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ABSTRACT The novel extremophilic yeast _Rhodotorula frigidialcoholis_, formerly _R_. JG1b, was isolated from ice-cemented permafrost in University Valley (Antarctic), one of coldest and

driest environments on Earth. Phenotypic and phylogenetic analyses classified _R. frigidialcoholis_ as a novel species. To characterize its cold-adaptive strategies, we performed mRNA and

sRNA transcriptomic analyses, phenotypic profiling, and assessed ethanol production at 0 and 23 °C. Downregulation of the ETC and citrate cycle genes, overexpression of fermentation and

pentose phosphate pathways genes, growth without reduction of tetrazolium dye, and our discovery of ethanol production at 0 °C indicate that _R. frigidialcoholis_ induces a metabolic switch

from respiration to ethanol fermentation as adaptation in Antarctic permafrost. This is the first report of microbial ethanol fermentation utilized as the major energy pathway in response to

cold and the coldest temperature reported for natural ethanol production. _R_. _frigidialcoholis_ increased its diversity and abundance of sRNAs when grown at 0 versus 23 °C. This was

consistent with increase in transcription of Dicer, a key protein for sRNA processing. Our results strongly imply that post-transcriptional regulation of gene expression and mRNA silencing

may be a novel evolutionary fungal adaptation in the cryosphere. SIMILAR CONTENT BEING VIEWED BY OTHERS MORPHOLOGICAL AND PHYSIOLOGICAL ADAPTATIONS OF PSYCHROPHILIC _PSEUDARTHROBACTER

PSYCHROTOLERANS_ YJ56 UNDER TEMPERATURE STRESS Article Open access 11 September 2023 A COLD SHOCK PROTEIN PROMOTES HIGH-TEMPERATURE MICROBIAL GROWTH THROUGH BINDING TO DIVERSE RNA SPECIES

Article Open access 16 March 2021 _SUBTERCOLA ENDOPHYTICUS_ SP. NOV., A COLD-ADAPTED BACTERIUM ISOLATED FROM _ABIES KOREANA_ Article Open access 15 July 2022 INTRODUCTION The majority of the

Earth’s biosphere exists at permanently cold temperatures, below 5 °C, and includes multiple cryoenvironments (<0 °C), many of which are characterized by some of the most dry, low

biomass, cold, and salty conditions on Earth. Despite these harsh conditions, microorganisms capable of metabolic activity and even growth in situ have been reported in these habitats and

include bacteria, archaea, algae, fungi [1,2,3]. These cold-adapted microorganisms are termed as psychrophilic (optimum temperature below 15 °C) or psychrotolerant (optimum above 15 °C) [4],

and are able to maintain viability for thousands of years in glacial ice [5, 6]. Microbial communities in these environments have to overcome numerous biochemical and physiological

challenges, including low water and nutrient availability, high oxidative stress, high solar irradiation, and multiple freeze-thaw cycles, coupled with a major decrease in membrane fluidity,

enzymatic activity, and protein folding, and with the creation of stable secondary inhibitory DNA/RNA structures [4, 7,8,9]. While numerous studies have investigated bacteria, archaea and

algae from Arctic and Antarctica environments [4, 10, 11], there has been a recent interest in fungi inhabiting these environments [12,13,14]. Many fungal species have been isolated and

characterized from a diversity of extreme environments, such as brines [15], Arctic glaciers, and Antarctica rocks and deserts [5, 16, 17]. Fungi play key roles in the cryosphere as they are

important facilitators of primary biomass production through endophytic and lichenic relationships [18, 19] and are involved in the nutrients recycling [20]. Although many basidiomycetous

yeasts are adapted to low temperatures and detected in a broad range of cold ecosystems [21, 22], their adaptation strategies to low temperatures are not fully understood. Molecular

mechanisms enabling yeast survival include the production of antifreeze and cold-active proteins, compatible solutes, and an increase in membrane fluidity [5, 23]. For example, the

heterotrophic _Rhodotorula_ yeasts are unicellular and pink-pigmented [24, 25] and have been isolated in many cold habitats [26]; numerous cold-adapted species, including _R. aurantiaca_,

_R. psychrophila_, _R. psychrophenolica_, _R. glacialis_, and _R. himalayensis_ [27, 28] have been identified. More recently, we isolated the putative novel psychrotolerant _Rhodotorula_

JG1b strain from ~150,000-year-old ice-cemented permafrost soil from University Valley, in the McMurdo Dry Valleys of Antarctica [24], one of the coldest and driest places on Earth [29].

_Rhodotorula_ JG1b was one of only six microorganisms isolated and sequenced form this ice-cemented permafrost. It was capable of growth at temperatures as low as −10 °C [1] and tolerated up

to 15% NaCl and 12% perchlorate [24]. In this study, _Rhodotorula_ JG1 was used as a model yeast to determine its adaptations to cold temperatures and how it survives in one of the coldest

environments on Earth. Our objectives were to determine the phylogenetic position of _Rhodotorula_ JG1b within the _Rhodotorula_ genus and to identify and characterize its metabolic activity

pathways and regulatory mechanisms in response to cold. To determine _R. frigidialcoholis’_ response to colder temperatures, we characterized this strain’s metabolic capabilities at 0 °C,

performed transcriptomic mRNAseq analysis in cultures grown at 0 and 23 °C, and assessed this species’ ethanol production capability at 0 °C compared to 23 °C. In addition, we analyzed the

short non-coding RNAs to determine their role in post-transcriptional gene regulation to cold adaptation. MATERIALS AND METHODS TAXONOMIC ANALYSIS _Rhodotorula_ JG1b was isolated from

ice-cemented permafrost soil in University Valley (Antarctica) (24). To determine the phylogenetic position of _Rhodotorula_ JG1b, we performed phylogenetic, phenotypic, metabolic, and

physiological characterization of this strain. Phylogenetic characterization and analyses were performed using the small and large subunits of the rRNA gene, the internal transcribed spacers

(ITS) 1 and 2, translation elongation factor 1-α (TEF), and cytochrome b as well as publicly available whole genome sequences of the _Rhodotorula_ genus (Supplementary Fig. S1). Detailed

methodology of the phylogenetic analyses is described in the Supplementary material (Supplementary File 1 and Supplementary Fig. S2). The morphology of the strain was determined by growth on

potato dextrose agar (PDA), malt extract agar (MEA), cornmeal agar (CMA), and yeast extract peptone dextrose (YPD) [30], after incubation at 15 °C for 14 days. Morphological characters of

pure cultures were observed on MEA with Nomarski interference contrast optics on an Olympus BX-51 microscope, colony color was described according to [31]. Growth at different temperatures

(0, 4, 15, 20, 24, 30 and 37 °C) was determined in microtiter plates in liquid medium yeast nitrogen base. Three inocula were used (A, B, C) of optical density 0.4, 0.2, and 0.02,

respectively, with 8 replicates/inocula. The growth curves were determined via OD at 590 nm. Assimilation of carbon and nitrogen sources were studied on three different sets of Phenotypic

MicroArray (PM; Biolog) plates, PM1 MicroPlate and PM2A MicroPlate for carbon metabolism, and PM3B MicroPlate for nitrogen metabolism, as described by Viti et al., incubated at 20 °C for up

to 14 days. Fermentation of D-glucose was tested in liquid medium with a 2% solution of sugar with Bromothymol blue and Durham tube [30], after an incubation of 12 days at 5, 15, 20, 30, and

37 °C. CULTURING AND GROWTH CONDITIONS For all subsequent experiments, _Rhodotorula_ JG1b was cultured on PDB agar (HIMEDIA, Shenzhen, China). Biological triplicates of _Rhodotorula_ JG1b

were grown in 50 ml liquid cultures to early exponential phase in PDB at 0 °C without shaking, and at 23 °C with shaking at 150 RPM. Growth was monitored using OD600 and converted to viable

cell numbers/ml based on CFU counts. Lack of shaking was necessary for the 0 °C treatment to allow growth at this low temperature since growth was not observed under shaking conditions at 0 

°C. We separately ensured that the 0 °C non-shaking cultures remained aerobic (11.5 ± 0.98 mg O2/l) during growth by monitoring O2 concentrations using a Piccolo2 Fiber-Optic Oxygen Meter

(Pyroscience, Aachen, Germany). O2 was not limited in this treatment, due to small culturing volume, slower growth rate at low temperature, and increase of oxygen solubility at lower

temperature [32]. PHENOTYPIC MICROARRAY ANALYSIS To physiologically characterize _Rhodotorula_ JG1b metabolic capabilities under cold conditions, its assimilation of different carbon and

nitrogen sources at 0 °C was assessed using the Biolog Phenotypic MicroArray (PM) technology (Biolog, Hayward, CA, United States). PM1 MicroPlate and PM2A MicroPlate were used for

determining carbon utilization, and PM3B MicroPlate was used for nitrogen utilization. The plates were inoculated following Viti et al. protocol [33] with the following modifications: the

initial inoculum was adjusted to OD600 0.200, and the Biolog Dye Mix G was used. The duplicate plates were incubated at 0 °C, and at room temperature of 23 °C for comparison. Absorbance

readings (590 nm) were taken at days 0, 1, 2, 6, 9, 14, 21, 28, 42, 56, 70, and 91 of incubation on a SpectraMax M2e microplate reader (Molecular Devices, San Jose, CA, United States). The

measurements were normalized by the subtraction of both the _T_0 and the negative control. Absorbance values >0.2 were considered as positive values as previously described [34]. RNA

EXTRACTION, LIBRARY PREPARATION, AND SEQUENCING To perform transcriptomic analysis, biological triplicate _Rhodotorula_ JG1b cultures of each condition were grown as described in section

“Culturing and growth conditions” to early exponential phase: OD600 0.95 at 0 °C; and OD600 6.20 at 23 °C. RNA was extracted using the Direct-zol RNA Miniprep Plus kit (#R2070) from Zymo

Research (Irvine, CA, United States) on cultures with an initial resuspended pellet of approximately 108 cells following the manufacturer’s instructions. An additional step of bead beating

was also performed where resuspended cell suspensions were bead beat with 1:3 sterile glass beads (0.1 mm) for 45 s using the Mini-BeadBeater-24 (Biospec Products, Bartlesville, OK, United

States) and centrifuged at 10,000 RCF for 2 min. Following a DNAse treatment with the TURBO DNAse kit (Invitrogen, Carlsbad, CA, United States), the RNA concentration was assessed with the

Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, United States). RNA integrity was analyzed with the RNA Nano 6000 Assay Kit on the 2100 Bioanalyzer system (Agilent

Technologies, Santa Clara, CA, United States). mRNA libraries were prepared following the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, United States) with an input

material of 1.2 µg of total RNA. Libraries band sizes were confirmed on a 4% agarose gel. We prepared small RNA (sRNA) (>200 nt) libraries for sequencing since we noted higher prevalence

of sRNAs in the 0 °C cultures compared to the 23 °C cultures (Supplementary Fig. S3). sRNA libraries were prepared following the NEBNext Multiplex Small RNALibrary Prep Workflow (New England

Biolabs, Ipswich, MA, United States). The cDNA constructs were purified with the Monarch PCR and DNA kit from New England Biolabs. Libraries band sizes were assessed with the Agilent High

Sensitivity DNA kit on the 2100 Bioanalyzer system. Library bands from 105 to 210 bp were selected using the Pippin Prep 3% Agarose Gel Cassette from Sage Science (Beverly, MA, United

States). mRNA sequencing was performed by RNASeq on the MiSeq system (Illumina) using the Reagent Kit v2 50 cycles and a 1 × 55 bp single-end configuration. sRNA sequencing was performed by

RNASeq on the MiSeq system (Illumina) using the Reagent Kit v2 300 cycles and a 2 × 151 bp paired-end configuration. mRNA and sRNA raw sequence files (fastq) were uploaded to GenBank with

accession id: PRJNA631292 BIOINFORMATICS The mRNA raw sequencing reads were quasi-mapped and quantified with Salmon software [35] to the genome of _Rhodotorula_ JG1b with the GenBank number

GCA_001541205.1, and JGI IMG Genome ID 276120717 [24]. The analysis of the resulting quantifications was performed in R using the package DESeq2 [36]. Low count genes with less than 10 reads

were removed before normalization of the samples. Hierarchical clustering and principal component analyses were used to check if the samples within the treatment (biological replicates)

were more similar than between the treatments. The _p_ value threshold for differential expression was 0.05. Log2 fold-change threshold was not used, but log2 fold-change shrinking was used

for data visualization and gene ranking. Genes with a log2 fold-change (log2FC) ratio ≥∣1.5∣, and a _p_ value <0.05 were considered as differentially expressed between the two growth

conditions. Predicted protein KEGG assignments for each differentially expressed gene were downloaded from BlastKOALA website [37], to obtain function and metabolic pathway assignments.

Heatmaps of the relative abundance of transcript expression of specific pathways were created. For those heatmaps, relative gene abundances of each sample were calculated by taking gene

abundance (corrected on sequencing depth and length) and subtracting the average abundance across all samples. sRNA raw sequence data were trimmed for adapter sequences and quantified using

the CLC Genomics Workbench version 12 (https://www.qiagenbioinformatics.com/). The short rRNA fragments (rRFs)/microRNAs (miRNAs) sequences (15–30 nt) were extracted and counted from the

total sRNA sequences using the CLC Genomics Workbench Small RNA Analysis tool. Identical short rRFs/miRNAs were grouped together and low count sequences with less than 50 reads were removed.

The short rRFs/miRNAs were normalized based on sequencing coverage and to the ratio of sRNA/Total RNA, and differential expression was analyzed, with a _p_ value threshold of 0.05. PCoA

analysis (Bray–Curtis distance) was performed with the CLC Genomics Workbench version 12. The genomic origin of the ten highest expressed short rRFs/miRNAs were predicted with BLAST [38] by

comparing to _Rhodotorula_ genomes, and their potential mRNA targets were predicted using the RNAhybrid algorithm with the short rRFs/miRNAs sequences and all possible transcribed mRNA

sequences as potential targets [39, 40]. The predicted protein mRNA targets of the short rRFs/miRNAs were identified with KEGG [37]. ETHANOL PRODUCTION ASSAY To corroborate the phenotypic

and transcriptomic results, _Rhodotorula frigidialcoholis’_ ethanol production capability was assessed. Triplicate _Rhodotorula_ JG1b cultures were grown in identical media and conditions as

with transcriptomic analysis, detailed in section “Culturing and growth conditions.” OD600 of the cultures was measured at regular time points for 83 days at 0 °C and for 17 h at 23 °C.

Aliquots (1 ml) of the cultures at each time point were analyzed for ethanol production using the EnzyChrom Ethanol Assay Kit (BioAssay System, Hayward, CA, United States). Analysis of the

ethanol production was done by comparing the trendline of ethanol concentration to the trendline of cell density over time. Ethanol concentrations were normalized to the ethanol evaporation

rate that was experimentally determined using sterile PDB broth inoculated with 0.05% of ethanol. RESULTS AND DISCUSSION TAXONOMY AND DESCRIPTION OF _RHODOTORULA FRIGIDIALCOHOLIS_ SP. NOV

The _Rhodotorula_ yeast strain JG1b isolated from the upper-elevation McMurdo Dry Valleys of Antarctica [24] was determined to be a novel species based on phylogenetic and phenotypic

analyses. Bayesian analysis of aligned, concatenated 18S, ITS, D1/D2 domains of 28S rRNA, and TEF sequences of species within the genus _Rhodotorula_ resulted in a consensus tree shown in

Supplementary Fig. S1A, while the phylogenetic tree based on available complete _Rhodotorula_ genomes is shown in Supplementary Fig. S1B. According to these analyses, the JG1b strain forms a

strongly supported monophyletic group within _Rhodotorula_ species and is highly supported as the sister clade of _R. mucilaginosa_ (bootstrap = 100%). Clustering analysis of the data

obtained from 190 carbon and 95 nitrogen assimilation assays confirmed that _Rhodotorula_ JG1b is distinct from other _Rhodotorula_ species included in the Biolog phenotyping experiment

(Supplementary Fig. S1C). Contrary to _R. mucilaginosa_ and _R. alborubescens_, _Rhodotorula_ JG1b is able to assimilate L-rhamnose, dulcitol, L-sorbose, and is unable to assimilate citrate

and D-cellobiose (Supplementary Table S1). Based on the results presented here, we strongly suggest that _Rhodotorula_ sp. JG1b is a novel species in the _Rhodotorula_ genus. Accordingly,

the species is taxonomically introduced herewith as _Rhodotorula frigidialcoholis_ D. Touchette & P. Zalar, sp. nov. (frigidus in Latin meaning cold, alcohilis in Latin genitive case of

alcohol; of cold alcohol, referring to ability of alcohol production in cold). Morphological characters of _R. frigidialcoholis_ are shown in Fig. 1. Holotype for _Rhodotorula

frigidialcoholis_designated herewith: EXF-10854H, permanently preserved and metabolically inactive deposit of the Culture Collection of Extremophilic Fungi Ex (EXF), University of Ljubljana,

Slovenia. Ex-type cultures: EXF-10854 and CBS 16468 (Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands). The strain EXF-10854 was isolated as JG1b from ~150,000-year-old

ice-cemented permafrost of University Valley, the upper-elevation McMurdo Dry Valleys of Antarctica in 2009 by Jacqueline Goordial  (24). DNA sequence accession numbers derived from type:

MT569975 (18S rRNA), MT569976 (D1/D2 of 28S rRNA), MT560678 (ITS), MT584855 (TEF1). MycoBank accession number for _Rhodotorula frigidialcoholis _is MB 835866. Diagnosis: After a 14-day

incubation at 15 °C on 5% MEA agar, the streaked cultures are slimy, liquid and shinning with entire margin, greyish red (7B6) (Fig. 1). The cells are subglobose, 3.5 × 4.5 µm, and

encapsulated, budding is unipolar (Fig. 1). Fermentation was not observed. Assimilated compounds are D-glucose, sucrose, raffinose, galactose, trehalose, maltose, melezitose, L-sorbose,

L-rhamnose, D-xylose, Dl-arabinose, D-ribose, glycerol, galactitol, D-mannitol, and xylitol. Not observed assimilated compounds are inulin, melibiose, lactose, methyl A-D-glucoside,

cellobiose, erythritol, myo-inositol, citrate, D-glucosamine, N-acetyl-D-glucosamine, nitrate, and nitrite. Hydrolysis of urea is positive. A complete list of assimilated and not assimilated

compounds is listed in the Supplementary Table S1. Growth from 0 to 37°C (Supplementary Fig. S4).  Strain JG1b was previously described as able to grow as low as −10 °C [1, 24], but appears

to have lost subzero growth capabilities through sub-culturing and long-term storage at −80 °C, as in this study _R. frigidialcoholis_ was only successfully grown at 0 °C while incubated in

a PDB 50 ml liquid culture, without shaking. An extended description of _Rhodotorula frigidialcoholis _ is detailed in the Supplementary material (Supplementary file 1) a complete list of

assimilated and not assimilated compounds in the Table S1. PHENOTYPIC MICROARRAY RESPONSE AT COLD TEMPERATURES To characterize _R. frigidialcoholis_ metabolic capabilities at low

temperature, its substrate utilization was assessed through the Biolog Phenotypic MicroArray that determines the ability of a microorganism to utilize a variety of sole carbon or nitrogen

sources [41]. Positive detection of metabolic activity relies on the transfer of electrons from the growth substrate, through the electron transport chain (ETC), to the tetrazolium redox

dye, transforming the dye into a violet colored reduced form [41, 42]. After 91 days of incubation, _R. frigidialcoholis_ utilized 35 on 190 substrates as a sole carbon source at 0 °C for

growth (Supplementary Table S2). Only two (L-Proline, L-Pyroglutamate) of the 95 nitrogen sources were utilized as sole nitrogen sources at 0 °C after 91 days of incubation (Supplementary

Table S2). Surprisingly, of the 35 positive carbon substrates, only six (d-Trehalose, d,l-α-Glycerol Phosphate, d-Mannose, Tween 20, Tween 40, and Tween 80) showed reduction of the dye and

parallel evidence of microbial growth (Supplementary Table S2). This was puzzling because in the remaining 29 carbon sources, we observed active growth in the wells (based on OD600 and

visual inspection) but without reduction of the dye; this growth was confirmed by streak plates. As the tetrazolium dye is reduced via the ETC [43], the observation of growth but no

reduction of the dye suggest that at 0 °C _R. frigidialcoholis_ potentially produces most of the energy for cellular growth through metabolic pathways other than ETC, such as glycolysis or

perhaps fermentation. Damage to the mitochondrial DNA and the ETC in _S. cerevisiae_ was previously shown to decrease tetrazolium dye reduction [44] while yeast mitochondria mutants have

respiratory dysfunction and an inability to reduce tetrazolium salts [45,46,47]. Therefore, growth of _R. frigidialcoholis_ coupled with a lack of tetrazolium dye reduction suggests that

cell respiration through the mitochondria and ETC was significantly reduced while growth was still maintained at 0 °C. MRNA TRANSCRIPTIONAL RESPONSES TO COLD TEMPERATURE To determine _R.

frigidialcoholis_ transcriptional response to cold, we performed comparative mRNA transcriptomic analysis on triplicate exponential phase cultures of _R. frigidialcoholis_ grown at 0 and 23 

°C. Overall, 1772 genes were significantly differentially expressed (_p_ < 0.05) between these two conditions, including 994 genes overexpressed, and 778 genes downregulated at 0 °C

compared to 23 °C (Supplementary Tables S3 and S4). Of these significantly differentially expressed genes, 52% were annotated with KEGG. The major global differences in transcript abundance

between 0 and 23 °C were found in genes related to carbohydrate metabolism, energy metabolism, lipid metabolism and signal transduction, glycan biosynthesis/metabolism, transcription

machinery, translation, and amino acid metabolism (Fig. 2). While protein levels are not always correlated with the transcript levels as they are also influenced by post-transcriptional,

translational, and degradation regulation, mRNA levels do partially correlate with protein abundance [48] and are informative to deduce overall metabolic processes [49]. CELLULAR MEMBRANE

AND SIGNAL TRANSDUCTION Maintenance in membrane fluidity through lipid membrane component modification is one of the best-known adaption strategies in microorganisms living in cold

environments [23]. Overall, _R. frigidialcoholis_ overexpressed genes involved in lipid metabolism at 0 °C compared to 23 °C (Figs 2 and 3 and Supplementary Table S5). Similar to previous

studies, genes related to unsaturated fatty-acids (FA) biosynthesis were overall overexpressed at 0 °C indicating increased unsaturated FA concentrations maintain membrane fluidity at cold

temperatures [50,51,52,53]. Relatively high amounts of unsaturated FAs were also reported in other psychrophilic basidiomycetous yeasts isolated from Antarctic and Patagonian ecosystems

[54,55,56]. Sphingolipid metabolism genes were also highly overexpressed at 0 °C compared to 23 °C in _R. frigidialcoholis_ (Supplementary Fig. S4) presumably leading to increased

biosynthesis of ceramide and phytoceramide. Phytoceramide synthesis genes were previously shown to be consistently up-regulated during low temperatures fermentation (12 °C) in _Saccharomyces

cerevisiae_ [57, 58]. Ceramide and other sphingolipid products can induce cell cycle arrest [59] indicating growth suppression in _R. frigidialcoholis_ may be a response to freezing

conditions in Antarctic permafrost habitats. Multiple _Rhodotorula_ species produce lipid antioxidants and photoprotective carotenoids [60,61,62], especially high concentrations of

β-carotene, torulene, and torularhodin. Carotenoids help maintain membrane rigidity in cold-adapted microorganisms as they balance out the higher percentage of unsaturated FAs [50, 63]. At 0

 °C, _R. frigidialcoholis_ overexpressed the phytoene desaturase gene (FC = 4.46, _p_ = 0.00) involved in the production of torulene and β-carotene precursors. This is corroborated by our

observation of a darker pink color in _R. frigidialcoholis_ grown at 0 °C compared to 23 °C. Carotenoids play an important role in UV and sunlight protection in yeast [61, 64, 65]. Thus, at

0 °C, _R. frigidialcoholis_ may increase carotenoid biosynthesis both to regulate membrane fluidity and as an adaption to solar irradiation or long-term background γ radiation in the extreme

University Valley permafrost environment. The PI3k-Akt, Hippo, TGFß, VEGF, RAS, MAPK, RAP1, FoxO, and Wnt cell signal transduction pathways genes were overexpressed at 0 °C (Fig. 2 and

Supplementary Table S4); these pathways are related to gene regulation, actin cytoskeleton formation, and cellular adhesion, and are activated by transmembrane receptors. While little is

known about cold response signal transduction modulation, it has been linked to freezing temperature survival in plants [66, 67]. Perception of cold in _Rhodotorula_ by cold-sensitive

membrane receptors may also lead to a cascade response, thus preparing it for cold temperatures. In yeasts, mitogen activated protein kinase cascades play major roles in gene transcription

and actin cytoskeletal organization [68]. Thus, _R. frigidialcoholis_ may overexpress genes coding for the formation of the actin cytoskeleton (cdc42, actin δ/γ subunits) at 0 °C

(Supplementary Table S4) as a way to transport lipids (biosynthesis of which is also overexpressed at 0 °C) to the membrane and maintain membrane fluidity (Figs 2 and 3 and Supplementary

Fig. S4). DIFFERENTIAL EXPRESSION OF HOMOLOGOUS PROTEINS BETWEEN 0 AND 23 °C We observed 19 differentially expressed homologous protein categories between _R. frigidialcoholis_ cultures at 0

and 23 °C (Supplementary Table S6). Eight of these differentially expressed homologous protein categories were related to membrane structure and transport across membranes. For example,

YAT, an amino acid transporter, had three homolog genes overexpressed at 0 °C, two homologs overexpressed at 23 °C, and two homologs that were not differentially expressed between the two

temperatures (Supplementary Table S6). Homologous proteins arise from gene duplication and sometimes gain new functions [69]. Gene duplication, genome redundancy, and paralogous (homologs)

genes increase the organism’s ability to grow under varied conditions (e.g., temperatures, substrates, pH, salinity) [2, 70, 71]. GLYCAN BIOSYNTHESIS AND METABOLISM A significant increase in

glycan biosynthesis genes was observed in _R. frigidialcoholis_ at 0 °C compared to 23 °C (Figs 2 and 3). Glycans are carbohydrate-based polymers associated with cellular protection and

storage [72] and are also important component of glycoproteins, including cell-surface membrane proteins, such as receptors and adhesion proteins [72, 73]. _Rhodotorula_ species produce

mannan, a mannose glycan extracellular polysaccharide (EPS) [74, 75]. Microbial EPS are components of microbial biofilms and constitute a protective matrix against the desiccation and

environmental fluctuations [76, 77], including freeze-thaw damage [78]. At 0 °C, _R. frigidialcoholis_ overexpressed genes involved in GDP-D-mannose synthesis (a glycan biosynthesis

precursor), and overexpressed multiple genes coding for glycosyltransferase and glycosidases (Supplementary Table S4), which regulate glycans [79]. These results indicate that _R.

frigidialcoholis_ increases EPS synthesis at 0 °C through overexpression of mannan and other glycoproteins as adaptation to desiccation and freeze-thaw cycles of the Antarctic University

Valley permafrost environment. TRANSCRIPTION, TRANSLATION, AND AMINO ACID METABOLISM _R. frigidialcoholis_ overexpressed multiple genes involved in the transcriptional machinery at 0 °C

compared to 23 °C (Figs 2 and 3), including the overexpression of genes coding for the three RNA polymerase complexes (RNA Polymerase I, II, and III and the RNA polymerase II transcription

factor) (Supplementary Table S4). A similar increase in the transcriptional machinery at 10 and 4 °C was reported in other yeasts [80,81,82,83]. Contrariwise, _R. frigidialcoholis_ decreased

expression of genes related to translation at 0 °C compared to 23 °C, such as the downregulation of genes involved in ribosomal protein synthesis and aminoacyl-tRNAs. In addition, _R.

frigidialcoholis_ overexpressed proteasome genes involved in protein degradation at 0 °C. Cold temperatures can reduce translation efficiency and protein synthesis by inducing formation of

secondary structures in DNA/RNA molecules and by inactivating ribosomes [4, 83, 84]. While amino acid biosynthesis genes were overall downregulated at 0 °C compared to 23 °C (Fig. 2), the

bacterium _Polaromonas_ sp. also downregulated genes involved in amino acid biosynthesis and transport [8]. Thus, at low temperatures, _R. frigidialcoholis_ may decrease amino acid synthesis

and transport to slow down translation and focus energy on cold acclimation, in parallel with increased protein degradation to possibly recycle amino acids. A notable exception was of

upregulation of the histidine pathway; this could be due to its role in membrane cold sensor histidine kinases [85] as was reported in the basidiomycetous yeast _Mrakia blollopis_, which

accumulated aromatic amino acids, such as histidine, at −3 °C compared to 10 °C [86]. CARBOHYDRATE METABOLISM AND ENERGY METABOLISM The majority of carbohydrate metabolism expression

differences between 0 and 23 °C were in genes of the citrate cycle, pentose phosphate pathway (PPP), and alcohol fermentation. At 23 °C, _R. frigidialcoholis_ significantly overexpressed

citrate cycle genes as well as glycolysis genes linked to oxaloacetate and acetyl-CoA syntheses which feed into the citrate cycle (Fig. 4 and Supplementary Table S5). In contrast, at 0 °C,

there was a significant increase in the expression of glycolysis genes feeding into ethanol production via fermentation and a significant increase in expression of the PPP genes (Fig. 4). At

0 °C, _R. frigidialcoholis_ overexpressed genes coding for xylulose reductase (FC = 2.40, _p_ = 0.00), non-oxidative PPP enzymes, and alcohol dehydrogenase (FC = 8.05, _p_ = 0.00), while it

downregulated gene coding for 6-phosphogluconate dehydrogenase (FC = 2.21, _p_ = 0.00) (Fig. 4). The increase in PPP promotes the production of erythrose-4-phospate, allowing energy (NADH)

conservation due to a reduction of the citrate cycle activity [87]. In yeast, including _Rhodotorula_, the PPP is involved in xylulose fermentation [88,89,90], while downregulation of

6-phosphogluconate dehydrogenase activity is correlated with increased production of ethanol via xylulose fermentation [91]. Xylulose is fermented by xylulose reductase [88] to xylitol, a

cryoprotectant and freezing point depressant in yeast [92]. The non-oxidative PPP enzymes can also increase the yield of ethanol fermentation through the conversion of pentose phosphate into

intermediates of the glycolysis pathway [90]. A similar upregulation of alcohol dehydrogenase was also observed in the Arctic permafrost bacterium, _Planococcus halocryophilus_, grown at

−15 °C [2]. These transcriptional changes in _R. frigidialcoholis_ suggest that it adapts to cold temperature by increasing xylitol production and redirecting PPP molecules to ethanol

fermentation, which would be beneficial for its survival in extreme cryoenvironments such as Antarctic University Valley. Antioxidant production through proline-linked PPP was proposed in

yeast [93] as an oxidative stress response to cold [87]. _R. frigoalchoholis_ can use L-Proline as a sole carbon and nitrogen source (Supplementary Table S2), without reducing the

tetrazolium redox dye, suggesting that L-Proline is used by _Rhodotorula_ in the proline-linked PPP, leading to decreased citrate cycle activity. This may result in a decrease of the ETC

activity and, consequently, the observed lack of tetrazolium dye reduction and a possible redirection of erythrose-4-phosphate to the fermentation. Overall, increased transcription of the

PPP genes at cold temperatures could help _R. frigidialcoholis_ to conserve NADH conservation, limit carbon loss through CO2, and enhance ethanol production to lower the freezing point.

Overall, a higher number of genes related to energy metabolism were downregulated at 0 °C compared to 23 °C (Fig. 2). In accordance with our carbohydrate metabolism results, this significant

change was related to an overall downregulation of the genes encoding for the ETC subunits at 0 °C (Fig. 5). The temperature effect on the ETC activity remains unclear, but some studies

confirm a general trend in decrease of ETC expression at lower temperatures in microorganisms [8, 94]. The University Valley permafrost bacterium, _Rhodococcus_ JG3, also downregulated

multiple ETC cytochromes when grown at −5 °C compared to 23 °C [8], and _Pseudomonas putida_ downregulated its NADH and ETC succinate dehydrogenase at 10 °C compared to 30 °C [94]. The ETC

gene expression patterns in _R. frigidialcoholis_ supports our hypothesis related to the lack of dye reduction in the PM assay at 0 °C as being due to the downregulation of the ETC and a

switch to fermentative metabolisms to produce energy. This is further supported by the observed switch in gene expression from the citrate cycle pathway at 23 °C to the fermentation ethanol

and xylitol pathways at 0 °C for primary energy production (Fig. 4). ETHANOL PRODUCTION IN _RHODOTORULA FRIGIDIALCOHOLIS_ _R. frigidialcoholis’_ switch in metabolism from mainly respiratory

at 23 °C to fermentative at 0 °C was experimentally confirmed as ethanol production was observed at 0 °C, at an average rate of 1.51 × 10−5 moles of ethanol, per liter, per day (Fig. 6A),

while ethanol production was not detected at 23 °C (Fig. 6B). Other _Rhodotorula_ species, _R. minuta, R. mucilaginosa_, and _R. pallida_, are capable of ethanol fermentation at 28 °C [95];

however, ethanol fermentation at low temperatures in _Rhodotorula_ has not been reported. Yeast ethanol production at cold temperatures was reported with _Saccharomyces cerevisiae_ in wine

(0 and 2 °C) and beer (6 °C) production, although in these studies, fermentation was facilitated by an addition of biocatalysts [96,97,98]. To our knowledge, _R. frigidialcoholis_ is able to

naturally produce ethanol at the lowest recorded temperature for microbial fermentation. _R. frigidialcoholis_ could then be studied on its potential to isolate cold-temperature active

enzymes that promote cellulosic biomass decomposition through fermentation [99], favorizing biofuel production with less energy. Ethanol fermentation has also previously been linked with an

increased production of carotenoids in yeast [100], which is consistent with _R. frigidialcoholis_ transcriptomic results of increased carotenoid gene expression. Combined together,

downregulation of citrate cycle and ETC genes (Figs 4 and 5), overexpression of fermentation genes (Fig. 4), growth without reduction of the tetrazolium redox dye (Supplementary Table S2),

and ethanol production at 0 °C but not at 23 °C suggest that _R. frigidialcoholis_ switches from a mainly respiratory metabolism when incubated at 23 °C to a mainly fermentative metabolism

when cultured in conditions similar to its natural environment at 0 °C. Ethanol production in _R. frigidialcoholis_ may be an ecologically significant adaptive response to subzero

temperatures by both acting as a freezing point depressant reducing intracellular freezing and/or to decrease its environment freezing point enhancing the formation of subzero liquid brine

vein microhabitats thought to exist within permafrost [101]. Ethanol production could also promote NADH conservation and limiting carbon loss by switching from the citrate cycle to ethanol

fermentation. This consequently may increase its survivability in the extremely cold Antarctic permafrost habitat from which it was isolated (Fig. 3). SMALL NON-CODING RNA EXPRESSION CHANGES

UNDER COLD TEMPERATURE Small non-coding RNA (<200 nucleotides; sRNA) have numerous functions, including protein synthesis (small rRNAs and tRNAs) and mRNA regulation through RNA

interference (RNAi) [102]. The roles of sRNA and RNAi are not well understood, but they have been linked to stress response [103, 104]. Based on quantification of the total RNA extracted

from _R. frigidialcoholis_ cultures, we initially observed a significantly higher (_p_ = 0.0003) proportion of sRNAs (<200 nt) in the 0 °C compared to 23 °C cultures (Supplementary Fig. 

S3). To determine how the sRNA were affected by low temperature in _R_. _frigoalcoholis_, we further analyzed the composition of sRNAs at 0 and 23 °C. On average, 53.40 ± 0.99% of the total

RNA was characterized as sRNAs in 0 °C stressed cultures compared to 27.70 ± 1.84% in 23 °C cultures. There are multiple types of sRNA molecules including miRNAs that are non-coding ~22 nt

sequences that play a role in gene regulation of eukaryote cells, including fungi [105, 106], and rRFs that are derived from rRNA [107]. We subsequently observed a higher proportion (_p_ = 

0.029) of short rRFs and miRNA (15–30 nt) in _R. frigoalcoholis_ at 0 °C compared to 23 °C (Supplementary Fig. S3). Specifically, 0.69 ± 0.34% of the total RNA was characterized as short

rRF/miRNAs in 0 °C stressed cultures compared to 0.02 ± 0.01% at 23 °C, resulting in a ~34-fold increase at 0 °C. In addition, we observed a higher diversity in short rRF/miRNAs at 0 °C

compared to 23 °C, based on PCoA analysis (Fig. 7 and Supplementary Fig. S3). Stability and non-random sequence distribution of rRFs in previous studies implies that they are not merely

products of rRNA turnover, but a functional part of cellular homeostasis, although they have historically been eliminated from sequencing datasets as contaminants [107]. rRFs can be produced

from stressed induced cleavage of tRNAs and rRNAs [107, 108] and globally downregulate translation by binding to ribosomes [109, 110] or by binding to mRNA, both triggering RNAi [107, 111,

112]. Thus, rRF/miRNAs production may be a stress response resulting in overall slower growth rate and metabolism. Strong links between cold adaptation in prokaryotes and fungi through miRNA

regulation have not yet been reported, although this mechanism was identified as a cold adaptation in plants [103]. rRFs have also been linked to cold and UV stress [107], and inhibition of

protein translation following DNA damage [113]. Another subset of rRFs are antisense ribosomal small interfering RNAs (risiRNAs) that downregulate pre-rRNA levels through the nuclear RNAi

pathway under cold and UV stress to maintain rRNA homeostasis [114, 115]. This phenomenon is triggered by production of erroneous rRNAs transcripts due to environmental stress [116]. Thus,

this RNAi pathway potentially decreases global translation by targeting rRNA thus slowing down the cell cycle and limiting production of erroneous proteins. We could not find any studies

clearly reporting the percent of rRFs/miRNAs of total RNA in microorganisms to compare with our results. However, we postulate that the 34-fold increase of rRF/miRNAs at 0 °C in _R.

frigidialcoholis_ may be a cold temperature response and result from a combination of three possible RNAi mechanisms (Fig. 3): (1) the rRFs were derived from rDNA triggered by DNA damage

[113, 115] due to cold stress, resulting in global suppression of mRNA translation, and consequently allowing _R. frigidialcoholis_ to slow down its cell cycle under cold temperature; (2)

the rRFs observed could act via the nuclear RNAi pathway to downregulate pre-rRNA levels thus suppressing global translation via a negative feedback loop that targets immature rRNA. This

pathway may be triggered by an increase in errors during rRNA transcription due to environmental stresses such as low temperature [114]. At 0 °C, we observed rRFs with sequence variability

suggesting that erroneous rRNA were potentially produced at a higher rate in _R. frigidialcoholis_ at 0 °C than 23 °C. (3) _R. frigidialcoholis_ may contain miRNA derived from mRNA and short

rRFs derived from rRNA. Both of these could target and suppress a variety of targeted mRNAs in response to cold, as previously observed in plants [103]. Using a RNAhybrid computational

method [40], the top putative mRNA targets for the ten most expressed short rRFs/miRNAs (Table 1) were predicted, based on the lowest energy of hybridization required to form a duplex [106].

Of the ten most abundant short rRFs/miRNAs, nine appear to originate from rRNA and one from a hypothetical protein (Table 1). These short rRFs/miRNAs were mainly overexpressed at 0 °C in

_R. frigidialcoholis_ and potentially target a wide variety of mRNAs as summarized in Table 1. However, we could not directly correlate the increase in expression of the short rRFs/miRNAs

with a decrease in expression of their putative mRNA targets, nor do the targets appear to be necessarily linked to cold adaptation. Absence of correlation between specific mRNA target

expression levels and short rRFs/miRNAs levels could potentially be due to (a) multiple targets per short rRFs/miRNAs, (b) short rRFs/miRNAs targeting rRNAs (instead of mRNAs) that results

in an overall global reduction of translation, or (c) RNAi only partially contributing to gene expression regulation. Through the action of two RNAse III-type proteins, Drosha and Dicer,

miRNAs are produced and recognize specific “target” mRNA that are then silenced via RNAi. Drosha is responsible for the primary transcript cleavage to create precursor miRNA, while Dicer,

along with Argonaute (Ago), cleaves the precursor miRNA to form a double strand miRNA. One of these miRNA strands is incorporated into the RNA-induced silencing complex that suppresses

translation [106]. Short rRFs have been associated with Ago proteins suggesting that rRFs may act like ribosomal derived miRNA as well [112]. Based on the transcriptomic results, the gene

coding for Drosha was not differentially expressed; however, Dicer (FC = 3.83, _p_ = 0.01) and RNA polymerase III subunits were significantly overexpressed at 0 °C (Supplementary Table S4).

This was consistent with a significantly higher proportion of short rRFs/miRNAs at 0 °C compared to 23 °C and a decrease in the transcript levels of the translational machinery (ribosome

proteins and tRNAs) at 0 °C (Fig. 2). Taken together, these results suggest that _R. frigidialcoholis_ may induce a short rRFs/miRNAs gene regulatory mechanism in response to cold that

triggers translational repression; however, molecular characterizations of the short rRFs/miRNAs, such as stem-loop RT-qPCR and northern blot analysis, are necessary to confirm the

involvement of RNAi in cold adaptation. CONCLUSIONS Transcriptomic analyses results suggest that _Rhodotorula frigidialcoholis_ adapts to cold temperatures in the Antarctic dry valley

permafrost through a variety of mechanisms including increasing expression of the PPP genes, increasing the production of carotenoids, sphingolipids, unsaturated fatty acid, and

exopolysaccharides while coupled with a reduction in expression of growth, transcriptional and translational machinery genes. We also identified novel cold adaptation features including a

switch from respiratory metabolism at 23 °C to fermentative metabolism when grown in a cold condition (0 °C), via downregulation of citrate cycle genes and the ETC genes. In parallel, _R.

frigidialcoholis_ overexpressed genes involved in ethanol and xylitol fermentation, resulting in ethanol production at the lowest known temperature described so far in any microorganism.

This switch may thus be an adaptation to save energy and delay intracellular freezing in cryoenvironments, such as the Antarctic permafrost. At low temperature, _R. frigidialcoholis_ also

produced a significantly higher proportion of sRNAs, specifically short rRFs/miRNAs, and overexpressed the Dicer gene, suggesting RNAi as a novel mechanism of cold adaptation in polar fungi.

Further characterization of the role of ethanol production and identification of mRNA targets of RNAi are needed to determine the roles they play in cold adaptation of yeasts inhabiting the

cryosphere. Taken together, our results indicate that _R. frigidialcoholis_ has evolved multiple mechanisms to survive in one of the coldest and driest cold environments on Earth.

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Download references ACKNOWLEDGEMENTS The authors acknowledge the valuable input from Dr Jennifer Ronholm and Dr Jacqueline Goordial for their critical feedback and for the setting up of the

project, and Dr Barry R. Bochner, from Biolog Inc. for his valuable advice with the Biolog Phenotypic MicroArray tests analysis. The authors acknowledge the support of the McGill Space

Institute. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Natural Resource Sciences, McGill University, Sainte-Anne-de-Bellevue, QC, Canada D. Touchette, I. Altshuler, I.

Raymond-Bouchard & L. G. Whyte * Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia C. Gostinčar, P. Zalar & N. Gunde-Cimerman * Lars Bolund

Institute of Regenerative Medicine, BGI-Qingdao, Qingdao, China C. Gostinčar * Agricultural Institute of Slovenia, Ljubljana, Slovenia J. Zajc * NASA Ames Research Center, Moffett Field, CA,

USA C. P. McKay Authors * D. Touchette View author publications You can also search for this author inPubMed Google Scholar * I. Altshuler View author publications You can also search for

this author inPubMed Google Scholar * C. Gostinčar View author publications You can also search for this author inPubMed Google Scholar * P. Zalar View author publications You can also

search for this author inPubMed Google Scholar * I. Raymond-Bouchard View author publications You can also search for this author inPubMed Google Scholar * J. Zajc View author publications

You can also search for this author inPubMed Google Scholar * C. P. McKay View author publications You can also search for this author inPubMed Google Scholar * N. Gunde-Cimerman View author

publications You can also search for this author inPubMed Google Scholar * L. G. Whyte View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS DT

wrote the manuscript, performed all laboratory experiments and data analysis. IA helped with the experimental design, laboratory experiments, data analysis, figure creation, writing and

manuscript editing. CG performed the bioinformatic pipeline analysis of the raw RNASeq data (mRNA). PZ and JZ described the novelty of the species. IR-B helped with experimental design and

manuscript editing. LGW and NG-C provided guidance, advise, and helped with the manuscript editing. CORRESPONDING AUTHOR Correspondence to L. G. Whyte. ETHICS DECLARATIONS COMPETING

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Gostinčar, C. _et al._ Novel Antarctic yeast adapts to cold by switching energy metabolism and increasing small RNA synthesis. _ISME J_ 16, 221–232 (2022).

https://doi.org/10.1038/s41396-021-01030-9 Download citation * Received: 23 June 2020 * Revised: 17 May 2021 * Accepted: 02 June 2021 * Published: 22 July 2021 * Issue Date: January 2022 *

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