Return to study ST002252 main page
MB Sample ID: SA216935
| Local Sample ID: | sample69 |
| Subject ID: | SU002338 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
| Taxonomy ID: | 3702 |
| Genotype Strain: | WT Col-0, atg7-2 (GABI_655B06), atg9-4 (SALK_145980) |
| Age Or Age Range: | 11-day old |
| Gender: | Not applicable |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002338 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
| Taxonomy ID: | 3702 |
| Genotype Strain: | WT Col-0, atg7-2 (GABI_655B06), atg9-4 (SALK_145980) |
| Age Or Age Range: | 11-day old |
| Gender: | Not applicable |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| sample69 | SA216935 | FL026315 | atg7-2 +N Root | Expeimental factors |
Collection:
| Collection ID: | CO002331 |
| Collection Summary: | Leaf and root tissues of Arabidopsis seedlings were collected from plants growing in hydroponic conditions. |
| Sample Type: | Plant |
| Collection Method: | Seeds were sterilized with 70% ethanol, followed by a 10-min incubation in 0.1% (v/v) Tween 20 (Thermo Fisher Scientific, Waltham, MA) and 50% (v/v) bleach solution. The seeds were then washed with sterile water, at least three times. Subsequently, the suspended seeds were vernalized by incubating at 4ºC in darkness for 2 days. After vernalization, the seeds were suspended in sterile 0.1% (w/v) agarose (VWR, Radnor, PA), and sown on 3.5 cm x 4 cm autoclaved stainless steel growth mesh (14 Mesh T304 Woven Stainless 0.017" wire diameter, TWP Inc. Berkeley, California), which were laid on ½ strength Murashige and Skoog (MS) solid medium composed of 2.15 g/L Murashige and Skoog Basal Salt Mixture (MilliporeSigma, Burlington, MA), 0.05% (v/v) Murashige and Skoog Vitamin Solution (MilliporeSigma), 1% (w/v) sucrose (Thermo Fisher Scientific), 6g/L Phytoblend Agar (Caisson Labs, Smithfield, UT), and 2mM MES (MilliporeSigma) at pH 5.7. Each 10 cm x 10 cm square Petri dish, containing 4 growth meshes were placed in a growth room maintained at 22 ºC under continuous illumination (50 ± 10 μE m−2 s−1) for a period of 5 days. Subsequently, the growth mesh, carrying the germinated seedlings, were sterilely moved onto a sterile 7.5 cm x 8.5 cm stainless steel platform mesh (10 Mesh Woven Stainless 0.025" wire diameter, TWP Inc.), which was in 11.4 cm × 8.6 cm × 6.4 cm Phytatray dish (MilliporeSigma) that contained sterile liquid medium, composed of ½ strength MS liquid media, which contained 10 mM NH4NO3 and 9.4 mM KNO3 (+N media). The volume of the medium was adjusted so that the growth mesh that carried the seeds was in contact with the surface of the medium, and thus as seedlings grew the root system extended into the liquid medium. After 1 day incubation in the +N liquid medium, half the growth meshes from each Phytatray dish were moved into a Phytatray dish that contained nitrogen-deficient liquid medium (-N medium, which contains no nitrogen salts). This medium was composed of 5% (v/v) Murashige and Skoog Basal Salt Micronutrient Solution (MilliporeSigma), 0.05% (v/v) Murashige and Skoog Vitamin Solution, 1.5 mM CaCl2, 0.75 mM MgSO4, 0.625 mM KH2PO4, 2.5 mM KCl, 2 mM MES and 1% (w/v) sucrose. After an additional 3-day incubation, the seedlings from both the +N and -N media were harvested by cutting the hypocotyls that were extending below the growth mesh, and leaf and root tissues were collected separately. |
| Collection Tube Temp: | The collected plant tissues (leaf or root) were transferred in to a 50 mL Teflon-lined screw-caped glass tube (Thermo Fisher Scientific) containing 3 mL preheated isopropanol (Thermo Fisher Scientific) containing 0.01 % (v/v) butylated hydroxytoluene (BHT) (MilliporeSigma) and 1 µM 1,2-didecanoyl-sn-glycero-3-phosphocholine (MilliporeSigma) as an internal standard. The tubes were incubated at 75 °C for 15 min to quench the action of any lipases. |
Treatment:
| Treatment ID: | TR002350 |
| Treatment Summary: | Plants were grown in either normal or nitrogen deficient media for last 3 days. -N condition was used for inducing of autophagy processes. |
| Treatment: | Nitrogen deficient media |
| Plant Light Period: | 24 hr |
| Plant Humidity: | 100% (Enclosed hydroponic growth condition) |
| Plant Temp: | 22 C |
| Plant Growth Stage: | 11-days |
Sample Preparation:
| Sampleprep ID: | SP002344 |
| Sampleprep Summary: | Lipids were extracted using a modification of a standard protocol. The collected plant tissues (leaf or root) were transferred in to a 50 mL Teflon-lined screw-caped glass tube (Thermo Fisher Scientific) containing 3 mL preheated isopropanol (Thermo Fisher Scientific) containing 0.01 % (v/v) butylated hydroxytoluene (BHT) (MilliporeSigma) and 1 µM 1,2-didecanoyl-sn-glycero-3-phosphocholine (PC 20:0)(MilliporeSigma) as an internal standard. The tubes were incubated at 75 °C for 15 min to quench the action of any lipases. Following the addition of 1.5 mL chloroform and 0.6 mL water the mixture was vigorously shaken at room temperature for 1 h. The clear liquid extract was transferred to another 50 mL tube using glass Pasteur pipettes, and the remnant tissue was further extracted for 30-minutes with another 4 mL chloroform/methanol (2:1) that contained 0.01% BHT. The clear liquid from this second extraction was removed and combined with the initial liquid extract. This chloroform/methanol (2:1) extraction was repeated three times, and the last extraction being incubated overnight. The residue tissue remaining after lipid extraction was dried at 105 °C, and the dry weight of each sample determined; each leaf tissue sample weighed approximately 20 mg, and each root tissue sampled weighed approximately 10 mg. All extract aliquots from each biological sample were combined into a single screw capped tube and stored at -80 °C under a nitrogen gas atmosphere. The solvent from each extract removed by evaporation with the aid of a stream of N2 gas, and the lipid residue was dissolved in 1 mL chloroform and transferred to 2.0 mL clear glass vial with Teflon-lined screw cap (Thermo Fisher Scientific). The solvent was again evaporated with N2 gas, and the vials were shipped, overnight on dry ice to Kansas Lipidomics Research Center (https://www.k-state.edu/lipid/) for lipidomics analysis. |
Combined analysis:
| Analysis ID | AN003679 |
|---|---|
| Analysis type | MS |
| Chromatography type | None (Direct infusion) |
| Chromatography system | N/A |
| Column | N/A |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Waters Xevo-TQ-S |
| Ion Mode | POSITIVE |
| Units | nmol/mg dry weight |
Chromatography:
| Chromatography ID: | CH002728 |
| Chromatography Summary: | No chromatography step |
| Instrument Name: | N/A |
| Column Name: | N/A |
| Chromatography Type: | None (Direct infusion) |
MS:
| MS ID: | MS003430 |
| Analysis ID: | AN003679 |
| Instrument Name: | Waters Xevo-TQ-S |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry on a Xevo TQ-S (Waters Co., Milford, MA) was used to analyze the intact lipids by direct infusion in positive ion mode with precursor and neutral loss scans (Peters, Li et al. 2010, Xiao, Gao et al. 2010, Li, Baughman et al. 2014), using the scans shown in the Table. Data processing was also performed as previously described. Response factors were applied to the MGDG and DGDG analyses to correct for differences in the response of the mass spectrometer to unsaturated galactolipid species compared to the saturated internal standards. Generally, phospholipid data do not require response factor corrections, as the biological compounds and the internal standard have similar response factors (and no corrections were applied). |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Lipidomics_MS.pdf |
