ORIGINAL RESEARCH published: 20 August 2020 doi: 10.3389/fpls.2020.01259 Glycerol Is an Osmoprotectant in Two Antarctic Chlamydomonas Species From an Ice-Covered Saline Lake and Is Synthesized by an Unusual Bidomain Enzyme † James A. Raymond 1*, Rachael Morgan-Kiss 2 and Sarah Stahl-Rommel 2 1 School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, United States, 2 Department of Microbiology, Miami University, Oxford, OH, United States Glycerol, a compatible solute, has previously been found to act as an osmoprotectant in Edited by: some marine Chlamydomonas species and several species of Dunaliella from hypersaline Linda Nedbalova´ , Charles University, Czechia ponds. Recently, Chlamydomonas reinhardtii and Dunaliella salina were shown to make Reviewed by: glycerol with an unusual bidomain enzyme, which appears to be unique to algae, that Aharon Oren, contains a phosphoserine phosphatase and glycerol-3-phosphate dehydrogenase. Here Hebrew University of Jerusalem, Israel David Dewez, we report that two psychrophilic species of Chlamydomonas (C. spp. UWO241 and ICE- Universite´ du Que´ bec à Montre´ al, MDV) from Lake Bonney, Antarctica also produce high levels of glycerol to survive in the Canada lake’s saline waters. Glycerol concentration increased linearly with salinity and at 1.3 M *Correspondence: NaCl, exceeded 400 mM in C. sp. UWO241, the more salt-tolerant strain. We also show James A. Raymond [email protected] that both species expressed several isoforms of the bidomain enzyme. An analysis of one †Present address: of the isoforms of C. sp. UWO241 showed that it was strongly upregulated by NaCl and is Sarah Stahl-Rommel thus the likely source of glycerol. These results reveal another adaptation of the Lake JES Tech, Houston, TX, United States Bonney Chlamydomonas species that allow them to survive in an extreme Specialty section: polar environment. This article was submitted to Keywords: Chlamydomonas, glycerol synthesis, phosphoserine phosphatase, glycerol-3-phosphate Marine and Freshwater Plants, dehydrogenase, Antarctica, Lake Bonney a section of the journal Frontiers in Plant Science Received: 18 June 2020 Accepted: 30 July 2020 INTRODUCTION Published: 20 August 2020 Citation: Glycerol is well-known for its ability to mitigate environmental stresses such as freezing and Raymond JA, Morgan-Kiss R and hyperosmotic conditions in a wide variety of organisms, including yeast (Wang et al., 2001), insects Stahl-Rommel S (2020) Glycerol Is an (Bentz and Mullins, 1999; Bennett et al., 2005), and vertebrates (Raymond, 1992; Layne, 1999). It is Osmoprotectant in Two Antarctic also well-known as an osmoprotectant in some algae, including salt pond isolates of Dunaliella Chlamydomonas Species From an (Ben-Amotz and Avron, 1973; Sussman and Avron, 1981) and several marine isolates of Ice-Covered Saline Lake and Is Chlamydomonas (Ahmad and Hellebust, 1986; Miyasaka and Ikeda, 1997; Miyasaka et al., 1998). Synthesized by an Unusual Bidomain Enzyme. As a compatible solute, glycerol prevents osmotic water loss and thus a build-up of intracellular salt Front. Plant Sci. 11:1259. concentrations. Two psychrophilic Chlamydomonas species, Chlamydomonas sp. UWO241 (Neale doi: 10.3389/fpls.2020.01259 and Priscu, 1995) (hereafter Chlamy-UWO) and Chlamydomonas sp. ICE-MDV (Li et al., 2016) Frontiers in Plant Science | www.frontiersin.org 1 August 2020 | Volume 11 | Article 1259 Raymond et al. Glycerol Synthesis in Antarctic Chlamydomonas (hereafter Chlamy-ICE) were isolated from Lake Bonney, a bidomain enzyme to produce glycerol. We also show that permanently ice-covered lake in Antarctica. The lake has a Chlamy-ICE is capable of producing high levels of glycerol steep halocline, in which the salinity reaches about 150 PSU under saline conditions and has similar bidomain enzymes. (equivalent to about 2.1 M NaCl, or 4.3 x the salinity of seawater) at a depth of 40 m (Spigel et al., 2018). Chlamy-UWO’s ability to survive in this environment has been intensively studied for over METHODS two decades. Among its adaptation are a photochemical apparatus well-suited for a cold, saline and low light environment Cells (Morgan et al., 1998), more fluid membranes (Morgan-Kiss et al., Chlamydomonas sp. UWO241 [Chlamy-UWO, also referred to 2002) and ice-binding proteins (IBPs) to prevent freeze-thaw injury as C. raudensis in some earlier studies, e.g., (Dolhi et al., 2013)] (Raymond and Morgan-Kiss, 2013). Less is known about Chlamy- and Chlamydomonas sp. ICE-MDV (Chlamy-ICE) were ICE which was isolated more recently: it also possesses IBPs previously isolated from the east lobe of Lake Bonney, (Raymond and Morgan-Kiss, 2017) but appears to have Antarctica. Chlamy-UWO was isolated from below the differences in acclimatory ability compared to Chlamy-UWO permanent chemocline in the deep photic zone (17 m) where (Cook et al., 2019). Much more is known about the the salinity is similar to the salinity of seawater but rapidly cold adaptations of a closely-related Antarctic pyschrophile increases with increasing depth (Spigel et al., 2018). Its natural Chlamydomonas sp. ICE-L (Cvetkovska et al., 2017). Because depth range is not known. Chlamy-ICE was recovered from a both Chlamy-UWO and Chlamy-ICE are adapted to a saline depth of 13 m, where the salinity is markedly lower (Li et al., environment, we wished to know whether they also produce 2016). Both strains were maintained in Bold’s basal medium glycerol as an osmoprotectant or cryoprotectant. (BBM) supplemented with the indicated amounts of NaCl in a − − Until recently, the pathways used by algae to produce glycerol temperature/light regime of 8°C/50 mmol · m 2 ·s1 at Miami have been a matter of speculation. Glycogen, or one of its products, University. Chlamydomonas sp. UWO241 is deposited in the dihydroxyacetone phosphate (DHAP), has been identified as the Bigelow Algal collection (CCMP1619). Chlamydomonas sp. ICE- source of glycerol in a number of organisms, including yeast, MDV is available upon request to RMK. (Modigetal.,2007), Arabidopsis (Caparros-Martin et al., 2007), a bacterium (Larrouy-Maumus et al., 2013), mountain pine beetle Glycerol Measurement (Fraser et al., 2017), and rainbow smelt (Raymond, 1995). In the Fresh cells were shipped overnight to the University of Nevada DHAP pathway, DHAP is converted to glycerol-3-phosphatate Las Vegas (UNLV) with ice packs. The temperature varied by less (G3P) by a nicotinamide-adenine dinucleotide (NAD+)-dependent than 2°C during shipment. For each sample, 1.50 ml of cell glycerol-3 phosphate dehydrogenase (GPDH), which is then culture was centrifuged in pre-weighed tubes. The supernatant converted to glycerol by a phosphoserine phosphatase (PSP). The was removed and the remaining medium was removed with a problem is that algae typically have several GPDHs and phosphatases, fine, drawn-out pipet. The tubes were reweighed to obtain the and attempts to identify the enzymes involved have been until pellet weights (about 3 mg), sealed, and stored at -25°C. For recently unsuccessful. glycerol measurement, the tubes were thawed, and the bottoms Dunaliella salina has a bidomain enzyme that includes an N- of the tubes were subjected to two freeze-thaw cycles in liquid terminal PSP and a C-terminal NAD+-dependent GPDH (He nitrogen to break the cells. The pellets were suspended in 1.00 ml et al., 2007). It was proposed as a candidate for glycerol DI water, vortexed to release glycerol and centrifuged at 16,000 x production directly from DHAP (He et al., 2007), but initial g for 2 min at 4°C to yield a clear supernatant. Subsequent testing attempts to confirm its activity were unsuccessful (He et al., of the pellet showed that virtually all of the glycerol was released 2009). However, this enzyme still seemed like the most likely from the cells. Glycerol was quantified enzymatically with Free source of glycerol. During our study of IBPs in Chlamy-UWO glycerol reagent (Sigma no. F6428), which develops a 540 nm (Raymond and Morgan-Kiss, 2013), we noticed that it produced (purple) color in the presence of glycerol. Intracellular glycerol glycerol as an osmoprotectant, i.e., glycerol production increased concentration (mM) in a cell pellet was calculated as Mg x 1,000/ with increasing salinity. We searched the Chlamy-UWO genome (MW x Mp x f), where Mg is the mass of glycerol released from for a Dunaliella-like bidomain enzyme and found at least two the pellet, MW is the molecular weight of glycerol (92 g mol-1), homologs. We then examined the expression of one of the Mp is the mass of the pellet, and f is fraction of the cell mass that homologs and found that it strongly increased with increasing is water. The latter was estimated as 0.7 based on measurements salinity, as reported in a master’s thesis (Stahl, 2014). This of Chlamydomonas pulsatilla (Ahmad and Hellebust, 1986). This suggested that this enzyme alone was capable of producing calculation assumed that the extracellular water content in the glycerol from DHAP pellets was zero. If extracelular water were present in the pellets, Recently, Morales-Sanchez et al. (2017) showed that a similar the actual glycerol concentrations would be higher than bidomain enzyme in Chlamydomonas reinhardtii was sufficient those reported. to produce glycerol directly from DHAP, and He Q. H. et al. (2020) finally confirmed that the Dunaliella salina bidomain Gene Sequences protein was sufficient to produce glycerol. Here, we report our Sequences of the enzymes described in this study were assembled test of the hypothesis that Chlamy-UWO uses a similar from transcriptome and genome data obtained from Chlamy- Frontiers in Plant Science | www.frontiersin.org 2 August 2020 | Volume 11 | Article 1259 Raymond et al. Glycerol Synthesis in Antarctic Chlamydomonas UWO (Raymond and Morgan-Kiss, 2013) and Chlamy-ICE viewer (Waterhouse et al., 2018) (http://www.yasara.org/).