SE149:/S1/M1/D1

Sample Set Information

ID	TSE1304
Title	A chloroplastic UDP-glucose pyrophosphorylase from Arabidopsis is the committed enzyme for the first step of sulfolipid biosynthesis.
Description	Plants synthesize a sulfur-containing lipid, sulfoquinovosyldiacylglycerol, which is one of three nonphosphorus glycerolipids that provide the bulk of the structural lipids in photosynthetic membranes. Here, the identification of a novel gene, UDP-glucose pyrophosphorylase3 (UGP3), required for sulfolipid biosynthesis is described. Transcriptome coexpression analysis demonstrated highly correlated expression of UGP3 with known genes for sulfolipid biosynthesis in Arabidopsis thaliana. Liquid chromatography-mass spectrometry analysis of leaf lipids in two Arabidopsis ugp3 mutants revealed that no sulfolipid was accumulated in these mutants, indicating the participation of UGP3 in sulfolipid biosynthesis. From the deduced amino acid sequence, UGP3 was presumed to be a UDP-glucose pyrophosphorylase (UGPase) involved in the generation of UDP-glucose, serving as the precursor of the polar head of sulfolipid. Recombinant UGP3 was able to catalyze the formation of UDP-glucose from glucose-1-phosphate and UTP. A transient assay using fluorescence fusion proteins and UGPase activity in isolated chloroplasts indicated chloroplastic localization of UGP3. The transcription level of UGP3 was increased by phosphate starvation. A comparative genomics study on UGP3 homologs across different plant species suggested the structural and functional conservation of the proteins and, thus, a committing role for UGP3 in sulfolipid synthesis.
Authors	Okazaki Y, Shimojima M, Sawada Y, Toyooka K, Narisawa T, Mochida K, Tanaka H, Matsuda F, Hirai A, Hirai MY, Ohta H, Saito K.
Reference	Plant Cell. 2009 Mar;21(3):892-909. doi: 10.1105/tpc.108.063925. Epub 2009 Mar 13.
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 the T-DNA insertion line for the ugp3-1, ugp3-2, and sqd1 mutants (SALK_020654, SALK_073806, and SALK_016799, respectively) were obtained from the ABRC. The T-DNA insertion site was confirmed by sequencing of PCR fragments. The primers used for this study are listed in Supplemental Table 1 online. The PCR fragment at the left border of the T-DNA was amplified using LBa1 and gene-specific primers (ugp3-1_Rv for SALK_020654, ugp3-2_Rv for SALK_073806, and sqd1_Rv for SALK_016799). Unless stated otherwise, plants were grown on agar-solidified Murashige and Skoog (MS) medium containing 1% (w/v) sucrose at 22°C under a 16-h-light/8-h-dark cycle. After an 18-d incubation, the aerial regions were harvested 6 h after the onset of the light phase. For lipid analyses of plants grown under phosphate-controlled conditions, wild type (Columbia-0 accession) plants, ugp3-1, ugp3-2, and sqd1 mutants were grown on phosphate-controlled medium with sufficient phosphate (10 mM) for 10 d and then transferred to either phosphate-sufficient (10 mM) or phosphate-depleted (0 mM) medium prepared as described by Härtel et al. (2000) and grown for another 10 d. For lipid analyses of plants grown under sulfate-controlled conditions, plants were grown on MS medium for 10 d and then transferred to either MS medium or sulfate-depleted (0 mM) MS medium by replacing MgSO4 with MgCl2 for another 2 d. For isolation of intact chloroplasts, plants were grown on soil at 22°C under a 16-h-light/8-h-dark cycle for 45 d.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M1
Title	LCMS-IT-TOF
Method Details ID	MS1
Sample Amount
Comment

Analytical Method Details Information

ID	MS1
Title	LC-MS Analysis of Lipid Extracts
Instrument	LC, Shimadzu LC-20AD system; MS, Shimadzu LCMS-IT-TOF
Instrument Type
Ionization	ESI
Ion Mode	positive and negative
Description	Total lipids were extracted according to the method of Bligh and Dyer (1959). Crude lipid extracts were dissolved in chloroform and subjected to LC-MS analysis using a Shimadzu LCMS-IT-TOF mass spectrometer combined with a Shimadzu LC-20AD HPLC system. 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. At the beginning of the gradient, the mobile phase was 100% solvent B for 3.33 min. Solvent B was linearly decreased to 60% over 6.67 min and successively decreased to 30% over 1.33 min. Solvent B was held at 30% for 3.33 min and then increased to 100% for reequilibration. The flow rate was held 0.18 mL min−1 for 3.33 min at the beginning of the gradient and linearly increased to 0.2 mL min−1 over 11.33 min. The flow rate was then increased to 0.4 mL min−1 at 14.66 min after the beginning of the gradient, maintained for 13.33 min, and then decreased to 0.18 mL min−1. Total elution time was 40 min. High-resolution ESI-MS were acquired in both positive and negative ion modes by switching the polarity during individual analyses. Conditions for measurement of ESI-MS were as follows: mass range, m/z 150 to 1600; interface voltage, 4.5 V; curved desolvation line temperature, 200°C; heat block temperature, 200°C; ion accumulation time, 10 ms; detector voltage, 1.80 kV; nebulizer gas, N2 (15 L·min−1). The collision-induced dissociation experiment was performed using Ar as the collision gas, with a relative collision energy of 50% and a relative collision gas flow of 50%.
Comment_of_details

Data Analysis Information

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

Data Analysis Details Information

ID	DS1
Title	Data analysis (LC-IT-TOF)
Description	Peak areas of individual lipid molecules were calculated based on the m/z values of their molecular-related ions or fragment ions. The ions used for calculation of peak areas are as follows: [M+HCOO]− for MGDG and DGDG, [M–H]− for SQDG and PI, [M+NH4–phosphoglycerol]+ for PG, [M+NH4–phosphoethanolamine]+ for PE, and [M+H]+ for PC. Because the lipid species with the same polar head were eluted at almost the same retention time (see Supplemental Figure 5 online), corrections for overlap of isotopic variants in higher mass lipids were applied. Ionization efficiency is largely influenced by the nature of the polar headgroups. Thus, the ionization efficiency is assumed to be almost identical when molecules have the same polar headgroups. Levels of individual lipid molecules in the wild-type plant and mutant lines were expressed as relative values against the sum of the peak areas of lipid molecules with the same polar headgroups in the wild type.
Comment_of_details