Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA255069

Local Sample ID:	Purslane_SP1_Adult_Leaf_Polar_Negative
Subject ID:	SU002637
Subject Type:	Plant
Subject Species:	Portulaca oleracea
Taxonomy ID:	46147

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Subject:

Subject ID:	SU002637
Subject Type:	Plant
Subject Species:	Portulaca oleracea
Taxonomy ID:	46147

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Purslane_SP1_Adult_Leaf_Polar_Negative	SA255069	FL032658	Leaf_Stressed	Group

Collection:

Collection ID:	CO002630
Collection Summary:	Leaves and roots from both treatments - five replicates per treatment - were collected for biomass and mineral analysis. Fifteen samples of substrate were collected for mineral analysis, five before salinization, and ten at the end of the experiment - five from control and five from stressed plants. Leaves and roots from both treatments - five replicates per treatment - were collected and immediately immersed in liquid nitrogen and then stored at -80 °C until extraction of metabolites or proteins.
Sample Type:	Plant

Treatment:

Treatment ID:	TR002649
Treatment Summary:	The B1 accession of purslane (Portulaca oleracea L.) used in this study belongs to the Purslane Collection at Embrapa Agroenergia. Seeds underwent disinfection following the same procedure described in Belo Silva et al. (2022), which consisted of soaking in 2% sodium hypochlorite and Tween® 20 for five minutes, under slow agitation, and then washing with sterile water and drying on sterilized filter paper. After being seeded on a culture medium (MS 1/2 strength, Phytagel 0.2%, and pH 5.8) (Murashige and Skoog, 1962), it was kept for germination in a Growth chamber Conviron mod. Adaptis 1000TC (Controlled Environments Ltd, Winnipeg, Canada) at 150 μmol/m2/s of light and 30°C. After 13 days, seedlings were individually transferred to 200 ml plastic cups containing 100 g of sterilized substrate - clay soil, vermiculite, and a commercial substrate (Bioplant®), 2:1:1 (v:v:v) ratio – and transferred to another Conviron® growth chamber mod. PGW40 at 25±2°C, 500±20 μmol/m2/s of light, 65±5% air relative humidity, and photoperiod of 16/8 h (light/dark), and kept there until the end of the experiments. The plants were allowed to acclimatize for three days, and the salinity stress started three weeks after the end of the acclimatization period. The salinization experiment consisted of two salinity levels (0.0 and 2.0 g of NaCl / 100 g of the substrate), with 16 replicates (plants) in a completely randomized design, and the stress lasted 12 days. During the entire experiment, plants were at field capacity. To avoid the loss of Na+ or Cl-, no leakage of the saline solution was allowed to get out of the plastic cup, as described previously in Belo Silva et al. (2022). The water lost due to evapotranspiration was replaced with deionized water daily, and the electric conductivity at field capacity (ECfc) and water potential in the substrate solution was measured once - on the 8th day of stress - for all replicates.

Treatment ID:

TR002649

Treatment Summary:

The B1 accession of purslane (Portulaca oleracea L.) used in this study belongs to the Purslane Collection at Embrapa Agroenergia. Seeds underwent disinfection following the same procedure described in Belo Silva et al. (2022), which consisted of soaking in 2% sodium hypochlorite and Tween® 20 for five minutes, under slow agitation, and then washing with sterile water and drying on sterilized filter paper. After being seeded on a culture medium (MS 1/2 strength, Phytagel 0.2%, and pH 5.8) (Murashige and Skoog, 1962), it was kept for germination in a Growth chamber Conviron mod. Adaptis 1000TC (Controlled Environments Ltd, Winnipeg, Canada) at 150 μmol/m2/s of light and 30°C. After 13 days, seedlings were individually transferred to 200 ml plastic cups containing 100 g of sterilized substrate - clay soil, vermiculite, and a commercial substrate (Bioplant®), 2:1:1 (v:v:v) ratio – and transferred to another Conviron® growth chamber mod. PGW40 at 25±2°C, 500±20 μmol/m2/s of light, 65±5% air relative humidity, and photoperiod of 16/8 h (light/dark), and kept there until the end of the experiments. The plants were allowed to acclimatize for three days, and the salinity stress started three weeks after the end of the acclimatization period. The salinization experiment consisted of two salinity levels (0.0 and 2.0 g of NaCl / 100 g of the substrate), with 16 replicates (plants) in a completely randomized design, and the stress lasted 12 days. During the entire experiment, plants were at field capacity. To avoid the loss of Na+ or Cl-, no leakage of the saline solution was allowed to get out of the plastic cup, as described previously in Belo Silva et al. (2022). The water lost due to evapotranspiration was replaced with deionized water daily, and the electric conductivity at field capacity (ECfc) and water potential in the substrate solution was measured once - on the 8th day of stress - for all replicates.

Sample Preparation:

Sampleprep ID:	SP002643
Sampleprep Summary:	For the metabolomics study, the following substances were acquired from Sigma Aldrich (Merck, USA): methanol UHPLC grade, acetonitrile LC-MS grade, methyl-tert-butyl-ether, formic acid LC-MS grade, and sodium hydroxide ACS grade. Water was obtained using a Milli-Q system (Millipore, USA). Metabolites were extracted using a well-established protocol, which provides polar and lipidic fractions from the same samples. In this protocol, we first ground the plant material (roots or leaves) in a ball mill (Biospec Products, USA), then added to a microtube containing 1 mL from a solution (1:3) of methanol and methyl-tert-butyl-ether at -20°C. Samples were incubated for 10 min at 4.0 °C, then ultrasonicated for another 10 min in an ice bath. A solution (1:3) of methanol and water was added to each microtube and then submitted to centrifugation (12,000 rpm at 4.0°C for 5 min). The polar (upper) and non-polar (lower) fractions were collected and vacuum-dried in a speed vac system overnight (Centrivap, Labconco, Kansas City, MO, USA). Four microliters of the extract were resuspended in 850 μL of the methanol and water (1:3) solvent mixture and then analyzed by UHPLC-MS.

Sampleprep ID:

SP002643

Sampleprep Summary:

For the metabolomics study, the following substances were acquired from Sigma Aldrich (Merck, USA): methanol UHPLC grade, acetonitrile LC-MS grade, methyl-tert-butyl-ether, formic acid LC-MS grade, and sodium hydroxide ACS grade. Water was obtained using a Milli-Q system (Millipore, USA). Metabolites were extracted using a well-established protocol, which provides polar and lipidic fractions from the same samples. In this protocol, we first ground the plant material (roots or leaves) in a ball mill (Biospec Products, USA), then added to a microtube containing 1 mL from a solution (1:3) of methanol and methyl-tert-butyl-ether at -20°C. Samples were incubated for 10 min at 4.0 °C, then ultrasonicated for another 10 min in an ice bath. A solution (1:3) of methanol and water was added to each microtube and then submitted to centrifugation (12,000 rpm at 4.0°C for 5 min). The polar (upper) and non-polar (lower) fractions were collected and vacuum-dried in a speed vac system overnight (Centrivap, Labconco, Kansas City, MO, USA). Four microliters of the extract were resuspended in 850 μL of the methanol and water (1:3) solvent mixture and then analyzed by UHPLC-MS.

Combined analysis:

Analysis ID	AN004175	AN004176
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Shimadzu Nexera X2	Shimadzu Nexera X2
Column	Waters ACQUITY UPLC BEH C8 (150 x 2.1mm,1.7um)	Waters ACQUITY UPLC BEH C8 (150 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Bruker maXis Impact qTOF	Bruker maXis Impact qTOF
Ion Mode	POSITIVE	NEGATIVE
Units	m/z	m/z

Chromatography:

Chromatography ID:	CH003093
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters ACQUITY UPLC BEH C8 (150 x 2.1mm,1.7um)
Column Temperature:	40 °C
Flow Gradient:	isocratic at the start (0 - 0.5 min) with 4% of B solvent, then at linear gradient (0.5 – 10 min) with 34% B and (10 – 15 min) with 100% B, and finally isocratic (15 – 18 min) with 100% B
Flow Rate:	400 μL min−1
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003922
Analysis ID:	AN004175
Instrument Name:	Bruker maXis Impact qTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	For external calibration, we used a sodium formate solution (10 mM NaOH solution in 50/50 v/v isopropanol/water containing 0.2% formic acid) directly injected through a 6-port valve at the beginning of each chromatographic run. UHPLC-MS data was acquired by the HyStar Application version 3.2 (Bruker Daltonics, Germany). Data pre-processing was performed using the software DataAnalysis version 4.4 (Bruker Daltonics, Germany), where raw data from the UHPLC-MS analysis were exported as .mzXML files.
Ion Mode:	POSITIVE

MS ID:	MS003923
Analysis ID:	AN004176
Instrument Name:	Bruker maXis Impact qTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	For external calibration, we used a sodium formate solution (10 mM NaOH solution in 50/50 v/v isopropanol/water containing 0.2% formic acid) directly injected through a 6-port valve at the beginning of each chromatographic run. UHPLC-MS data was acquired by the HyStar Application version 3.2 (Bruker Daltonics, Germany). Data pre-processing was performed using the software DataAnalysis version 4.4 (Bruker Daltonics, Germany), where raw data from the UHPLC-MS analysis were exported as .mzXML files.
Ion Mode:	NEGATIVE