Characterization of triacylglycerol secretion with shikonin derivatives in Lithospermum erythrorhizon
Creators: name: Yazaki, Kazufumi
name: Tatsumi, Kanade
Publisher: name: Zenodo
Year: 2021

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, and the Supporting Figures and a Table are deposited to 'Zenodo'. The Excel file 'result03_positive_181119_Yazaki_Lab_5' is the data of LC-MS for Figure 5a, b of the above paper, and another Excel file 'Tatsumi_Figure5_GC-FID_data' are the raw data for the quantitative analysis of fatty acids shown in Figure 5c. Followings are the explanations to the Supporting Figures and a Table. Transmission electron micrographs of cultured <i>L. erythrorhizon</i> cells fixed with the HPF/FS method show characteristic structures on in apoplastic spaces (Supplemental Figure 1). Electron-dense small particles are also observed in the cell wall. Lipidomic analysis detected many lipid molecules, and relative signal intensity of each lipid class is summarized in Supplemental Figure 2. The values of fraction in LS Dark cells (shikoinn-non-producing) were set to 1 for each lipid class. Black dots represent individual values of each repeat (n = 3; LS Dark, n =4; M9 Dark and M9 Light). PC, phosphatidylcholine; lysoPC, lysophosphatidylcholine; PE, phosphatidylethanolamine; lysoPE, lysophosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol. Subcellular localization of a key biosynthetic enzyme of shikonin, LePGT, was evaluated in <i>Nicotiana benthamiana</i> (Supplemental Figure 3). Confocal microscopic images of LePGT1-mGFP (A), LePGT2-mGFP (B), and GFP-h (C) transiently expressed in epidermal cells of <i>N. benthamiana</i> leaves by agroinfiltration. Bars = 10 µm. Lipid fraction from <i>L. erythrorhizon</i> cells cultured in M9 was analyzed by normal phase thin-layer chromatography (TLC) (Supplemental Figure 4). Triacylglycerol was visualized by iodine vapor (right TLC). Lipids extracted from hairy roots of <i>L. erythrorhizon</i> were analyzed by normal phase TLC (Supplemental Figure 5). The TLC was developed with n-hexane: diethyl ether (7: 3, v/v). (Left) Without staining; (right) after staining with 80% (v/v) aqueous acetone containing 0.01% (w/v) primuline. Yellow arrowheads indicate TAG spots. Primers used for LePGT2 gene construction are shown in Supplemental Table 1.
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<i> </i>et al<i>.,</i> 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&amp;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.