Methanol-Promoted Lipid Remodelling during Cooling Sustains Cryopreservation Survival of Chlamydomonas reinhardtii

RESEARCH ARTICLE Methanol-Promoted Lipid Remodelling during Cooling Sustains Cryopreservation Survival of Chlamydomonas reinhardtii Duanpeng Yang1,3, Weiqi Li1,2* 1 Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China, 2 Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China, 3 University of Chinese Academy of Sciences, Beijing, China a11111 * Abstract OPEN ACCESS Citation: Yang D, Li W (2016) Methanol-Promoted Lipid Remodelling during Cooling Sustains Cryopreservation Survival of Chlamydomonas reinhardtii. PLoS ONE 11(1): e0146255. doi:10.1371/ journal.pone.0146255 Editor: Andrew Webber, Arizona State University, UNITED STATES Received: September 10, 2015 Accepted: December 15, 2015 Published: January 5, 2016 Copyright: © 2016 Yang, Li. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Cryogenic treatments and cryoprotective agents (CPAs) determine the survival rate of organisms that undergo cryopreservation, but their mechanisms of operation have not yet been characterised adequately. In particular, the way in which membrane lipids respond to cryogenic treatments and CPAs is unknown. We developed comparative profiles of the changes in membrane lipids among cryogenic treatments and between the CPAs dimethyl sulfoxide (DMSO) and methanol (MeOH) for the green alga Chlamydomonas reinhardtii. We found that freezing in liquid nitrogen led to a dramatic degradation of lipids, and that thawing at warm temperature (35°C) induced lipid remodelling. DMSO did not protect membranes, but MeOH significantly attenuated lipid degradation. The presence of MeOH during cooling (from 25°C to −55°C at a rate of 1°C/min) sustained the lipid composition to the extent that membrane integrity was maintained; this phenomenon accounts for successful cryopreservation. An increase in monogalactosyldiacylglycerol and a decrease in diacylglycerol were the major changes in lipid composition associated with survival rate, but there was no transformation between these lipid classes. Phospholipase D-mediated phosphatidic acid was not involved in freezing-induced lipid metabolism in C. reinhardtii. Lipid unsaturation changed, and the patterns of change depended on the cryogenic treatment. Our results provide new insights into the cryopreservation of, and the lipid metabolism in, algae. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by grants from the National Natural Science Foundation of China (31070262), Kunming Institute of Botany (KSCX2-EW-J-24), the Germplasm Bank of Wild Species and the CAS Innovation Program of Kunming Institute (540806321211), as well as the 100-Talents Program of CAS. Competing Interests: The authors have declared that no competing interests exist. Introduction Cryopreservation is commonly used for storing viable cells, tissues, organs or organisms at ultralow temperatures, usually involving immersion in liquid nitrogen at −196°C. Using this procedure, organisms can be preserved with their morphological, physiological, biochemical and genetic properties unchanged [1]. As a consequence of the development of cryopreservation, cryobanking was established as a means of protecting biodiversity, important (valuable or endangered) organisms and genetic resources [2]. The methods of cryopreservation for plants PLOS ONE | DOI:10.1371/journal.pone.0146255 January 5, 2016 1 / 17 Lipids Remodelling in Cryopreservation of Chlamydomonas reinhardtii Abbreviations: ACL, acyl chain length; C, Control (normal growth); CC’LT, Control-Cooling-freezing in Liquid nitrogen and Thawing; CC’LTR, ControlCooling-freezing in Liquid nitrogen-Thawing and Recovery growth; CC’T, Control-Cooling and Thawing; CLT, Control-freezing in Liquid nitrogen and Thawing; CC’LR, Control-Cooling-freezing in Liquid nitrogen-maintenance at room temperature for 30 minutes and Recovery growth; CPA, cryoprotective agent; CM, culture medium; DAG, diacylglycerol; DBI, double bond index; DGDG, digalactosyldiacylglycerol; DGTS, diacylglyceryl-N,N,N-trimethylhomoserine; DMSO, dimethyl sulfoxide; ESI-MS/MS, electrospray ionization tandem mass spectrometry; LN, liquid nitrogen; MeOH, methanol; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PLD, phospholipase D; PS, phosphatidylserine; SPSS, Statistical Product and Service Solutions; TAG, triacylglycerol; TAP, Trisacetate-phosphate. include two-step cooling: vitrification and encapsulation-dehydration [3]. Herein, we focus on two-step cooling. When using this method, materials are first cooled with a cryoprotective agent (CPA) at a slow and constant rate (0.2–1°C/min) to between −35°C and −75°C; then they are dipped into liquid nitrogen for storage [4]. Two-step cooling is an effective method for cryopreserving Chlamydomonas reinhardtii, which is an important model organism; the viability on revival is usually >40% [4]. The use of a CPA is essential for successful cryopreservation. Such agents include methanol (MeOH) and dimethyl sulfoxide (DMSO). MeOH is usually used when cryopreserving freshwater and terrestrial algal strains, whereas DMSO is more effective than MeOH for cryopreserving marine algae [5]. DMSO is a good CPA for higher plants, but CPA that contains 2–10% MeOH works well for algae. Cryopreservation of C. reinhardtii is also important for industrial applications [6]. The cryopreservation of this organism has been documented intensively, in terms of cryoprocedure and the choice of CPA [2]. However, despite the importance of cryopreservation and the attention that has been paid to which methods are effective, little is known about the cellular changes that cryopreserved organisms undergo and how these changes sustain survival. Cryopreservation is a complicated process, in which ice nucleation and glass transition are two determinative events for the survival of cryopreserved organisms. In most biological systems, the temperature of homogeneous ice nucleation is at, or around, −40°C [7]. At this temperature, water molecules form an “ice embryo” of a critical size that then grows into a crystal. The glass transition temperature is around −130°C to −137°C. At this temperature the “glass state” is formed, called vitrification, in which the crystals solidify together [8]. The ice nucleation and glass transition can be modulated by cryoprotective strategies, mainly by changing the CPA and cooling rate. In general, an effective CPA should penetrate the cell and be nontoxic at its working concentration [2]. Glycerol, DMSO, low molecular we (...truncated)