Summary of Study ST002252

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 PR001440. The data can be accessed directly via it's Project DOI: 10.21228/M8KQ6P This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

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.

Study ID	ST002252
Study Title	Lipidomics analysis on Arabidopsis autophagy mutants
Study Summary	Autophagy is an essential cellular process in eukaryotes that degrades and recycles macromolecules and organelles. Defects in autophagy is known to affect metabolism, including the lipidome. Genetic approaches have identified a series of AuTophaGy-related (ATG) genes in Arabidopsis. In this study we used WT (ecotype Col-0) and two Arabidopsis autophagy-defective mutants, atg7 and atg9 to perform a multi-omics study on the effect of nitrogen starvation treatment, which induces autophagy. Specifically, we have quantified ~100 lipids from leaf and root tissues of WT, atg7 and atg9 mutant plants, under either autophagy-inducing conditions (-N) or normal nitrogen conditions (+N). The lipid species we quantified include: DGDG, MGDG, LPC, LPE, PE, LPG, PC, PA, PG, PI, and PS. Our study sheds lights on the understanding of the relationships between autophagy and metabolism, especially lipid metabolism.
Institute	Iowa State University
Department	Biochemistry Biophysics, and Molecular Biology
Laboratory	Nikolau Lab
Last Name	Ding
First Name	Geng
Address	2252 Molecular Biology BLDG, Pammel Drive
Email	gengding@iastate.edu
Phone	515-294-0347
Submit Date	2022-02-21
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	MS(Dir. Inf.)
Release Date	2023-02-21
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001440
Project DOI:	doi: 10.21228/M8KQ6P
Project Title:	Lipidomics analysis on Arabidopsis autophagy mutants
Project Summary:	We are interested in how autophagy, as an essential cellular process, affects the lipid metabolism in plants such as Arabidopsis. Specifically, we applied autophagy inducing treatments to two autophagy deficient mutants (atg7 and atg9) and WT Arabidopsis plants, and we quantified about 100 different lipids using ESI triple-quadrupole MS. The lipid species we quantified include: DGDG, MGDG, LPC,LPE, PE, LPG, PC, PA, PG, PI, and PS.
Institute:	Iowa State University
Department:	Biochemistry Biophysics, and Molecular Biology
Laboratory:	Nikolau Lab
Last Name:	Ding
First Name:	Geng
Address:	2252 Molecular Biology BLDG, Pammel Drive, Ames, Iowa, 50011, USA
Email:	gengding@iastate.edu
Phone:	515-294-0347

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:

Subject type: Plant; Subject species: Arabidopsis thaliana (Factor headings shown in green)

mb_sample_id	local_sample_id	Expeimental factors
SA216931	sample34	atg7-2 +N Leaf
SA216932	sample58	atg7-2 +N Leaf
SA216933	sample70	atg7-2 +N Leaf
SA216934	sample82	atg7-2 +N Leaf
SA216939	sample72	atg7-2 -N Leaf
SA216940	sample60	atg7-2 -N Leaf
SA216941	sample84	atg7-2 -N Leaf
SA216942	sample36	atg7-2 -N Leaf
SA216935	sample69	atg7-2 +N Root
SA216936	sample57	atg7-2 +N Root
SA216937	sample33	atg7-2 +N Root
SA216938	sample81	atg7-2 +N Root
SA216943	sample83	atg7-2 -N Root
SA216944	sample59	atg7-2 -N Root
SA216945	sample35	atg7-2 -N Root
SA216946	sample71	atg7-2 -N Root
SA216947	sample54	atg9-4 +N Leaf
SA216948	sample78	atg9-4 +N Leaf
SA216949	sample30	atg9-4 +N Leaf
SA216953	sample32	atg9-4 -N Leaf
SA216954	sample80	atg9-4 -N Leaf
SA216955	sample56	atg9-4 -N Leaf
SA216950	sample53	atg9-4 +N Root
SA216951	sample29	atg9-4 +N Root
SA216952	sample77	atg9-4 +N Root
SA216956	sample55	atg9-4 -N Root
SA216957	sample31	atg9-4 -N Root
SA216958	sample79	atg9-4 -N Root
SA216909	QC11	Quality control samples
SA216910	QC07	Quality control samples
SA216911	QC09	Quality control samples
SA216912	QC08	Quality control samples
SA216913	QC06	Quality control samples
SA216914	QC10	Quality control samples
SA216915	sample50	WT +N Leaf
SA216916	sample62	WT +N Leaf
SA216917	sample26	WT +N Leaf
SA216918	sample74	WT +N Leaf
SA216923	sample76	WT -N Leaf
SA216924	sample64	WT -N Leaf
SA216925	sample52	WT -N Leaf
SA216926	sample28	WT -N Leaf
SA216919	sample49	WT +N Root
SA216920	sample25	WT +N Root
SA216921	sample61	WT +N Root
SA216922	sample73	WT +N Root
SA216927	sample27	WT -N Root
SA216928	sample63	WT -N Root
SA216929	sample75	WT -N Root
SA216930	sample51	WT -N Root

Showing results 1 to 50 of 50

Showing results 1 to 50 of 50

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.

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