DOI: 10.5061/dryad.kprr4xh5d
title: Characterization of triacylglycerol secretion with shikonin derivatives in Lithospermum erythrorhizon

description: This dataset contains data from electron microscopy and biochemical
 analyses described in the paper: “Tatsumi, K., et al. Excretion of
 triacylglycerol as a matrix lipid facilitating apoplastic accumulation of
 a lipophilic metabolite shikonin” submitted to Journal of Experimental
 Botany. The raw data underlying the paper are given as separate excel
 files, which are deposited to ‘Data’. The Excel file
 ‘result03_positive_181119_Yazaki_Lab_5.xlsx’ is the data of LC-MS for
 Figure 3 of the above paper. Another Excel file
 ‘GC-FID_rawdata-processed.xlsx’ is the raw data for the quantitative
 analysis of fatty acids by GC-FID as well as the processed data shown in
 Figure 5 and Supporting Figure S7. The third Excel file
 ‘lipidome_data_processed.xlsx’ is the data of lipidome analysis shown in
 Supporting Figures S3, S5, and S6.
description: Transmission electron microscopy The method of
 transmission electron microscopy is as follows. Cultured cells were
 treated with 5 mM aluminum chloride for 3 h before chemical fixation of
 shikonin derivatives, as described (Tatsumi et al., 2016). High-pressure
 freezing and the freeze substitution method (HPF/FS) were performed as
 follows. Cells cultured in M9 or LS medium containing 3% sucrose were
 added to flat specimen carriers and frozen using a high-pressure freezing
 machine (Leica EM PACT, Leica Microsystems, Wetzlar, Germany). The frozen
 samples were transferred to 2% osmium tetroxide in anhydrous acetone at
 -80 °C and incubated at -80 °C for 6 days (120 h). These samples were
 warmed gradually from -80 to -30 °C over 5 h, warmed again from -30 to 4
 °C over 3.4 h, and held at 4 °C for 2 h (Cryo Porter CS-80CP, Scinics
 Corporation, Tokyo, Japan). Subsequently, the samples were washed with
 acetone, and embedded in Epon812 resin (TAAB, Aldermaston, England).
 Ultrathin sections (60-80 nm) were cut with a diamond knife on an
 ultramicrotome (Leica EM UC7, Leica Microsystems) and placed on
 formvar-coated copper grids. The ultrathin sections were stained with 4%
 uranyl acetate followed by lead citrate solution and observed with a
 JEM-1400 (JEOL Ltd., Tokyo, Japan) transmission electron microscope at 80
 kV. Some sections were not stained with lead citrate to prevent excess
 staining. LC-MS analysis The liquid
 cultures were filtered through Miracloth (Merck Millipore) to separate the
 culture medium (30 ml) from the cells or root tissues. The cultured medium
 was partitioned with 15 ml of 2 : 1 (v/v) chloroform : methanol to yield
 organic phase (medium fraction). The harvested wet cells/tissues were
 rinsed with 15 ml of 2 :1 (v/v) chloroform-methanol and 30 ml distilled
 water by prompt everting of the glass vessel to recover the cell surface
 lipids (surface fraction). The remaining cells/tissues were completely
 dried and the lipids extracted with 2 ml of 2 :1 (v/v) chloroform-methanol
 to yield the cell fraction. Each fraction was evaporated under nitrogen
 stream before chromatographic analyses. Before LC-MS analysis, lipid
 extracts were roughly separated into polar and non-polar lipids by
 thin-layer chromatography (TLC) using silica plates (TLC silica gel 60,
 Merck Millipore) developed with chloroform because the high amount of
 shikonin derivatives hampered the chemical analysis of TAG and polar
 lipids by LC-MS. Lipid samples, except for shikonin derivatives that could
 be recognized by their red color, were recovered from TLC plates and
 extracted with chloroform or methanol from the silica gel. The lipids were
 subjected to LC-q-TOF-MS (Waters, Boston, MA) analysis with an Acquity
 UPLC HSS T3 column (Waters), as described (Okazaki et al., 2013; Okazaki
 and Saito, 2018). Lipidomic analysis was performed using the data set
 recorded in the positive ion mode. Levels of lipid species were normalized
 relative to the intensity of the internal standard PC (20:0). To compare
 the amount of each lipid class among samples, the level of each lipid
 class, which is the sum of individual lipid species belonging to the
 class, was standardized by using the mean of cell fraction in LS Dark
 cultured cells. GC-FID analysis To
 quantify TAG, TAG was purified by preparative TLC developed with 6 : 4
 (v/v) n-hexane : diethyl ether. Following derivatization to fatty acid
 methyl esters using a fatty acid methylation kit (Nacalai Tesque), TAG was
 quantified by capillary gas chromatography GC-2014 (Shimadzu, Kyoto,
 Japan) with a J&W DB-23 capillary column (GL Science, Tokyo,
 Japan) as described (Kajikawa et al., 2015; 2016), with heptadecanoic acid
 (C17:0) used as the internal standard.
description: Followings are explanations to the Excel files.
 Excel file name: result03_positive_181119_Yazaki_Lab_5
 Analysis of TAG molecules secreted by L. erythrorhizon cells
 cultured under three different conditions; LS medium (dark), M9 medium
 (dark), and M9 medium (light). Lipids of plant cells were extracted into
 three parts, i.e., medium, cell surface, and the rest of the cell
 fraction. Abbreviations of each lipid molecule is as
 follows. PC, phosphatidylcholine; lysoPC, lysophosphatidylcholine; PE,
 phosphatidylethanolamine; lysoPE, lysophosphatidylethanolamine; PG,
 phosphatidylglycerol; PI, phosphatidylinositol; SQDG,
 sulfoquinovosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG,
 digalactosyldiacylglycerol; TAG, triacylglycerol; DAG, diacylglycerol;
 Acyl-ACP, acyl-acyl carrier protein; FAS, fatty acid synthase.
 Excel file name: GC-FID_rawdata-processed.xlsx
 Quantitative analysis of TAG in three fractions of L.
 erythrorhizon cells (T-TOM): medium, cell surface, and inside cells. L.
 erythrorhizon cells were cultured under three different conditions, i. e.,
 LS medium (dark), M9 medium (dark) and M9 medium (light). Quantitative
 analysis of TAG in hairy root of L. erythrorhizon (hairy root), tobacco
 cultured cells (BY-2) and intact tobacco plant (tabacum) are given in
 separate sheets. Fatty acid composition of TAG recovered from three
 fractions (medium, cell surface, and inside cells) of L. erythrorhizon
 cells is also shown. Excel file name:
 lipidome_data_processed.xlsx Data of lipidome analysis
 by LC-MS are summarized. Three fractions of L. erythrorhizon cells
 (T-TOM): medium, cell surface, and inside cells were analyzed, and those
 cells were cultured under three different conditions; LS medium (dark), M9
 medium (dark), and M9 medium (light). The data of each lipid molecule are
 given in separate sheets.