SE150:/S1/M1/D1

Sample Set Information

ID	TSE1305
Title	A new class of plant lipid is essential for protection against phosphorus depletion.
Description	Phosphorus supply is a major factor responsible for reduced crop yields. As a result, plants utilize various adaptive mechanisms against phosphorus depletion, including lipid remodelling. Here we report the involvement of a novel plant lipid, glucuronosyldiacylglycerol, against phosphorus depletion. Lipidomic analysis of Arabidopsis plants cultured in phosphorus-depleted conditions revealed inducible accumulation of glucuronosyldiacylglycerol. Investigation using a series of sulfolipid sulfoquinovosyldiacylglycerol synthesis-deficient mutants of Arabidopsis determined that the biosynthesis of glucuronosyldiacylglycerol shares the pathway of sulfoquinovosyldiacylglycerol synthesis in chloroplasts. Under phosphorus-depleted conditions, the Arabidopsis sqd2 mutant, which does not accumulate either sulfoquinovosyldiacylglycerol or glucuronosyldiacylglycerol, was the most severely damaged of three sulfoquinovosyldiacylglycerol-deficient mutants. As glucuronosyldiacylglycerol is still present in the other two mutants, this result indicates that glucuronosyldiacylglycerol has a role in the protection of plants against phosphorus limitation stress. Glucuronosyldiacylglycerol was also found in rice, and its concentration increased significantly following phosphorus limitation, suggesting a shared physiological significance of this novel lipid against phosphorus depletion in plants.
Authors	Okazaki Y, Otsuki H, Narisawa T, Kobayashi M, Sawai S, Kamide Y, Kusano M, Aoki T, Hirai MY, Saito K.
Reference	Nat Commun. 2013;4:1510. doi: 10.1038/ncomms2512.
Comment

PubMed

Sample Information

ID	S1
Title	Arabidopsis thaliana
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	Seeds of T-DNA insertion lines for sqd2-1 (SALK_070595) and sqd2-2 (SALK_139798) were purchased from ABRC at Ohio State University. The T-DNA insertion sites of these lines were confirmed by sequencing PCR fragments (Supplementary Fig. S16). A PCR fragment at the left border of the T-DNA of sqd2-1 was amplified using LBa1 and SALK_070595_Fw, and the right border of the T-DNA of sqd2-2 was amplified using SALK_070595_Fw and RBa1 (Supplementary Table S1). The sqd1, ugp3-1 and ugp3-2 mutants were isolated in our previous study based on the results of genome PCR17 and used again in this research. Seeds of the sqd2 mutant in the Ws-0 background (provisionally named sqd2-3 in this study) and its wild-type were kind gifts from Professor Benning.
Sample Preparation Details ID	SS1
Comment

Sample Preparation Details Information

ID	SS1
Title	Sample Preparation
Description	Surface-sterilized seeds were sown on agar-solidified Murashige and Skoog medium containing 1% (w/v) sucrose at 22 °C under 16 h light/8 h dark cycles. Photon flux density was 50 μmol m–2 sec–1. The seedlings were kept on agar for 14 days before transfer to plates with controlled phosphate (Pi) concentrations. For P-limitation experiments, sterile Arabidopsis medium was used but at half concentration and containing 0.8% (w/v) agar, 20 mM MES (pH 6.0) and different concentrations of KH2PO4 as indicated. For lipid analysis, leaves were harvested 6 h after the onset of the light phase, frozen in liquid nitrogen and stored at −80 °C until lipid extraction.
Comment_of_details

Analytical Method Information

ID	M1
Title	Lipidomics by HILIC-MS
Method Details ID	MS1
Sample Amount	1 μL
Comment

Analytical Method Details Information

ID	MS1
Title	Lipidomics by HILIC-MS
Instrument	LC, Shimadzu LC-20AD system; MS, Shimadzu LCMS-IT-TOF
Instrument Type
Ionization	ESI
Ion Mode	negative
Description	Plant lipids were extracted following the method of Bligh and Dyer with slight modifications, and directly subjecting the extract to LC-MS analysis. The lipidomic data were recorded on an LC-MS-IT-TOF mass spectrometer using electrospray ionization, combined with an LC-20AD HPLC system (Shimadzu). Parameters for MS were as follows: scan range, m/z 150–1,600; loop time, 0.6 s; CDL and heat block temperature, 160 °C; nebulizer gas (N2), 1.5 l min–1. Metadata (from 'Supplementary Data Set.xls') Chromatography instrument description: Shimadzu, LC-20AD HPLC system, operated by LCMSsolution v.3.6. Auto-injector: Shimadzu, SIL-20AC, LCMSsolution v.3.6., 1microL injection, 1 wash per injection (0.2 mL MeCN) Separation column and pre/guard column: Shimadzu, Atlantis HILIC Silica (pore size: 3 microm) 2.1 by 100 mm Separation parameter: A two-solvent system was used to generate the mobile phase: solvent A, methanol-water (95:5, v/v) containing 0.2% ammonium formate (pH 5.9); solvent B, acetonitrile-methanol-water (95:2:3, v/v/v) containing 0.2% ammonium formate (pH 5.9). The pH of both solvents A and B was adjusted by adding 30% NH4OH to the mixtures of solvents containing 0.2% (v/v) formic acid.; elution program: 100% solvent B (0-3.33 min), 100~65% solvent B (3.33~10 min), 65~30% (10~11.33 min), 30% solvent B (11.33~14.66 min), 100% solvent B (14.66~40 min). The flow rate was held at 0.2 mL/min, and it was then increased to 0.4 mL/min at 14.66 min after the beginning of the gradient, maintained for 13.33 min, and then decreased to 0.2 mL/min. Total elution time was 40 min. Instrument description: Shimadzu, LCMS-IT-TOF, operated by LCMSsolution v.3.6. Sample introduction and delivery: From LC Ionization source: ESI negative. interface voltage, 4.5 V; curved desolvation line temperature, 200C; heat block temperature, 200C; ion accumulation time, 10 msec; nebulizer gas, N2 (15 L/min). Mass analyzer description and acquisition mode: ion trap-time-of-flight operated at scan mode Data acquisition parameters: detection mode: scan (m/z 150-1600; event time: 0.2 s; profile). Data file format: Original data file (.lcd)
Comment_of_details

Data Analysis Information

ID	D1
Title	Data Processing and Analysis
Data Analysis Details ID	DS1
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS1
Title	Data processing and analysis
Description	Data processing Peak picking and alignment were performed using the Profiling Solution software (version 1.0.76.0; Shimadzu). The profiling solution parameters were as follows: ion m/z tolerance, 20 mDa; ion RT tolerance, 0.1 min; ion intensity threshold, 2e4; detect isomer valley, 20%; allow some ion without isotope peak, ON. The data matrix containing ~480 ions consisting of negatively detected ions was exported from the profiling solution software and normalized relative to the intensity of the [M+HCO2]− ion of the internal standard (1,2-didecanoyl-glycero-3-phosphocholine). Annotation was based on the theoretical m/z values of each lipid species and the retention time of identified compounds with the same polar head. Statistics for the analysis of lipidomic data Normalized LC-MS data were pareto-scaled and subjected to OPLS-DA using SIMCA-P (version 11.0.0.0; Umetrics). Tukey' s test and Welch' s t-test were performed using their respective functions (TukeyHSD and t-test) in the statistical software R version 2.14.0 ( http://www.r-project.org/). HCL and jackknife testing were performed on MultiExperiment Viewer (MeV) version 4.8 ( http://www.tm4.org/mev/). For HCL, Pearson’s correlations were used to analyse the distance metric. Average linkage clustering was selected for the linkage method. The data set used for HCL is available as ‘Supplementary Data Set.xls’. Metadata also can be found in the same file.
Comment_of_details