896HKR396h
SA09378996HKR596h
SA09379096HUKR596h
SA09379196HUKR296h
SA09379296HUKR196h
SA09379396HUKR396h
SA09379496HUKR496h
SA09379596HKR196h
SA09379696HKR296h
Showing results 1 to 56 of 56

Collection:

Collection ID:CO001358
Collection Summary:Durum wheat seeds (Triticum durum L. cv. Opera) were germinated in Petri dishes (9 cm ) in a growth chamber at 25°C, 70% humidity and with a photoperiod of 16 : 8 (light : dark) and light intensity of 90 mol m-2 s-1 supplied by a cool white fluorescent lamp (Polylux XL FT8, 55W 8440). Immediately after germination uniform seedlings were transferred to a 4.5 l hydroponic system and grown in a modified Hoagland solution formulated as follows: KNO3 (1 mM); MgSO4 (100 µM); CaSO4 (400 µM); KCl (5 µM); K2SO4 (200 µM); KH2PO4 (175 µM); H3BO3 (2.5 µM); MnSO4 (0.2 µM); ZnSO4 (0.2 µM); NaMoO4 (0.05 µM); CuSO4 (0.05 µM); Fe-EDTA (200 µM).
Sample Type:Plant
Storage Conditions:-80℃
Collection Vials:2ml Eppendorf Screwcapped Round Bottom tubes
Storage Vials:2ml Eppendorf Screwcapped Round Bottom tubes
Collection Tube Temp:4 C
Additives:None

Treatment:

Treatment ID:TR001378
Treatment Summary:Seedlings (10 day old) were treated for 0 h (T0), 6 h (T1), 12 h (T2), 24 h (T3) and 96 h (T4) with nitrate (1 mM) and/ or umbelliferone (100 uM)
Treatment Protocol Comments:Durum weath seeds were directly sown in hydroponic systems the 11/March/2018 and allowed to establish for 10 days. Nutritional Regime:Plant were grown in hydroponic solution composed as follow: KNO3 (1 mM); MgSO4 (100 µM); CaSO4 (400 µM); KCl (5 µM); K2SO4 (200 µM); KH h2PO4 (175 µM); H h3BO3 (2.5 µM); MnSO4 (0.2 µM); ZnSO4 (0.2 µM); NaMoO4 (0.05 µM); CuSO4 (0.05 µM); Fe-EDTA (200 µM). Plant Watering Regime: plants were cropped in hydroponic solution daily changed
Treatment:Umbelliferone (Inhibitor: 100 uM), Time (0, 6, 12, 24, 48, 96 h)
Treatment Compound:Umbelliferone (Inhibitor: 100 uM)
Treatment Route:Roots- Hydropony
Treatment Dose:100 uM
Treatment Dosevolume:Umbelliferone (Inhibitor: 100 uM), Nitrate (1 mM)
Treatment Doseduration:Time (0, 6, 12, 24, 48, 96 h)
Treatment Vehicle:Hoagland Solution
Plant Growth Location:Growth chambers belonging to the University Mediterranea of Reggio Calabria
Plant Light Period:18h/6h light/dark
Plant Humidity:60% Relative Humidity
Plant Temp:25°C
Plant Watering Regime:See protocol comments
Plant Nutritional Regime:See protocol comments
Plant Estab Date:2018-03-11
Plant Harvest Date:2018-03-25
Plant Growth Stage:Seedlings collected at the formation of second true leaves
Plant Metab Quench Method:Immediately after collection snap frozen in liquid Nitrogen
Plant Harvest Method:Full plants harvested manually (5 plants for replicate)
Plant Storage:Snap frozen and powdered plant material stored at -80°C

Sample Preparation:

Sampleprep ID:SP001371
Sampleprep Summary:Freshly homogenized (100 mg) plant material were obtained for each biological sample (plant) and replicates. These were transferred to a 2 ml microcentrifuge round bottom screw cap tubes (Eppendorf). Extraction was done by adding 1400 µl of methanol (at -20°C) and vortexing for 10 s after addition of 60 µl ribitol (0.2 mg/ml stock in dH2O) as an internal quantitative standard for the polar phase. Samples were transferred in a thermomixer at 70C and were shaken for 10 min (950 rpm) and were then further centrifuged for 10 min at 11000 g. The supernatants were collected and transferred to glass vials where 750 µl CHCl3 (-20°C) and 1500 µl dH2O (4°C) were sequentially added. All the samples were vortexed for 10 s and then centrifuged for another 15 min at 2200 g. Upper polar phase ((150 µl ) for each replicate were collected, transferred to a 1.5 ml tube and were dried in a vacuum concentrator without heating. Before freezing and storing at -80°C, the tubes were filled with argon and placed in a plastic bag with silica beads (for avoiding moisture and hydration during short-term storage). Before derivatization, stored samples, were placed in a vacuum concentrator for 30 minutes to eliminate any trace of humidity. Then, to the dried samples, 40 µl methoxyamine hydrochloride (20 mg/ml in pyridine) were added and incubated for 2 h in a Thermomixer (950 rpm) at 37 C. Methoxyaminated samples were then silylated by adding 70 µl of MSTFA to the aliquots. Samples were further shaken for 30 min at 37 C.
Sampleprep Protocol ID:NA
Sampleprep Protocol Filename:NA
Sampleprep Protocol Comments:NA
Processing Method:Freshly homogenized (100 mg) plant material were obtained for each biological sample (plant) and replicates. These were transferred to a 2 ml microcentrifuge round bottom screw cap tubes (Eppendorf). Extraction was done by adding 1400 µl of methanol (at -20°C) and vortexing for 10 s after addition of 60 µl ribitol (0.2 mg/ml stock in ddH2O) as an internal quantitative standard for the polar phase. Samples were transferred in a thermomixer at 70C and were shaken for 10 min (950 rpm) and were then further centrifuged for 10 min at 11000 g. The supernatants were collected and transferred to glass vials where 750 µl CHCl3 (-20°C) and 1500 µl ddH2O (4°C) were sequentially added. All the samples were vortexed for 10 s and then centrifuged for another 15 min at 2200 g. Upper polar phase (150 µl ) for each replicate were collected, transferred to a 1.5 ml tube and were dried in a vacuum concentrator without heating. Before freezing and storing at -80°C, the tubes were filled with argon and placed in a plastic bag with silica beads (for avoiding moisture and hydration during short-term storage).
Processing Storage Conditions:On ice
Extraction Method:Freshly homogenized (100 mg) plant material were obtained for each biological sample (plant) and replicates. These were transferred to a 2 ml microcentrifuge round bottom screw cap tubes (Eppendorf). Extraction was done by adding 1400 µl of methanol (at -20°C) and vortexing for 10 s after addition of 60 µl ribitol (0.2 mg/ml stock in ddH2O) as an internal quantitative standard for the polar phase. Samples were transferred in a thermomixer at 70C and were shaken for 10 min (950 rpm) and were then further centrifuged for 10 min at 11000 g. The supernatants were collected and transferred to glass vials where 750 µl CHCl3 (-20°C) and 1500 µl ddH2O (4°C) were sequentially added. All the samples were vortexed for 10 s and then centrifuged for another 15 min at 2200 g. Upper polar phase (150 µl ) for each replicate were collected, transferred to a 1.5 ml tube and were dried in a vacuum concentrator without heating. Before freezing and storing at -80°C, the tubes were filled with argon and placed in a plastic bag with silica beads (for avoiding moisture and hydration during short-term storage).
Extract Enrichment:NA
Extract Cleanup:NA
Extract Storage:4℃
Sample Resuspension:Dried for Derivatization
Sample Derivatization:Methoxyaminatin + trimethylsilylation (MSTFA + 1% TMCS)
Sample Spiking:60 µl ribitol (0.2 mg/ml stock in ddH2O)
Subcellular Location:NA

Combined analysis:

Analysis ID AN002145
Analysis type MS
Chromatography type GC
Chromatography system Trace GC 1310, Thermo Fisher Scientific
Column TG-5MS
MS Type EI
MS instrument type GC-ITQ
MS instrument name ISQ LT, Thermo Fisher Scientific
Ion Mode POSITIVE
Units Relative Abundance

Chromatography:

Chromatography ID:CH001570
Chromatography Summary:The derivatized extracts were injected into a TG-5MS capillary column (30 m x 0.25 mm x 0.25 µm) (Thermo Fisher Scientific, Waltham, MA, USA) using a gas chromatograph apparatus (Trace GC 1310, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a single quadrupole mass spectrometer (ISQ LT, Thermo Fisher Scientific, Waltham, Massachusetts, US). Injector and source were set at 250°C and 260°C temperature, respectively. One µl of sample was injected in splitless mode with a helium flow of 1 ml/min using the following programmed temperature: isothermal 5 min at 70 °C followed by a 5°C/ min ramp to 350 °C and a final 5 min heating at 330°C.
Methods ID:NA
Instrument Name:Trace GC 1310, Thermo Fisher Scientific
Column Name:TG-5MS
Column Temperature:programmed temperature: isothermal 5 min at 70 °C followed by a 5°C/ min ramp to 350 °C and a final 5 min heating at 330°C
Flow Rate:1 ml/min
Injection Temperature:250
Internal Standard:Adonitol (0.0002mg/ml of pure water) 60 μL per sample
Retention Index:Alkanes mixture C10-C40
Retention Time:Alkanes mixture C10-C40
Oven Temperature:programmed temperature: isothermal 5 min at 70 °C followed by a 5°C/ min ramp to 350 °C and a final 5 min heating at 330°C
Transferline Temperature:250
Sample Syringe Size:10 μL
Randomization Order:YES
Chromatography Type:GC

MS:

MS ID:MS001997
Analysis ID:AN002145
Instrument Name:ISQ LT, Thermo Fisher Scientific
Instrument Type:GC-ITQ
MS Type:EI
MS Comments:Mass spectra were recorded in electronic impact (EI) mode at 70 eV, scanning at 40-600 m/z range, scan time 0.2 sec. Mass spectrometric solvent delay was settled as 9 min.
Ion Mode:POSITIVE
Fragmentation Method:EI-MS
Helium Flow:1 ml/min
Ion Source Temperature:260
Ionization Energy:70 eV
Mass Accuracy:Unit Resolution
Precursor Type:Full scan
Source Temperature:260
Scan Range Moverz:40-600
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