Effect of Ethephon as an Ethylene-Releasing Compound on the

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Effect of ethephon as an ethylene-releasing compound on the metabolic profile of Chlorella vulgaris So-Hyun Kim, Sa Rang Lim, Seong-Joo Hong, Byung-Kwan Cho, Hookeun Lee, Choul-Gyun Lee, and Hyung-Kyoon Choi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00541 • Publication Date (Web): 23 May 2016 Downloaded from http://pubs.acs.org on May 25, 2016

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Journal of Agricultural and Food Chemistry

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[Article]

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Effect of ethephon as an ethylene-releasing compound on the metabolic profile of

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Chlorella vulgaris

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So-Hyun Kim,†,∥ Sa Rang Lim, †,∥ Seong-Joo Hong,‡ Byung-Kwan Cho,§ Hookeun Lee,#

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Choul-Gyun Lee,‡ Hyung-Kyoon Choi*,†

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†

College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea.

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‡

Institute of Industrial Biotechnology, Department of Biological Engineering, Inha University,

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Incheon 402-751, Republic of Korea.

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§

Department of Biological Sciences, KAIST, Daejeon 305-701, Republic of Korea.

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#

College of Pharmacy, Gachon University, Incheon 406-840, Republic of Korea.

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∥

These authors equally contributed to this work.

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*Corresponding author.

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Tel: +82-2-8205605; Fax: 82-2-8123921; E-mail: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT

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In this study, Chlorella vulgaris (C. vulgaris) was treated with ethephon at low (50 µM) and

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high (200 µM) concentrations in medium and harvested at 0, 7, and 14 days, respectively. The

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presence of ethephon led to significant metabolic changes in C. vulgaris, with significantly

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higher levels of α-tocopherol, γ-aminobutyric acid (GABA), asparagine, and proline, but

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lower levels of glycine, citrate, and galactose relative to control. Ethephon induced increases

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in saturated fatty acids but decreases in unsaturated fatty acids. The levels of highly saturated

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sulfoquinovosyldiacylglycerol species and palmitic acid-bounded phospholipids were

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increased on day 7 of ethephon treatment. Among the metabolites, the productivities of α-

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tocopherol (0.70 µg/L/day) and GABA (1.90 µg/L/day) were highest for 50 and 200 µM

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ethephon on day 7, respectively. We propose that ethephon treatment involves various

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metabolic processes in C. vulgaris and can be an efficient way to enrich the contents of α-

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tocopherol and GABA.

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KEYWORDS: C. vulgaris, metabolomics, ethephon, α-tocopherol, GABA, fatty acids, lipids

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■ INTRODUCTION

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Chlorella vulgaris is a single-celled green alga of size 5–10 µm that is known to be one of

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the fastest growing microalgae.1 C. vulgaris is a potential candidate for livestock feeding and

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human food because it comprises a high proportion of proteins (50–70%), with also

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significant amounts of lipids (2–22%) and carbohydrates (8–26%), and appreciable amounts

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of vitamins and minerals.2 Kang and Sim (2004) revealed that an extract of C. vulgaris

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exerted various therapeutic effects, such as degradation of toxic materials, control of

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arteriosclerosis, and immunoprotection activity, anticancer activity, and growth-stimulating

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activity of intestinal bacteria.3

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Ethylene is a gaseous phytohormone that is regarded as a powerful natural regulator of

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plant metabolism. Ethylene is considered to play major roles in regulating plant defense

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responses against pathogens, pests, and abiotic stresses.4 Ethylene reportedly induced the

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formation of defense-related secondary metabolites such as phytoalexins and phenolic

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compounds in Vitis vinifera and Malus domestica, respectively.5,6 However, little is known

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about the functional role or physiological impacts of ethylene in microalgae. It has been

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found that endogenous ethylene is involved in the regulation of cell development in the

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filamentous charophyte Spirogyra pratensis and the programmed cell death of

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Chlamydomonas reinhardtii.7,8

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Ethylene cannot be applied directly to plants due to its relatively low solubility. However,

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the synthetic growth regulator 2-ehloroethylphosphonic acid (ethephon) is available in the

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liquid state. Ethephon can provide a plant with ethylene directly when the pH of the solution

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increases after it contacts plant tissue.9 Ethephon is often used to promote fruit ripening and

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improve fruit quality.10-12 Furthermore, ethephon has several advantages, including a low

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residue in the environment and being relatively inexpensive.13 An ethephon preparation was 3



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therefore chosen for use in the present study to elucidate the effects of exogenous ethylene on

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C. vulgaris.

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To our knowledge there has been no research focused on the effects of exogenous ethylene

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on the global metabolic networks of microalgae. Notably, the total lipid content and the fatty

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acid (FA) compositions have been reported extensively, whereas the individual lipid

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molecular species of microalgae with combinations of various fatty acyl chains and

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characteristic polar head groups are insufficiently known. Determining the individual lipid

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molecules of microalgae is becoming important not only for understanding the biological

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functions of lipids in algal cells but also in providing novel targets for metabolic engineering

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with the aim of increasing the production of biofuels and biopharmaceuticals from

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microalgae.14

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The present study is the first to have applied a comprehensive metabolomics approach

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including alcohols, amino acids, organic acids, sugars, FAs, and intact lipid species to

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characterize C. vulgaris cultivated in exogenous ethephon at both low (50 µM) and high

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(200 µM) concentrations. The objectives of the study were to characterize the effects of

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ethephon on metabolite profiles of C. vulgaris, and especially to determine if ethephon

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treatment is a feasible strategy for enhancing of production of useful compounds from C.

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vulgaris.

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■ MATERIALS AND METHODS

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Chemicals and Reagents. Ethephon, Bold Modified Basal Freshwater Nutrient Solution,

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37 component fatty acid methyl esters (FAME) mixture (C4-C24) standard, methyl

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nonadecanoate, methanolic hydrochloride, myristic-d27 acid, methoxylamine hydrochloride,

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α-tocopherol standard, γ-aminobutyric acid standard (GABA), butylated hydroxytoluene 4


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(BHT), HPLC-grade methyl-tert-butyl ether (MTBE), and pyridine were purchased from

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Sigma-Aldrich (St Louis, MO, USA). BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide)

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containing 1% TMCS (trimethyl chlorosilane) was purchased from Alfa Aesar (Ward Hill,

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MA, USA). HPLC-grade methanol, chloroform and water were purchased from Fisher

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Scientific (Pittsburgh, PA, USA). HPLC-grade hexane was purchased from Honeywell

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Burdick & Jackson (Muskegon, MI, USA).

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Strains and Culture Conditions. C. vulgaris strains (AG10032) were supplied by the

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Korean Collection for Type Cultures (KCTC) (Biological Resource Center, Daejeon,

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Republic of Korea). Whole cells of C. vulgaris were grown in a shaking incubator

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(NEX220SRL, Nexus Technologies, Seoul, Republic of Korea) at 20°C and 120 rpm under

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illumination from fluorescent lamps at 51–54 µmol/m2s and a 16:8-h light/dark cycle. The

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Bold Modified Basal Freshwater Nutrient Solution was used as a culture medium with 50

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times dilution. The culture medium consisted of 11.42 mg/L of H3BO3, 25.0 mg/L

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CaCl2.2H2O, 0.49 mg/L of Co(NO3)2·6H2O, 1.57 mg/L of CuSO4·5H2O, 50.0 mg/L of EDTA

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(free acid), 4.98 mg/L of FeSO4·7H2O, 75.0 mg/L of MgSO4·7H2O, 1.44 mg/L of

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MnCl2·4H2O, 0.71 mg/L of

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mg/L of KI, 175.0 mg/L of KH2PO4, 75.0 mg/L of K2HPO4, 25.0 mg/L of NaCl, 250.0 mg/L

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of NaNO3, 0.002 mg/L of Na2O3Se, 0.001 mg/L of SnCl4, 0.0022 mg/L of VOSO4·3H2O, and

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8.82 mg/L of ZnSO4·7H2O. The 40 mL of stock cultures were transferred into 500 mL

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Erlenmeyer flask filled with 200 mL of the culture medium and grown for either 7 days or 14

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days.

MoO3, 0.003 mg/L of NiCl2·6H2O, 31.0 mg/L of KOH, 0.003

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Preparation of Ethephon and Sampling. Stock solutions of ethephon (200 µM) were 5



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prepared in distilled water and sterilized through 0.2 µm membrane filter (cellulose

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acetate membrane, Advantec, Tokyo, Japan). Ethephon treatments were applied to C. vulgaris

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culture on day 0, at concentrations 50 µM and 200 µM. All samples were cultivated in

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quadruplet for both control and ethephon treatment conditions, and harvested from

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independent culture flasks. To harvest C. vulgaris cells, 240 mL cell suspension of each flask

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was transferred to 15 mL tubes, and settled by centrifugation at 1,800 g for 10 min (UNION

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32R PLUS, Hanil Science Industrial, Incheon, Korea). The supernatant was discarded, and

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the pellet was washed three times with distilled water to remove component of culture

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medium. After washing, the harvested algae cells were freeze-dried by using a lyophilizer

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(Bondiro Ilshin Lab., Seoul, Republic of Korea), and kept at －80°C until further analysis.

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Growth Measurement. The growth measurement of C. vulgaris was conducted every day

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by optical density (OD) at 750 nm using spectrophotometer (xMark™ Microplate

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Absorbance Spectrophotometer, Bio-Rad, Hercules, CA, USA). All measurements were

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conducted in triplicate. The data obtained from the OD at 750 nm converted to cell dry

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weight (DW). The correlation of the measurements is described by the equation y = 0.617 x –

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0.0577 (R2 = 0.9433) for the wavelength of 750, where y represents the DW (g/L) and x is the

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absorbance value at 750 nm.

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Metabolites Analysis. Ten milligrams of freeze-dried C. vulgaris were separately

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weighted in microfuge tubes (Eppendorf, Hamburg, Germany), and extracted with 1 mL of

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methanol. The samples were vortexed for 30 sec and sonicated for 30 min. After sonication,

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the supernatant were collected separately from each sample and filtered through a 0.45 µm

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PTFE membrane (Minisart SRP 15, Sartorius, Germany). For derivatization, 100 µL of C. 6


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vulgaris extract was transferred into a GC vial and dried with nitrogen gas for 15 min. 30 µL

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of 20,000 µg/mL methoxylamine hydrochloride in pyridine for oximation, 50 µL of BSTFA

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[N,O-bis(trimethylsilyl) trifluoroacetamide) containing 1% TMCS (trimethyl chlorosilane)]

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for trimethylsilylation (TMS) derivatization,

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pyridine as an internal standard were separately added to the dried samples. The samples

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were then incubated for 60 min at 65°C.

and 10 µL of 1000 µg/mL myristic-d27 acid in

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For gas chromatography-mass spectrometry (GC-MS) analysis, a 1.0 µL of derivatized

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sample was injected by an Agilent 7683B autosampler (Agilent Technologies, CA, USA) into

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Agilent 7890A GC, equipped with Agilent 5975C mass spectrometric detector, on a 30 m ×

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0.25 mm i.d. × 0.25 µm film thickness fused-silica capillary colum bonded with DB5-MS (5%

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phenyl-methylpolysiloxane) stationary phase with split injection of 1:10. The inlet

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temperature was set at 250°C. The GC was operated at carrier gas (helium) flow rate of 1.0

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mL/min. The temperature of auxiliary, ion source, and quadrupole temperature was adjusted

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to 280, 230, and 150 °C, respectively. The mass range was 50-700Da, and data were obtained

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in full scan mode. The initial oven temperature was set at 70°C and was programmed to

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increase to 135°C (at 5°C/min) and then to 140°C (at 1°C/min) and then

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3°C/min) and then to 225°C (at 6°C/min) and then to 300°C (at 10°C/min; hold 8 min).

to 200°C (at

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The identification of comprehensive metabolites was performed using the GC-MS

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fragment spectra of each component. The National Institute of Standards and Technology

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(NIST)

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http://www.hmdb.ca/) and Golm Metabolome Database (GMD; gmd.mpimp-golm.mpg.de/)

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were used for identification of metabolites. The mass spectral of the compounds were

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accepted when the matching quality was more than 70% compared to the NIST-Wiley Mass

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Spectra Library and when mass fragment of compound was corresponded with database of

mass

spectral

search

program,

Human

Metabolome

7


Database

(HMDB;


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HMDB or GMD. For relative quantification of metabolites, raw GC-MS data were imported

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into Expressionist MSX v2013.0.39 software (Genedata, Basel, Switzerland) for pre-

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processing. Peak detection was performed and a list of molecular features [mass-to-charge

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ratio (m/z), retention time, and intensity] was obtained for each chromatogram. Data were set

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up with intensity of base peak. Normalization was performed by dividing the base peak

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intensity of each compound by that of internal standard and all value was multiplied by 100.

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Significant differences of comprehensive metabolite levels were detected by one-way

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analysis of variance (ANOVA) and post-hoc Tukey’s tests (p < 0.05) using the SPSS

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statistics 21 software (IBM, Somers, NY, USA).

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Fatty Acids Methyl Esters Analysis. Five milligrams of freeze-dried C. vulgaris treated

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with ethephon were separately weighted in glass vials with polytetrafluoroethylene (PTFE)-

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lined caps (Agilent Technologies). For solubilization and transesterification, 200 µL of

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chloroform/methanol (2:1, v/v) and 300 µL of 1.25 M methanolic hydrochloride were added

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to the freeze-dried C. vulgaris samples and incubated for 60 min at 85°C. FAMEs were

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extracted with hexane at room temperature for 60 min. Fifty microliter of methyl

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nonadecanoate (800 µg/mL in hexane) as an internal standard was added to the hexane extract

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and the mixture was vortexed.

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For the analysis of FAMEs, a VF-WAXms column (30 m × 0.25 mm i.d. × 0.25 µm,

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Agilent Technologies) was used, and the split ration was 5:1. The mass range was 40-400Da,

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and data were gathered in full scan mode. The initial oven temperature was set at 50°C for 3

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min before being increased to 130°C at 20°C/min, 178°C at 4°C/min, and 200°C at 1°C/min.

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Calibration standards of FAME mixture (C4-C24) were analyzed in triplicate. The peak

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area ratios (each peak area of FAME mixture/peak area of internal standard) versus 8


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concentrations were used for obtaining the calibration curve. The FAMEs in C. vulgaris were

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identified by comparing the retention times with methylated standards. The notation in this

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study represents each FA as CX:Y, where X is the carbon number of the FA chain and Y is the

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number of double bonds. The amounts of individual fatty acids were calculated by regression

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equation and expressed as mg FA/g DW. The regression equation, R2 value, LOD, and LOQ

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were represented in Table S1 (Supporting Information). Significant differences in FAs were

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detected by ANOVA and post-hoc Tukey’s tests (p < 0.05).

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Lipid Analysis. Total lipids extraction was performed as reported by Matyash et al.15 with

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some modifications. Briefly, 2 mg of freeze dried C. vulgaris was placed in a 2 mL vial

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(Eppendorf). Three hundred microliters of methanol and 1 mL of MTBE was added and

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vortexed. Afterwards, incubated for 1 h at room temperature in a shaker, and then phase

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separation was induced by adding 250 µL of water. The mixture was centrifuged at 1,000 g

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for 10 min and 900 µL of the upper organic phase was collected separately from each sample.

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Then the lower phase was extracted again as was done in the above procedure with 400 µL of

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MTBE/methanol/water (10:3:2.5) mixture and then combined with the first upper phase.

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Then the extracts were passed through with a 0.2-µm PTFE syringe filter (Whatman,

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Maidstone, UK). The filtrate was taken and then dried with nitrogen gas. Dried lipid extracts

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were dissolved in 90 µL of chloroform/methanol (2:1) mixture and 100 µL methanol-

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chloroform (9:1, v/v) containing 7.5 mM ammonium acetate was added. 10 µL of the internal

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standards 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine with a concentration of

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3.4 nM was added and the solution was thoroughly mixed. 30 µL of the solution was loaded

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into a 96 well plate (Eppendorf) and then sealed with aluminum foil (Eppendorf).

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Lipids were analyzed using a linear ion-trap mass spectrometer (LTQ-XL, ThermoFisher 9



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Scientific, San Jose, CA, USA), combined with an automated nanoelectrospray system

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(Triversa NanoMate System, Advion Biosciences, Ithaca, NY, USA) both in positive- and

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negative-ion modes. Ten microliters of lipid extract from the 96-well plate was aspirated and

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infused into MS system through an electrospray ionization (ESI) chip (Advion Biosciences)

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containing the nozzles with 5.5-µm i.d.. A spray voltage of 1.4 kV and a delivery pressure of

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0.4 psi of nitrogen gas were applied, and Chipsoft 8.3.1 software (Advion Biosciences) was

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used to control the ion source. The analysis of lipid extract was performed in duplicate and

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the data acquisition was conducted for 2 min in profile mode. The scan range was set at m/z

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400–1,200 and m/z 500–1,300 in positive- and negative-ion modes, respectively. The mass

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spectrometer settings include: a capillary voltage of 31 V (positive ion mode), –33 V

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(negative ion mode), a tube lens voltage of 250 V (positive ion mode), –117 V (negative ion

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mode), and a capillary temperature of 200°C. The target automatic gain control values for full

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MS and multistage MS were adjusted at 30,000 and 1,000, respectively. MS/MS conditions

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include: a normalized collision energy of 35%, an isolated width of 1.5 m/z units, and a

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charge state of 1. In dynamic exclusion parameters, a repeat duration, an exclusion duration,

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and an exclusion list size were 60 s, 60 s, and 50, respectively.

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The identification of lipids and phytyl derivatives was based on authentic reference

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MS/MS spectra by Melo et al.14 and an in-house library. Also the spectra were confirmed

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with databases including LIPID MAPS (http://www.lipidmaps.org/) and LipidBlast by Kind

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et al.16 For relative quantification of lipid species, the conversion of raw data files (*.raw)

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into *.mzXML format conducted by using ProteoWizard MSConvert.17 Mass spectra were

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imported into the Expressionist MSX v2013.0.39 software (Genedata) for further data

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processing. The spectra scans were averaged, smoothed, and aligned in m/z. Then, the data

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matrices were exported from Expressionist MSX. The peak intensities of each lipid 10


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molecules were normalized with the peak intensity of internal standard. ANOVA with post-

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hoc Tukey’s tests Tukey’s tests were used to assess the statistical significance in lipid

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molecules. The level of statistical significance was set at p < 0.05.

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Quantification of α-Tocopherol and GABA by GC-MS. Internal standard calibration

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was used for quantification of α-tocopherol and GABA in C. vulgaris. Quantitative analysis

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was carried out with GC-MS under the same conditions described in 2.5. The initial oven

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temperature was set at 70°C before being increased to 200°C at 5°C/min, 300°C at 20°C/min

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for 8 min. Calibration standards of α-tocopherol and GABA were analyzed in triplicate. The

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peak area ratios (each peak area of standard mixture/peak area of internal standard) versus

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concentrations were used to obtain the calibration curve. The amount of α-tocopherol and

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GABA were calculated by the regression equation of the calibration curve, and the regression

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equation, R2 value, LOD, and LOQ were listed in Table S2 (Supporting Information).

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■ RESULTS AND DISCUSSION

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Effects of Ethephon on Cell Growth. Figure 1 illustrates the growth curves of C. vulgaris

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exposed to ethephon at concentrations of 0 (control), 50, and 200 µM for 14 days in culture

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flasks. All of the growth profiles have a similar pattern with three phases: lag phase,

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exponential phase, and stationary phase. However, the DW was significantly higher for C.

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vulgaris exposed to 200 µM ethephon (2.90 g/L) than for control and when applying 50 µM

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ethephon (both in 2.76 g/L and 2.80 g/L, respectively) after 14 days of cultivation.

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[Figure 1 near here]

251 11



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It is known that the growth control associated with exposure to ethylene depends on the

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ethylene concentration, tissue type, and plant species. Interestingly, relatively high ethylene

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concentrations exert growth-inhibiting effects on most terrestrial species such as cucumber,

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Arabidopsis thaliana, and wheat, while growth-stimulating effects are seen in semiaquatic

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species such as Rumex palustris.18 In present study, we found that treatment with ethephon at

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200 µM—but not at 50 µM—enhanced the growth of C. vulgaris slightly. Consequently, it is

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suggested that an exogenous ethephon concentration of 200 µM can be considered

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appropriate to apply to C. vulgaris cells when it is used as bioresource for producing various

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bioproducts without growth retardation.

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Metabolic Profiling of Ethephon-Treated C. vulgaris. As listed in Table S3 (Supporting

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Information), 24 metabolites were detected: alcohols (myo-inositol and xylitol), amino acids

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(alanine, asparagine, aspartic acid, glutamic acid, glycine, isoleucine, lysine, phenylalanine,

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proline, serine, threonine, tyrosine, valine, and GABA), organic acids (citric acid, fumaric

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acid, malic acid, and succinate), sugars (galactose and glucose), and other compounds

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(furanone, neophytadiene, and α-tocopherol).

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Figure 2 presents the metabolic pathway of C. vulgaris. The metabolites are distributed in a

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broad metabolic pathway, and their levels differed significantly between the control and

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ethephon-treated groups. Based on the observed changes in metabolite levels, 200 µM

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ethephon resulted in extensive changes in the metabolic profile of C. vulgaris, while 50 µM

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ethephon induced changes only in aspartic acid, glycine, and proline. One of the main

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metabolic changes found in the group treated with 200 µM ethephon was that the levels of

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xylitol, GABA, citrate, furanone, α-tocopherol, and glucose were significantly increased on

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day 7, while those of glycine, proline, and valine were decreased. We also detected that the 12


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levels of asparagine, glutamic acid, lysine, serine, threonine, and GABA were clearly

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elevated in 200-µM-ethephon-treated C. vulgaris on day 14, while those of aspartic acid,

278

glycine, phenylalanine, citrate, succinate, and galactose were decreased.

279 280


281 282

The glycine levels in C. vulgaris were significantly lower on both days 7 and 14 in

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ethephon treatment groups than in control group (Figure 1). It is assumed that the reduction

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of glycine level is related to the scavenging mechanism to lipid peroxidation. Xu et al. found

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that treatment of Houttuynia cordata with excessive ethephon induced a higher accumulation

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of hydrogen peroxide (H2O2), resulting in lipid peroxidation.19 As one of the antioxidant

287

enzymes, glutathione peroxidase (GPx) is commonly used to catalyze the reduction of lipid

288

hydroperoxides.20,21 Glutathione (GSH) is a cofactor for the GPx activation that is

289

synthesized from glycine, cystein, and glutamic acid.22 Therefore, it is conceivable that the

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glycine is consumed for GSH biosynthesis.

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The α-tocopherol was significantly higher in 200 µM ethephon treated C. vulgaris than in

292

control on day 7 (Figure 1). Similarly, increased level of α-tocopherol in mango by

293

exogenous ethylene has been reported.23 It is well known that α-tocopherol is a lipophilic

294

antioxidant to protect the lipids in cellular membranes against oxidative damage. Previous

295

study has suggested that ethylene perception and signaling may be involved in α-tocopherol

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biosynthesis for stress tolerance in A. thanliana.24 Thus, it was assumed that high ethephon

297

concentration stimulated the biosynthesis of α-tocopherol in C. vulgaris.

298

It was observed an increased level of GABA and decreased levels of intermediates in the

299

tricarboxylic acid cycle including succinate and citrate in ethephon treated C. vulgaris on day 13



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14 (Figure 1). It has been shown previously that ethephon treatment also induced the GABA

301

accumulation in Oryza sativa root.25 GABA is metabolized via GABA shunts composed of

302

three enzymes: (1) glutamate decarboxylase (GAD), which converts glutamate to GABA, (2)

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GABA transaminase, which changes GABA into succinic semialdehyde, and (3) succinic

304

semialdehyde dehydrogenase (SSADH), which oxidizes succinic semialdehyde to produce

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succinate.26 Bouche et al. reported that a GABA shunt suppressed the accumulation of H2O2

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generated in a lipid peroxidation condition by regulating GAD positively and SSADH

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negatively.27 Therefore, the decline of GABA and elevation of succinate and citrate levels

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might be due to the activation of a GABA shunt to protect C. vulgaris against ethephon-

309

induced lipid peroxidation. However, the hypothesis of ethephon treatment on metabolites

310

mentioned above obviously requires further study on related gene or enzymes involving

311

metabolism of those metabolites.

312

α-Tocopherol and GABA are known to influence a wide range of mechanisms that affect

313

human health and disease. Coombes et al. reported that α-tocopherol exerts protective effects

314

in preventing cardiovascular disease and decreasing the erythrocyte osmotic fragility and

315

plasma malonyldialdehyde.28 GABA is a nonprotein amino acid that is the predominant

316

inhibitory neurotransmitter of the central nervous system in animals, and is involved in the

317

regulation of cardiovascular functions and the perception of pain and anxiety.29 Therefore, we

318

conducted a quantitative analysis to identify the most appropriate ethephon treatment for

319

enhancing the production of α-tocopherol and GABA from C. vulgaris.

320

Table 1 lists the concentrations and productivities of α-tocopherol and GABA in C.

321

vulgaris extract. The α-tocopherol concentration ranged from 4.03 to 8.62 µg/L, with

322

productivity of 0.48–0.70 µg/L/day, while the GABA concentration ranged from 5.21 to

323

13.29 µg/L, with productivity of 0.37–1.90 µg/L/day. The concentrations and productivities 14


Page 14 of 40

Page 15 of 40


324

of both α-tocopherol and GABA increased with the ethephon concentration in the medium,

325

while the cell growth remained largely unchanged. The highest productivity of α-tocopherol

326

(0.70 µg/L/day) was observed on day 7 for 50-µM-ethephon-treated C. vulgaris. In contrast,

327

the highest productivity of GABA (1.90 µg/L/day) was exhibited on day 7 for 200-µM-

328

ethephon-treated C. vulgaris. Therefore, we suggest that harvesting C. vulgaris cells on day 7

329

after applying ethephon treatment could be a promising strategy for the highest productivities

330

of both α-tocopherol and GABA.

331 332

[Table 1 near here]

333 334

Fatty Acids Profiling of Ethephon-Treated C. vulgaris. The absolute concentrations and

335

percentage compositions of FAs from C. vulgaris treated with ethephon at different

336

concentrations (0, 50, and 200 µM) are listed in Table 2. The nine FAs identified in

337

C. vulgaris comprised three saturated fatty acids (SFAs) [hexadecanoic acid (C16:0),

338

heptadecanoic acid (C17:0), and octadecanoic acid (C18:0)], two monounsaturated fatty acid

339

(MUFA) [trans-octadecenoic acid (C18:1t) and cis-octadecenoic acid (C18:1c)], and four

340

polyunsaturated fatty acids (PUFAs) [hexadecadienoic acid (C16:2), hexadecatrienoic acid

341

(C16:3), octadecadienoic acid (C18:2), and octadecatrienoic acid (C18:3)]. The dominant FA

342

was C18:3 (58–61%), followed by C16:0 (11–12%), and C18:2 (7–10%), with the minor FAs

343

being C17:0 (6–7%), C18:1c (1–3%), and C18:1t (1–3%) followed by C18:0 (0.5–0.6%).

344

This FA composition was consistent with the findings of Dejoye et al.30

345

The results of ANOVA tests shown in Figure 3 and presented in Table 2 indicate that the

346

levels of MUFAs and PUFAs were significantly decreased and that of SFAs was increased in

347

C. vulgaris when ethephon was applied. This effect was obvious in day 14 with the treatment 15



Page 16 of 40

348

of 200 µM ethephon, where the content of C17:0 was significantly increased and the contents

349

of C18:1c, C18:2, C18:3, and C16:3 were significantly decreased compared with the control

350

group. Meanwhile, exposing C. vulgaris to 50 µM of ethephon resulted in changes in C18:1c

351

only, which level was significantly lower on day 7 but significantly higher on day 14.

352 353

[Figure 3 and Table 2 near here]

354 355

Ethylene is closely linked to membrane breakdown involving free radicals. Arora et al.

356

suggested that the synthesis of ethylene induces lipid peroxidation in plant cell membranes.31

357

The PUFAs present in membrane lipids are particularly vulnerable to reactive oxygen species.

358

When lipid peroxidation occurs, PUFAs are converted into several reactive species such as

359

aldehydes (including MDA and 4-hydroxyl-2-nonenal), alkanes, lipid epoxides, and

360

alcohols.32 Therefore, a decreased level of PUFA may imply the presence of ethephon-

361

induced lipid peroxidation.

362 363

Lipid Profiling of Ethephon-Treated C. vulgaris. Table S4 (Supporting Information)

364

summarizes the various lipids detected from C. vulgaris, and their corresponding spectra are

365

illustrated in Figure S1 (Supporting Information). The main classes of glycerolipids in C.

366

vulgaris

367

monogalactosyldiacylglycerol (MGDG) (5 species), digalactosyldiacylglycerol (DGDG) (4

368

species), and sulfoquinovosyldiacylglycerol (SQDG) (15 species), and phospholipids being

369

represented by phosphatidylglycerol (PG) (4 species) and phosphatidylinositol (PI) (3

370

species). Also, the following four phytyl derivatives were detected: chlorophyll a, chlorophyll

371

b, hydroxychlorophyll a, and pheophytin a.

were

measured,

with

galactolipids

16


being

represented

by

Page 17 of 40


372

The largest proportion of lipid species analyzed in this study contained 34 acyl carbons,

373

indicating a combination of 16-C and 18-C chains. It has been reported that the dominance of

374

34-C species in most glycerolipid classes is due to palmitic acid (16:0) and oleic acid (18:1)

375

being the main products of FA synthesis.33 MGDG was the only class containing 36 acyl

376

carbons (two 18-C chains), while SQDG was the only lipid class containing 32 acyl carbons

377

(two 16-C chains). The less-abundant species contained odd numbers of acyl carbons, such as

378

15-C or 17-C chains in MGDG and SQDG classes. The 16-C acyl chains showed a low level

379

of desaturation in phospholipids, whereas a high level of desaturation of 16-C acyl chains

380

was observed in galactolipids.

381

To investigate the changes in the lipids and phytyl derivatives of C. vulgaris induced by

382

ethephon treatment, the statistical significance of the relative intensities of all compounds

383

was assessed using ANOVA tests (with a cutoff of p < 0.05); the results are illustrated in

384

Figure 4 and listed in Table S5 (Supporting Information). Exposing C. vulgaris to 50 µM

385

ethephon resulted in changes only in MGDG 18:3/16:2, whereas 200 µM ethephon caused

386

remarkable changes in various lipid classes. The lipid species that responded to 200 µM

387

ethephon were present at higher levels than in the control condition on day 7. Among these

388

increasing species on day 7 of ethephon treatment, three SQDG species (SQDG 16:0/16:0,

389

SQDG 16:0/17:0, and SQDG 18:0/16:0) contain saturated acyl chains. Moreover, all of the PI

390

species (PI 18:1/16:0, PI 18:2/16:0, and PI 18:3/16:0) and PG 18:1/16:0 that were increased

391

in this condition contained saturated 16-C. Sylvestre and Paulin (1987) reported that

392

suppressing an increase in ethylene inhibited the increase in the level of saturation of the acyl

393

chains in lipids.34 This is in agreement with the increased level of saturation of acyl chains in

394

lipid species from C. vulgaris after ethephon treatment in the present study. Meanwhile, the

395

single increased species of the MGDG class (MGDG 18:3/18:3) in this condition is highly 17



396

desaturated.

397 398


399 400

On the other hand, 200 µM ethephon induced decreases in the levels of lipid species on

401

day 14, with the notable exception of MGDG. In MGDG, most species that responded to

402

ethephon showed higher levels than in the control condition on day 14. Interestingly, in other

403

lipid classes (PI, PG, DGDG, and SQDG) were commonly decreased on day 14 of ethephon

404

treatment. Wade and Bishop (1978) reported the fatty acid composition of the glycolipids and

405

phospholipids in banana fruit pulp tissue during ripening induced by ethylene, in which there

406

was a decrease in the levels of palmitic acid (C16:0) and oleic acid (C18:1) and an increase in

407

the level of linolenic acid (C18:3).35 This distribution of the fatty acyl chains is in accordance

408

with the results on C. vulgaris on day 14, where C18:3-enriched MGDG was increased and

409

lipid species (PI, PG, DGDG, and SQDG) containing C16:0 and C18:1 as their acyl chains.

410

In terms of phytyl derivatives, only hydroxychlorophyll a showed the higher level in

411

200 µM ethephon on day 7. Ethylene involves the senescence of the leaves in plants and

412

accelerates processes characteristic of leaf senescence, such as declines in chlorophyll and

413

photosynthesis.36 No significant decrease in phytyl derivatives was found in the present study,

414

which indicates that 200 µM ethephon did not lead to major damage to chloroplasts.

415

Microalgae have attracted considerable attention since they can serve as an alternative

416

source of lipids for the production of biofuels and biopharmaceuticals. There have been many

417

efforts to enhance lipid production of microalgae based on controlling the nutritional or

418

cultivation conditions, with nutrient starvation being the most commonly employed strategy.

419

However, nutrient deficiency has been reported to also retard cell growth.37 Furthermore, it 18


Page 18 of 40

Page 19 of 40


420

may slow down photosynthesis in microalgal cells because the commonly used limiting

421

nutrients such as nitrates and phosphates are essential for photosynthesis in microalgae. For

422

example, Li et al. observed that the chlorophyll content of Neochloris oleoabundans declined

423

sharply after the external nitrogen pool in the medium was exhausted.38 We found that

424

treating C. vulgaris with ethephon appeared to enrich the lipids without impairing cell growth

425

or damaging the pigment essential for photosynthesis on day 7, with C. vulgaris showing

426

exponential growth. We therefore suggest that ethephon treatment in C. vulgaris could be a

427

promising strategy for enhancing lipid production.

428

Ethephon is a widely used ethylene-relaeasing plant regulator in agriculture. However, its

429

mechanism of action on microalgae in metabolomic level has been unrevealed. To the best of

430

our knowledge, it is the first report that revealed the effect of ethephon on various metabolites

431

present in C. vulgaris. We demonstrate that treatment with ethephon in C. vulgaris induced

432

extensive changes on levels of metabolites including alcohols, amino acids, organic acids,

433

sugars, fatty acid and lipids. Notably, ethephon treatment induced high production of α-

434

tocopherol and GABA in C. vulgaris. In addition, ethephon could be an excellent alternative

435

regulator for controlling lipid composition of micoalgae for the biofuel production. This

436

study provides a foundation for comprehensive understanding the response pathways of C.

437

vulgaris to exogenous ethephon at the metabolic level for the microalgal-based production of

438

biofuels and biopharmaceuticals.

439 440

■ ABBREVIATIONS USED

441

GABA, γ-aminobutyric acid; FA, fatty acid; FAME, fatty-acid methyl ester; GC, gas

442

chromatography; MS, mass spectrometry; nanoESI, nanoelectrospray ionization; GPx,

443

glutathione peroxidase; GSH, glutathione; GAD, glutamate decarboxylase; SSADH, succinic 19



444

semialdehyde dehydrogenase; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated

445

fatty acid; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; SQDG,

446

sulfoquinovosyldiacylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; DAG,

447

diacylglycerol; LPA, lyso-phosphatidic acid; PA, phosphatidic acid.

448 449

■ ACKNOWLEDGEMENTS

450

This research was supported by the Basic Core Technology Development Program for the

451

Oceans and the Polar Regions of the National Research Foundation (NRF) funded by the

452

Ministry of Science, ICT & Future Planning (no. NRF-2016M1A5A1027464) and by the

453

National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP)

454

(NRF-2015R1A5A1008958).

455

20


Page 20 of 40

Page 21 of 40


456

Supporting information

457

Supporting Information Available: Representative spectra of lipid profiles from C. vulgaris

458

derived from nanoESI-MS (Figure S1); Regression equation, correlation coefficient (R2

459

values), LOD, and LOQ of FAMEs standard for FAs in C. vulgaris quantification (Table S1);

460

Regression equation, correlation coefficient (R2 values), LOD, and LOQ of α-tocopherol and

461

GABA standard (Table S2); Comprehensive metabolic profiles of C. vulgaris cultivated

462

under various concentrations of ethephon over three different time periods (Table S3);

463

Identification of lipid species from C. vulgaris by nanoESI-MS (Table S4); Relative levels of

464

lipid compounds in C. vulgaris cultivated under various concentrations of ethephon over

465

three different time periods (Table S5). This material is available free of charge via the

466

Internet at http://pubs.acs.org.

467 468

Notes

469

The authors declare no competing financial interest.

470

21



471

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472

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wastewater effluent by Chlorella vulgaris. Tsinghua Sci. Technol. 2010, 15, 391–396.

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glutathione peroxidase activities in trisomy 16 fetal mice and human trisomy 21

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hydroxyphenylpyruvate dioxygenase leads to increased tocopherol levels during ripening in

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mango. J Exp Bot, 2011, 62, 3375–3385.

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of tocopherol biosynthesis in Arabidopsis thaliana. FEBS letters, 2009, 583, 992–996.

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(25) Reggiani, R. A role for ethylene in low-oxygen signaling in rice roots. Amino acids,

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(29) Park, K.B.; Oh, S.H. Production of yogurt with enhanced levels of gamma-aminobutyric

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acid and valuable nutrients using lactic acid bacteria and germinated soybean

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extract. Bioresour. Technol. 2007, 98, 1675–1679.

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(30) Dejoye, C.; Vian, M.A.; Lumia, G.; Bouscarle, C.; Charton, F.; Chemat, F. Combined

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extraction processes of lipid from Chlorella vulgaris microalgae: microwave prior to

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supercritical carbon dioxide extraction. Int. J. Mol. Sci. 2011,12, 9332–9341.

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(31) Arora, A.; Sairam, R.; Srivastava, G. Oxidative stress and antioxidative system in plants.

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Curr. Sci. 2002, 82, 1227–1238.

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(33) Ohlrogge, J.; Browse, J. Lipid biosynthesis. Plant Cell. 1995, 7, 957–970.

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(34) Sylvestre, I.; Paulin, A. Accelerated ethylene production as related to changes in lipids

559

and electrolyte leakage during senescence of petals of cut carnations (Dianthus

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caryophyllus). Physiol. Plantarum. 1987, 70, 530–536.

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(35) Wade, N.L.; Bishop, D.G. Changes in the lipid composition of ripening banana fruits and

562

evidence for an associated increase in cell membrane permeability. Biochim. Biophys. Acta.

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1978, 529, 454–460.

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(36) Mattoo, K.M.; Aharoni, N. Ethylene and plant senescence. In Senescence and Aging in

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(37) Courchesne, N.M.D.; Parisien, A.; Wang, B.; Lan, C.Q. Enhancement of lipid production

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2009, 141, 31–41.

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(38) Li, Y.; Horsman, M.; Wang, B.; Wu, N.; Lan, C.Q. Effects of nitrogen sources on cell

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growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol. Biot.

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Chlamydomonas. In The Molecular Biology of Chloroplasts and Mitochondria in

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Chlamydomonas; Rochaix, J.D., Goldschmidt-Clermont, M., Merchant, S., Eds.; Kluwer

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Academic Publisher, Dordrecht, Netherlands, 1998; Vol. 7, pp. 415–431.

Trémolières,

A.

Glycerolipids:

Composition,

biosynthesis

577

26


and

function

in

Page 27 of 40


578

■ FIGURE CAPTIONS

579

Figure 1. Growth curve of C. vulgaris grown under various concentrations of ethephon

580

during cultivation. Data are mean values and the vertical bars represent the standard deviation

581

from triplicate experiment.

582 583

Figure 2. The metabolic change and pathway of C. vulgaris cultivated under various

584

concentrations of ethephon over three different time periods. The proposed metabolic

585

pathway was based on the KEGG database (http://www.genome.jp/kegg/). Data are shown as

586

the mean ± SD values for eight measurements (n = 8, biological four replicate and

587

experimental duplicate). ANOVA and post-hoc Tukey’s tests (p < 0.05) were conducted. The

588

* and + indicate significant differences of the responsiveness changes in 50 and 200 µM-

589

ethephon, respectively. The ‡ indicates significant differences in both 50 and 200 µM-

590

ethephon groups. The error bars represent SD values.

591 592

Figure 3. Fatty acids change and pathway of C. vulgaris cultivated under various

593

concentrations of ethephon over three different time periods. The proposed metabolic

594

pathway was based on the KEGG database (http://www.genome.jp/kegg/). Data are shown as

595

the mean ± SD values for eight measurements (n = 8, biological four replicate and

596

experimental duplicate). ANOVA and post-hoc Tukey’s tests (p < 0.05) were conducted. The

597

* and + indicate significant differences of the responsiveness changes in 50 and 200 µM-

598

ethephon, respectively. The ‡ indicates significant differences in both 50 and 200 µM-

599

ethephon groups. The error bars represent SD values.

600 601

Figure 4. Graphs showing lipid species changes in C. vulgaris treated with 27



Page 28 of 40

602

a different concentration of ethephon. Data are shown as the mean ± SD values for eight

603

measurements (n = 8, biological four replicate and experimental duplicate). ANOVA and

604

post-hoc Tukey’s tests (p < 0.05) were conducted. The * and + indicate significant differences

605

of the responsiveness changes in 50 and 200 µM-ethephon, respectively. The ‡ indicates

606

significant differences in both 50 and 200 µM-ethephon groups. The error bars represent SD

607

values. The arrows represent the lipid metabolism based on previous findings from studies of

608

Chlamydomonas.39 Proposed metabolic pathway of phytyl derivatives was based on the

609

KEGG

610

digalactosyldiacylglycerol;

611

monogalactosyldiacylglycerol; PA, phosphatidic acid; PG, phosphatidylglycerol; PI,

612

phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol

database

(http://www.genome.jp/kegg/). LPA,

DAG,

lyso-phosphatidic

28


diacylglycerol;

DGDG,

acid;

MGDG,

Page 29 of 40


613

Table 1. Concentration (µg/L) and productivity (µg/L/day) of α-tocopherol and GABA in C. vulgaris cultivated under various concentrations of

614

ethephon over three different time periods. Day 7 Compound α-tocopherol GABA

0day Concentration (µg/L) Productivity (µg/L/day) Concentration (µg/L)

4.08±0.50 9.34±3.71

Control 4.03±0.58 a 0.58±0.082 a 8.16±1.08 a

Productivity (µg/L/day)

-

1.14±0.15 a

Day 14 50 µM 4.93±0.35 b 0.70±0.050 b 11.57±1.24 b

200 µM 4.20±0.30 a 0.60±0.043 a 13.29±1.60 c

Control 6.74±0.80 0.48±0.057 5.21±0.63 A

50 µM 8.62±0.52 0.62±0.037 8.28±1.50 B

1.62±0.18 b

1.90±0.23 c

0.37±0.045 A 0.59±0.11 B

200 µM 8.41±2.54 0.60±0.18 11.79±2.43 C 0.84±0.17 C

615 616

Data are shown as the mean ± SD values for 8 measurements (biological four replicate and experimental duplicate). Lower case letters-superscripts (a-c)

617

denote significant differences (p < 0.05) among three groups (control, 50 µM, and 200 µM) on 7day. Capital letter-superscripts (A-C) denote significant

618

differences (p < 0.05) among four groups (control, 50 µM, and 200 µM) on 14day.

29



Page 30 of 40

619

Table 2. Fatty acid profiles, absolute concentrations (mg FA/g dried cell), and percent composition (% of total identified FA) in C. vulgaris

620

cultivated under various concentrations of ethephon over three different time periods. CX:Y

Fatty acids

RT

mg FA/g DW (% composition) 0Day

Day 7

Day 14

Control C16:0

C16:2

C16:3

C17:0

C18:0

C18:1t

C18:1c

C18:2

Hexadecanoic acid

Hexadecadienoic acid

Hexadecatrienoic acid

Heptadecanoic acid

Octadecanoic acid

trans-Octadecenoic acid

cis-Octadecenoic acid

Octadecadienoic acid

20.44

21.98

23.78

21.73

26.32

26.93

27.19

28.60

50 µM ab

200 µM

8.36±0.0043

a

Control

8.37±0.0047

b

8.36±0.0036

50 µM A

8.35±0.0020 B

8.37±0.0047

8.36±0.0068

(11.29)

(12.29)

(12.73)

(11.73)

(12.11)

(12.39)

(12.9)

2.30±0.0029

2.30±0.0039

2.29±0.0034

2.30±0.0030

2.30±0.0021 AB

2.30±0.0029 A

2.29±0.0093 B

(2.99)

(3.35)

(3.44)

(3.21)

(2.97)

(2.98)

(3.35)

A

8.36±0.0026

200 µM A

4.58±0.0022 B

4.60±0.0047

4.59±0.0073

4.59±0.0051

4.59±0.0063

4.59±0.0027

(3.10)

(3.37)

(3.49)

(3.22)

(3.33)

(3.41)

(3.54)

2.21±0.14

2.28±0.21

2.26±0.19

2.29±0.10

2.05±0.14 A

2.01±0.064 A

2.17±0.077 B

(6.20)

(6.75)

(6.99)

(6.43)

(6.65)

(6.80)

(7.07)

0.43±0.038

0.40±0.020 a

0.38±0.025 a

0.49±0.11 b

0.35±0.018 A

0.38±0.012 B

0.34±0.027 A

(0.58)

(0.59)

(0.58)

(0.69)

(0.51)

(0.56)

(0.52)

1.22±0.13

1.42±0.48

1.35±0.46

ab

1.04±0.37

a

2.36±1.06

b

4.59±0.0032

A

2.56±0.39

1.77±0.20

(3.46)

(2.59)

(1.58)

(3.30)

(1.76)

(2.11)

(2.08)

2.05±0.21

1.51±0.20 a

1.20±0.14 b

1.31±0.24 ab

1.86±0.14 A

2.03±0.089 B

1.51±0.13 C

(2.76)

(2.21)

(1.83)

(1.83)

(2.69)

(3.01)

(2.33)

8.06±0.39

6.23±0.75 ab

5.46±1.05 a

6.68±0.65 b

6.79±0.37 A

6.39±0.28 A

4.70±0.50 B

(10.87)

(9.16)

(8.30)

(9.36)

(9.83)

(9.47)

(7.26)

30


Page 31 of 40

C18:3


Octadecatrienoic acid

ΣSFA

ΣMUFA

ΣPUFA

31.19

43.58±1.50

40.62±3.17

40.12±2.49

42.96±2.92

41.52±1.92 A

39.99±1.30 AB

39.46±0.77 B

(58.76)

(59.68)

(61.07)

(60.21)

(60.14)

(59.27)

(60.94)

11.01±0.17

11.05±0.23 ab

11.00±0.18 a

11.15±0.19 b

10.76±0.16 A

10.75±0.07 A

10.86±0.08 B

(14.85)

(16.27)

(16.77)

(15.68)

(15.60)

(15.94)

(16.78)

3.08±0.21

3.45±0.49

2.86±0.58

4.61±0.52

3.27±0.34

(6.22)

(4.81)

a

2.24±0.50

b

(3.40)

3.66±1.28

c

(5.07)

(4.45)

58.54±1.76

53.74±3.90

52.46±2.76

56.53±3.56

55.20±2.11

(78.93)

(78.92)

(79.83)

(79.25)

(79.95)

(5.11) A

53.27±1.46 (78.96)

(4.39) B

51.04±1.25 B (78.82)

621 622

Data are shown as the mean ± SD values for 8 measurements (biological four replicate and experimental duplicate). Lower case letters-superscripts (a-c)

623

denote significant differences (p < 0.05) among three groups (control, 50 µM, and 200 µM) on 7day. Capital letter-superscripts (A-C) denote significant

624

differences (p < 0.05) among four groups (control, 50 µM, and 200 µM) on 14day. RT, retention time; SFA, saturated fatty acid; MUFA, monounsaturated

625

fatty acid; PUFA, polyunsaturated fatty acid

31



626

Figure 1

627 628

32


Page 32 of 40

Page 33 of 40

629


Figure 2

630

33



631

Figure 3

632 34


Page 34 of 40

Page 35 of 40

633


Figure 4

634 35



635

[Graphic for table of contents]

636

36


Page 36 of 40

Page 37 of 40


Figure 1. Growth curve of C. vulgaris grown under various concentrations of ethephon during cultivation. Data are mean values and the vertical bars represent the standard deviation from triplicate experiment. 254x190mm (300 x 300 DPI)



Figure 2. The metabolic change and pathway of C. vulgaris cultivated under various concentrations of ethephon over three different time periods. The proposed metabolic pathway was based on the KEGG database (http://www.genome.jp/kegg/). Data are shown as the mean ± SD values for eight measurements (n = 8, biological four replicate and experimental duplicate). ANOVA and post-hoc Tukey’s tests (p < 0.05) were conducted. The * and + indicate significant differences of the responsiveness changes in 50 and 200 µM-ethephon, respectively. The ‡ indicates significant differences in both 50 and 200 µM-ethephon groups. The error bars represent SD values. 190x254mm (300 x 300 DPI)


Page 38 of 40

Page 39 of 40


Figure 3. Fatty acids change and pathway of C. vulgaris cultivated under various concentrations of ethephon over three different time periods. The proposed metabolic pathway was based on the KEGG database (http://www.genome.jp/kegg/). Data are shown as the mean ± SD values for eight measurements (n = 8, biological four replicate and experimental duplicate). ANOVA and post-hoc Tukey’s tests (p < 0.05) were conducted. The * and + indicate significant differences of the responsiveness changes in 50 and 200 µM-ethephon, respectively. The ‡ indicates significant differences in both 50 and 200 µM-ethephon groups. The error bars represent SD values. 254x190mm (300 x 300 DPI)



Figure 4. Graphs showing lipid species changes in C. vulgaris treated with a different concentration of ethephon. Data are shown as the mean ± SD values for eight measurements (n = 8, biological four replicate and experimental duplicate). ANOVA and post-hoc Tukey’s tests (p < 0.05) were conducted. The * and + indicate significant differences of the responsiveness changes in 50 and 200 µM-ethephon, respectively. The ‡ indicates significant differences in both 50 and 200 µM-ethephon groups. The error bars represent SD values. The arrows represent the lipid metabolism based on previous findings from studies of Chlamydomonas.39 Proposed metabolic pathway of phytyl derivatives was based on the KEGG database (http://www.genome.jp/kegg/). DAG, diacylglycerol; DGDG, digalactosyldiacylglycerol; LPA, lysophosphatidic acid; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol 254x190mm (300 x 300 DPI)


Page 40 of 40

Effect of Ethephon as an Ethylene-Releasing Compound on the

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