FEMS Yeast Research, 15, 2015, fov008
doi: 10.1093/femsyr/fov008
Advance Access Publication Date: 27 February 2015
Research Article
RESEARCH ARTICLE
Uptake of inorganic phosphate is a limiting factor
for Saccharomyces cerevisiae during growth
at low temperatures
Isabel Vicent1,# , Alfonso Navarro1,†,# , Jose M. Mulet2,∗,# , Sukesh Sharma3
and Ramón Serrano2
1
Instituto de Universitario de Ingenierı́a de Alimentos para el Desarrollo. Universidad Politécnica de Valencia,
46022 Valencia, Spain, 2 Instituto de Biologı́a Molecular y Celular de Plantas. Universidad Politecnica de
Valencia- Consejo Superior de Investigaciones Cientı́ficas, 46022 Valencia, Spain and 3 Department of
Biochemistry, Panjab University, 160014 Chandigarh, India
∗ Corresponding author. IBMCP, Universitat Politècnica de València, Camino de Vera S/N, 46022 Valencia, Spain. Tel: +34-96-3877775;
Fax: +34-96-3877859; E-mail: [email protected]
Present address: Sección Departamental de Microbiologı́a, Facultad de Medicina y Odontologı́a, Universidad de Valencia. Avda Blasco Ibáñez 15, 46010
Valencia, Spain.
#
I.V., A.N., and J.M.M contributed equally to this work.
One sentence summary: Phosphate uptake is a limiting factor for yeast growth at low temperatures.
Editor: Isak Pretorius
†
ABSTRACT
The fermenting ability of Saccharomyces at low temperatures is crucial for the development of alcoholic beverages, but the
key factors for the cold tolerance of yeast are not well known. In this report, we present the results of a screening for genes
able to confer cold tolerance by overexpression in a laboratory yeast strain auxotrophic for tryptophan. We identified genes
of tryptophan permeases (TAT1 and TAT2), suggesting that the first limiting factor in the growth of tryptophan auxotrophic
yeast at low temperatures is tryptophan uptake. This fact is of little relevance to industrial strains which are prototrophic
for tryptophan. Then, we screened for genes able to confer growth at low temperatures in tryptophan-rich media and found
several genes related to phosphate uptake (PHO84, PHO87, PHO90 and GTR1). This suggests that without tryptophan
limitation, uptake of inorganic phosphate becomes the limiting factor. We have found that overexpression of the previously
uncharacterized ORF YCR015c/CTO1 increases the uptake of inorganic phosphate. Also, genes involved in ergosterol
biosynthesis (NSG2) cause improvement of growth at 10◦ C, dependent on tryptophan uptake, while the gluconeogenesis
gene PCK1 and the proline biosynthesis gene PRO2 cause an improvement in growth at 10◦ C, independent of tryptophan
and phosphate uptake.
Keywords: cold stress; phosphate transport; CTO1; tryptophan uptake
INTRODUCTION
Yeasts, and particularly those of the Saccharomyces genus, have
been associated with human industry since the origin of civilization. Saccharomyces strains were selected among other organ-
isms for the production of alcoholic beverages or food processing
due to its ability to efficiently convert sugars into ethanol, carbon dioxide and many secondary metabolites that lead to balanced flavor and aroma in the final product. Industrial use of
Saccharomyces cerevisiae forces yeast physiology to adapt to an
Received: 8 January 2015; Accepted: 23 February 2015
C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Yeast Research, 2015, Vol. 15, No. 3
extremely changing environment and to cope with osmotic and
oxidative stress, nutrient depletion, high hydrostatic pressure,
ethanol toxicity, shear stress during yeast handling and separation and competition with other yeasts or bacteria (Teixeira,
Mira and Sa-Correia 2011). In addition, during alcoholic beverage
production, temperature control is needed as raising temperatures increases yeast performance but diminishes final product
quality. Temperature also increases the level of glycerol production, pyruvate, alpha-ketoglutaric acid and other metabolites,
that in some cases correlate with unwanted organoleptic characteristics. Another problem is that high temperature can favor the growth of bacteria or other food spoilage yeasts (Smits
and Brul 2005). Most of these problems can be prevented by
fermentation at low temperatures. In addition, there are some
products for which fermentation at low temperature is crucial, for instance, proper lager beer fermentation must be carried out between 10 and 15◦ C. In winemaking, fermentation at
lower temperature correlates with fresh character and fruity
notes in wines and diminishes the risk of bacterial contamination and the production of volatile acids (Molina et al. 2007).
Lowering the temperature of fermentation improves product
quality, but also increases the time required to complete fermentation, and therefore, the economic cost and the energy requirements. There is a clear interest from the industry to develop yeast strains with enhanced capability to ferment under
low temperatures, especially for those selected for their flavor
characteristics, that are not necessarily able to grow well under
cold temperatures (Saerens, Duong and Nevoigt 2010).
Yeast physiological and molecular responses to high temperature or heat shock have been extensively studied (Verghese et al. 2012), but our knowledge of the genes whose function becomes limiting at low temperatures is very poor. Upon
a downshift in temperature, there is a transcriptional response
and an upregulation of cold-inducible proteins, but they are
neither conserved nor shared by such a wide range of organisms as the heat shock proteins (HSPs). It is known that cold
induces biochemical, biophysical and physiological changes to
cells. Cold strengthens the interactions between the two strands
of DNA and the secondary structure of mRNA, so transcription
and translation are impaired (Jones and Inouye 1996; Farewell
and Neidhardt 1998). There is also a decrease in the fluidity of
the lipid bilayer of membranes and an increase in their rigidity
which decreases transport through cell membrane (Los and Murata 2004) and increases passive permeability (Hoekstra, Golovina and Buitink 2001).The speed of protein folding decreases,
and conformational instability increases. Cold also can induce
protein denaturation (Fersht 1999) and there is a general decrease of the enzymatic activity (Gerday et al. 1997).
Sensing of cold in yeast is still an open question. It is considered that cold sensing depends on the membrane osmosensor Sln1p, (Panadero et al. 2006) but not exclusively. There is also
a role of the Mox1 transcription factor which regulates the expression of the DAN/TIR genes (Abramova et al. 2001). Mga2 has
also been proposed to be a cold sensor for the activation of OLE1
(Nakagawa et al. 2002). Other well-characterized signal transduction pathways have also been related to cold sensing or signaling such as the cAMP-PKA pathway, the PKC pathway and the
TOR pathway (Aguilera, Randez-Gil and Prieto 2007; CórcolesSáez et al. 2012), although in most cases evidence is indirect.
On the other hand, the transcriptional response of yeast under low temperatures is well characterized and proceeds in different phases (Schade et al. 2004). In the early response, there
is an induction of genes related to transcription, RNA processing and RNA helicases (Winzeler et al. 1999). Also genes able to
alter membrane fluidity as the delta-9-desaturase OLE1 (Nakagawa et al. 2002; Martin, Oh and Jiang 2007) and those involved
in phospholipid biosynthesis such as INO1 and OPI3 appear to
be induced indicating that an increase in membrane biosynthesis is important for survival under cold temperatures (Murata
et al. 2006). Other genes related to lipid signaling such as ERG10
are upregulated, but its overexpression does not improve growth
under cold conditions (Loertscher et al. 2006).
In the late response, there is an induction of the genes involved in general stress response as a consequence of the reduced enzymatic activity, membrane transport and misfolded
proteins. A downshift of the temperature increases the expression of some genes codifying HSPs. Specifically: HSP12, HSP26,
HSP42 and HSP104 are induced at 10 (Sahara, Goda and Ohgiya
2002; Schade et al. 2004) and 4◦ C (Homma, Iwahashi and Komatsu 2003; Murata et al. 2006). HSP42 and HSP104 are also induced at 0◦ C (Kandror et al. 2004). There are also changes in
carbohydrate accumulation, such as trehalose (Kandror et al.
2004) and glycerol (Panadero et al. 2006). Trehalose accumulation has been related to its role as membrane and protein stabilizer (Singer and Lindquist 1998; Elbein et al. 2003), and glycerol
has a protective role against the dehydration that occurs in the
processes of freezing/thawing due to membrane breaking. In the
late response, there is also an increase in the expression of genes
encoding antioxidant enzymes, both related to the glutathione–
glutaredoxin system and with H2 O2 detoxification (Schade et al.
2004).
There is a clear interest in the use of biotechnology for the development of new yeast strains able to ferment at low temperatures. With this goal in mind, it would be very useful to know not
only the physiological changes but also the genes whose function becomes limiting under cold conditions. The yeast overexpression approach is a powerful technique to screen for genes
able to confer tolerance under stress conditions (Serrano et al.
2003) that has been used to identify genes conferring tolerance
to salt stress (Gaxiola et al. 1992; Mendizabal et al. 1998; Rios et al.
2013) or even adaptation to low temperatures (Hernandez-Lopez
et al. 2011). Here, we have used this technique to identify the
limiting factors for growth at 10◦ C of a yeast laboratory strain
auxotrophic for tryptophan and found that in normal medium
tryptophan uptake is limiting while in tryptophan-rich medium
the main limiting factor is inorganic phosphate uptake. We have
also found other genes unrelated to tryptophan or phosphate
uptake that can improve growth at 10◦ C upon overexpression.
MATERIALS AND METHODS
Yeast culture conditions
Standard methods for yeast use and manipulation were used.
YPD medium contained 2% glucose, 2% peptone and 1% yeast
extract. The content of tryptophan in YPD medium was 0.1
mg ml−1 and that of inorganic phosphate 2 mM (Difco Manual). SD (synthetic minimal medium) contained 2% glucose, 0.7%
yeast nitrogen base (Difco) without amino acids, 50 mM succinic acid adjusted to pH 5.5 with Tris, and the amino acids
and nitrogenated bases required by the strains. SG medium
was prepared as described in Guthrie and Fink (1991). The
tryptophan-rich YPD medium contained 0.2% w/v (2 mg ml−1 ) of
tryptophan added from a concentrated stock sterilized by filtration. The phosphate-rich YPD medium contained 10 mM sodium
phosphate, added from an autoclaved concentrated stock of
sodium phosphate buffer (pH 5.8). In both cases, the supplement
was added after autoclaving and prior to gelification. For the
Vicent et al.
tryptophan uptake measurements, we used the minimal 165
medium described in Ramos and Wiame (1979).
Yeast strains
Diploid strain W303 (ML-39 in our collection) was constructed in
our laboratory by Dr Martin Leube by mating of strains W303-1A
(MATa can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1) and W303-1B
(MATα can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1) from Wallis
et al. (1989). Diploid strain RS452 was constructed in our laboratory by transformation of strain BWG1–7A (MATa ade1-100 ura352 leu2-3,112 his4-519) (Guarente, Yocum and Gifford 1982) with
URA3 plasmid CY173 containing the HO gene (Russell et al. 1986).
This induces the formation of autodiploids and after isolation of
one we selected loss of the plasmid after growth in rich medium.
Diploids were identified by their capability to sporulate.
Strains W303 and RS452 are very similar laboratory strains
mostly differing in the auxotrophy for tryptophan in the first
case. We have also utilized a derivative of the W303 strain transformed with a plasmid containing the TRP1 gene to correct the
tryptophan auxotrophy. This plasmid was isolated in our screening (see Fig. 1A) and as indicated under results this strain exhibits similar cold tolerance as strain RS452.
3
Growth assays in solid and liquid media
Growth assays on solid media were performed by spotting serial dilutions of saturated cultures onto the indicated plates.
For the elaboration of the growth curves, 50 ml of the indicated
medium in a 100 ml Erlenmeyer flask was inoculated with cells
from a preculture to an initial optical density at 600 nm (OD600 )
of 0.01. These cultures were maintained with shaking (200 rpm)
at the indicated temperature. At the specified intervals, aliquots
were taken and optical density determined. Measurements were
done in a DU 730 spectrophotometer (Beckman Coulter, USA).
Results are the average of three independent experiments, and
error bars represent standard deviations. Experimental values
of the growth curves were adjusted to an exponential equation
(y = y0 2x ) to determine the lag phase and the generation time.
Amplification and screening of the genomic library
The yeast genomic library used in the screening was amplified from the library described in Rose and Broach (1990) using standard protocols. The average size of the insert is 10 kb
and is inserted in the yeast shuttle vector YEp24 (ampr , 2μm origin, URA3; New England Biolabs, USA). Diploid strain W303 was
transformed with the aforementioned library and selected on SD
Figure 1. Identification of the genes conferring growth at 10◦ C by overexpression. (A and B) Cultures of transformants of diploid strain W303 carrying the empty
episomal plasmid (YEp24) or the different plasmids containing either the original genomic fragments (YCT plasmids) or the genes identified to be responsible for the
phenotype (YEp plasmids with the genes indicated in each lane) were grown in selective (SD) medium until saturation at 28◦ C. Serial dilutions of each strain (1/10,
1/100 and 1/1000) were spotted onto YPD medium and incubated at 28 or 10◦ C. Growth was recorded after 3 days (28◦ C) or 8 days (10◦ C). (C) Growth curves in liquid
media of W303 transformed with the empty episomal plasmid (YEp24) (filled squares), YEp24TAT1 (filled diamonds), YEp24TAT2 (filled triangles) and YEp24TRP1 (filled
circles). (D) Growth curve in liquid media of W303 transformed with the empty episomal plasmid (YEp24) (filled squares), YEp24NSG2 (empty circles), YEp24PRO2
(empty diamonds) and YEp24PKC1 (empty triangles). In C and D cells were inoculated at an initial cell density of 104 colony-forming units (cfu)/ml from saturated
precultures and growth was at 10◦ C with continuous shaking. OD600 measurements were taken at the indicated time (in hours). Each curve represents the average
values of three independent experiments. Error bars represent standard deviation.
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FEMS Yeast Research, 2015, Vol. 15, No. 3
plates containing all the nutritional requirements except uracil
at 28◦ C. Batches of transformed cells were recovered, and plated
on YPD (0.002% w/v tryptophan content) incubated at 10◦ C or
on YPD plus tryptophan (0.2% w/v tryptophan content) incubated at 8◦ C. The total number of transformed colonies screened
was about 106 . In all cases, colonies exhibiting improved growth
were selected and plasmids were isolated. The process for selection and re-evaluation of tolerant colonies was performed
as described in Mulet et al. (2004) Isolated plasmids were used
for transforming W303 strains, and the ability to confer cold
tolerance was re-checked. Those unable to confer tolerance or
conferring very low tolerance were discarded at this step. Plasmids conferring enhanced low-temperature growth when compared to control strains were subjected to partial sequencing
to obtain nucleotide sequences at the ends of the inserts. The
number of times that the same plasmid was independently isolated was recorded. The total number of independent plasmids
screened was about 106 . The complete sequence of each clone
was inferred by comparison of these sequences with the Saccharomyces Genome Database (http://www.yeastgenome.org).
DNA manipulation and plasmid construction
Isolation of plasmid DNA from positive clones was performed using the protocol described in Rose and Broach
(1990). DNA sequencing was performed using the commercial kit ‘Dye Terminator Cycle Sequencing Ready reaction Kit
v3.1’ (Applied Biosystems Corporate, USA), using the following
primers: YEp24Bd (5-TCGCTACTTGGAGCCACTATC and YEp24Br
5-CAGCAACCGCACCTGTGGC and an ‘ABI GeneAmp PCR System
9700 thermal cycler’ (Applied Biosystems Corporate, USA). Fragment separation and analysis was performed using an automatic sequencing equipment ‘ABI Prism 3100’ (Applied Biosystems Corporate, USA). Subcloning of the different genes included in the original genomic clones into yeast episomal
plasmid YEp24 was performed by standard PCR amplification
(Sambrook and Russell 2001), treatment with the appropriate
restriction enzymes and ligation. The sequence of all oligonucleotides used for subcloning and a description of the plasmids
constructed in this work can be found in Table S1 (Supporting
Information).
Analysis of TAT1, TAT2 and PHO90 expression
Yeast cells were grown as indicated in each experiment. Total
RNA was isolated using the method described in Burke, Dawson and Stearns (2000). cDNA was synthesized from 50 μg of
RNA using the commercial kit SuperScript II RT-PCR (Invitrogen Corporation, USA). Analysis of gene expression was performed using real time quantitative PCR. To normalize, we used
the ACT1 gene given that its expression is not affected by low
temperature (Homma, Iwahashi and Komatsu 2003). Amplification was performed from 50 ng of cDNA using a LightCycler 2.0 (Roche Diagnostics GmbH, Germany) and the protocol recommended by the manufacturer. Oligonucleotides used
for the amplification are described in Table S1 (Supporting
Information).
Tryptophan transport assay
Yeast was grown at 28 or 10◦ C until an OD600 of 0.4–0.6 in 50
ml of minimal 165 medium, and then cells were resuspended
in the same medium without tryptophan to a final volume of
12 ml and starved for 2 h for the assay at 28◦ C and 16 h for the
10◦ C assay. After the starvation time, an aliquot was taken to
determine the colony-forming units. The transport assay was
initiated by the addition of L-3 H-tryptophan (20 μg ml−1 and 2
μCi ml−1 final concentrations; GE healthcare, UK) and cold tryptophan (20 μg ml−1 final concentration), both from concentrated
stock solutions. Cells were maintained in a bath with shaking
at the assay temperature and at the indicated times aliquots
were taken. Cells were collected by vacuum filtration through
a 0.45-μm-pore-size 25 mm diameter nitrocellulose filter (Millipore HAWP) and washed twice in the filter with 15 ml icecold 10 mM tryptophan. Moist filters were transferred to Filter Count solution (Perkin Elmer, USA). Radioactivity was measured using a Wallac 1409 liquid scintillation counter (Perkin
Elmer, USA).
Phosphate transport assay
Yeast was grown at 8◦ C until an OD600 of 0.4–0.6 in 20 ml of
YPD, then cells were centrifuged, resuspended in 5 ml water and starved for 24 h at 8◦ C. The transport assay was performed by transferring the cells to a buffer containing 50 mM
succinic acid adjusted to pH 5.5 with Tris, 2% glucose, 50 mM
KCl and 0.2 mM of inorganic phosphate. Cells were maintained in a bath with shaking at the assay temperature, and at
the indicated times 1 ml aliquots were taken. Cells were pelleted by centrifugation and supernatant was collected to determine its inorganic phosphate concentration as described in
Serrano (1983).
RESULTS
Screening of a yeast genomic library
The genomic library in a yeast episomal plasmid that we used in
the present screening was described in Mulet et al. (1999) and has
been extensively used to identify halotolerance genes (Ferrando
et al. 1995; Mulet et al. 1999). One technical problem of this kind
of screening is the presence of spontaneous recessive mutations
which confer tolerance to the studied stress (our unpublished
observations). To minimize this effect, we used a W303 diploid
strain constructed in our laboratory by mating cells from MATa
and MATα strains (Fan, Cheng and Klein 1996). This diploid strain
is unable to grow at 10◦ C, so we transformed our genomic library
in this genetic background and screened for plasmids able to
confer the capacity to grow under this suboptimal temperature.
In the first round, we identified 224 strains. 33 of them were discarded because the phenotype could not be reproduced in a drop
test assay. In order to confirm that the phenotype was due to the
plasmid and not to a chromosomal mutation, we recovered the
plasmid from the remaining 191 strains, retransformed W303
diploid cells and rechecked the phenotype. Nine plasmids could
not confirm the phenotype. The remaining 182 plasmids were
sequenced. Results are shown in Table 1 and Fig. 1. The phenotype could be reproduced in several growth media such as YPD,
SD and SG media (data not shown). Most of the genomic clones
contained several genes or ORFs, but the most represented genes
were related to tryptophan transport (TAT1 and TAT2, encoding low- and high-affinity tryptophan permeases) or tryptophan biosynthesis (TRP1, the gene mutated in the auxotrophic
strain). Overexpression of TAT2 was already described to rescue
the cold sensitivity phenotype of a cse2–1 mutant (Chen, Xiao
and Fitzgerald-Hayes 1994). We confirmed that the phenotype
was dependent on these three genes by subcloning them in the
same episomal plasmid, retransforming the W303 strain and
Vicent et al.
5
Table 1. Sequencing data of the isolated clones. The genes in bold are those responsible of the cold-tolerance phenotype as demonstrated by
subcloning. Brief description obtained from SGD (Saccharomyces Genome Database, www.yeastgenome.org).
Clone name
Times isolated
Chrom.
Coordinates
Size (bp)
Genes included
Brief description
YCT 19.2
129
XV
284 694–293 783
9989
TAT2
Tryptophan and tyrosine high-affinity
permease
Protein of unknown function
ORF, uncharacterized
Syntaxin-like t-SNARE; facilitates
t-SNARE complex formation.
Phosphoribosylanthranilate isomerase
that participates in tryptophan
biosı́ntesis.
Dubious ORF
Dubious ORF
Transcriptional regulator involved in
activation of the GAL genes in response
to galactose.
Major cell wall mannoprotein with
possible lipase activity
High-affinity leucine permease
A acid transporter for Val, Leu, Iso, and
Tyr; low-affinity Trp and His transporter.
Similar to bacterial and human
glycosyltransferases
Phosphoenolpyruvate carboxykinase,
gluconeogenesis enzyme
Ubiquitin-specific protease that cleaves
ubiquitin from ubiquitinated proteins.
Protein of unknown function that
interacts with Pex14p.
Protein O-mannosyltransferase
Protein involved in regulating the
endocytosis of plasma membrane
proteins.
Gamma-glutamyl phosphate reductase,
participates in proline biosı́ntesis.
Integral inner nuclear membrane.
Negative regulator of SPS.
Essential component of
GPI-mannosyltransferase II.
Protein involved in regulation of sterol
biosynthesis.
YOL019w
YOL019w-A
TLG2
YCT 19.14
46
IV
459 314–467 529
8215
TRP1
YDR008c
YDR010C
GAL3
YCT 19.9
3
II
371 621–380 400
8779
TIP1
BAP2
TAT1
ALG14
YCT 80
2
XI
627 388–635 542
8154
PCK1
UBP11
ESL2
YCT 188
1
XV
915 000–924 527
9527
PMT3
LBD19
PRO2
YCT 32
1
XIV
338 060–344 598
6538
ASI2
PGA1
NSG2
observing the phenotype (Fig. 1A). It is known that the expression of the cell wall mannoprotein TIP1 is upregulated by cold
(Kondo and Inouye 1991), and BAP2 encodes a transporter of
branched amino acids (Grauslund et al. 1995). We subcloned
these genes that were present in the same genomic fragment
as TAT1 in order to check whether they were contributing to
the observed phenotype of the genomic clone. Our results indicate that the phenotype is entirely dependent on the low-affinity
tryptophan permease encoded by TAT1, and that overexpression
of BAP2 or TIP1 did not confer tolerance to cold in our experimental conditions (Fig. 1A). For the genomic clone YCT80, we
did not have any hint on which gene could be giving the tolerance (Table 1), so we subcloned the three ORFs included in the
fragment and found that the tolerance was entirely dependent
on the phosphoenolpyruvate carboxykinase PCK1 gene (Fig. 1B).
In plasmid YCT32 (Table 1), the gene encoding Nsg2, a protein
involved in the regulation of sterol biosynthesis was the only
one that could reproduce the phenotype upon overexpression
(Fig. 1B, data not shown for IGO1 and YNL155w).
The identified genes improve growth at 10◦ C in liquid
media
The hitherto described that screening was performed on solid
media. However, growth in liquid media represents better normal environment of yeast in the production of wine or beer.
We wanted to confirm if our results could also be reproduced
in liquid media. For this purpose, we performed growth-curve
experiments at 10◦ C in rich YPD media. All the genes confirmed
the phenotype in liquid media. The growth improvement conferred by the overexpression of the selected clones was very similar between the different clones when compared to the strain
overexpressing the empty plasmid. This control strain exhibited at 10◦ C a lag time of about 200 h and the rate of growth
was very slow, with duplication time of more than 20 h within
the measured interval. On the other hand, strains transformed
with cold-tolerance genes had lag times of about 100 h and duplication times of 5–6 h during growth at 10◦ C (Fig. 1C and D).
Experiments longer than 10–11 days were complicated by the
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FEMS Yeast Research, 2015, Vol. 15, No. 3
appearance of spontaneous cold-tolerant mutants: at variance
with the starting strain, the cells in these old cultures could start
grow at low temperatures with lags of only 50–70 h (data not
shown). This appearance could be explained by the reversion of
the trp1–1 mutation (Stearns, Ma and Botstein 1990).
Quantification of tryptophan uptake under cold
conditions
The results of Table 1 indicate a clear overrepresentation (94%
of the positives) of genes involved in tryptophan uptake or
biosynthesis. TAT2 was previously reported to confer tolerance
to growth under hyperbarometric conditions and also at 10◦ C
(Abe and Horikoshi 2000), supporting the soundness of our experimental design. We measured the uptake of radiolabeled
tryptophan in order to quantify the effect of cold on transport
of this amino acid. Overexpression of TAT2 is able to maintain
this uptake at 10◦ C to levels similar to the control strain at 28◦ C
(Fig. 2A and C). TAT1 is less effective, but the difference with
the control strain is still significant (Fig. 2B and C). The effect
of cold on tryptophan uptake becomes clear when we measure
the initial uptake rate at 10 or 28◦ C (Fig. 2C). In the wild type,
the uptake rate falls from 11.3 at 28◦ C to 1.06 at 10◦ C (pmol
tryptophan × 106 cells−1 × min−1 ). Overexpression of TAT1 or
TAT2 increases the initial uptake rate at 10◦ C (from 1.06 ± 0.02
to 4.6 ± 0.4 and 9.21 ± 0.02, respectively) and at 28◦ C (from 11.3
± 0.2 to 30.0 ± 3 and 33.5 ± 0.04, respectively). The growth improvement can be explained by the fact that at 10◦ C the uptake
in strains overexpressing TAT1 and TAT2 is much higher than
in control.
Phosphate uptake becomes limiting under cold
condition in tryptophan-rich medium
It has been previously reported that tryptophan uptake is a
limiting factor under cold conditions (Abe and Horikoshi 2000;
Hernandez-Lopez et al. 2011). Our first screening confirmed this
and in addition, it allowed us to identify three new genes (PRO2,
PCK1 and NSG1) that confer cold tolerance under tryptophanlimiting conditions. We have also quantified above the effect
of cold on tryptophan uptake. In order to identify physiological targets affected by cold independently of tryptophan uptake, we redesigned the experimental conditions of our screening adding an excess of tryptophan (0.2% or 2 mg ml−1 ) to the
growth medium. By doing this, we eliminated the main limiting
factor and we could unveil other targets. As tryptophan addition
improves yeast growth at 10◦ C, we dropped the screening temperature to 8◦ C to strengthen the conditions for tolerance. We
used the same genomic library as in the previous screening. In
the first round, 85 candidates were selected, but only in 48 of
them the phenotype was dependent on the plasmid. The cold
tolerance conferred by each independent set of isolated plasmids is shown in Fig. 3, and the sequencing results are presented
in Table 2. The most represented plasmid was YCT2 1.8, isolated
24 times (Fig. 3). This plasmid contained four different genes
(Table 2). The subcloning experiments indicated that overexpression of the gene encoding the high-affinity phosphate transporter PHO84 or the GTP-binding protein GTR1, also involved
in phosphate transport (Bun-Ya, Harashima and Oshima 1992),
could confer some tolerance to 8◦ C, but less than the original
plasmid containing the genomic fragment (Fig. 3A). Only overexpression of both genes could restore cold tolerance to an extent
equivalent to the genomic fragment, indicating that the original tolerance was due to an additive effect of GTR1 and PHO84.
Figure 2. Tryptophan uptake is impaired when cells are grown at 10◦ C. (A) Cultures of the W303 strain transformed with the empty episomal plasmid (YEp24)
(squares) or with the episomal plasmid containing TAT2 (triangles) were grown
at 28◦ C (filled) or 10◦ C (empty), and uptake of radiolabeled tryptophan monitored
using a scintillation counter. Results of a typical experiment are shown. (B) Cultures of the W303 strain transformed with the empty episomal plasmid (YEp24)
(squares) or with the episomal plasmid containing TAT1 (diamonds) were grown
at 28◦ C (filled) or 10◦ C (empty), and uptake of radiolabeled tryptophan monitored
using a scintillation counter. Results of a typical experiment are shown. (C) Statistical data of the initial rate of tryptophan uptake (pmol × 106 cells−1 × min−1 )
of W303 strains overexpressing the empty episomal plasmid (Yep24) or the plasmid containing the TAT1 or TAT2 genes. The averages of three determinations
with the standard deviations are shown.
In other clones, the genes responsible for the phenotype were
the low-affinity inorganic phosphate transporter PHO87 (plasmid set YCT2 1.10) and the low-affinity phosphate transporter
PHO90 (plasmid set YCT2 1.19) (Fig. 3A and Table 2). A separate group of clones contained plasmid YCT2 1.23 that was isolated twice. The subcloning experiments demonstrated that the
Vicent et al.
7
Figure 3. Identification of the genes conferring growth at 8◦ C in tryptophan-rich medium. (A) Cultures of W303 transformants carrying the empty episomal plasmid
(YEp24) or the different plasmids containing either the original genomic fragments (YCT plasmids) or the genes identified to be responsible for the phenotype (YEp
plasmid with the genes indicated in each line) were grown in selective (SD + tryptophan) medium until saturation. Serial dilutions of each strain (1/10, 1/100 and
1/1000) were spotted onto YPD medium and incubated at 28 or 8◦ C. Growth was recorded after 3 days (28◦ C) or 8 days (8◦ C). (B) Growth curve in liquid media of
W303 transformed with the empty episomal plasmid YEp24 (squares), YEp24PHO87 (circles), YEp24CTO1 (diamonds), YEp24PHO84.GTR1 (triangles) and YEp24PHO90
(crosses). Cells were inoculated at an initial cell density of 104 colony-forming units (cfu)/ml from saturated precultures and grown at 8◦ C with continuous shaking.
OD600 measurements were taken at the indicated time (in hours). Each curve represents the average values of three independent experiments. Error bars represent
standard deviation.
Table 2. Sequencing data of the isolated clones in tryptophan-rich medium. The genes in bold are those responsible of the cold-tolerance
phenotype as demonstrated by subcloning. Brief description obtained from SGD (Saccharomyces Genome Database, www.yeastgenome.org).
Clone name
Times isolated
Chrom.
Coordinates
Size (bp)
Genes included
Brief description
YCT2 1.8
24
XIII
23 733–31 368
7 635
PHO84
High-affinity inorganic phosphate (Pi)
transporter and low-affinity manganese
transporter
Dubious ORF
Cytoplasmic GTP-binding protein involved in
phosphate transport
NADH: ubiquinone oxidoreductase
Exosome non-catalytic core component
Putative ribokinase
Low-affinity inorganic phosphate (Pi)
transporter
GTP/GDP exchange factor for Rsr1p (Bud1p)
required for both axial and bipolar budding
patterns
Exosome non-catalytic core component
Homeobox-domain protein that represses
a-specific genes in haploids
Transcriptional co-activator involved in
regulation of mating-type-specific gene
expression
Dubious ORF
Low-affinity phosphate transporter
ORF, dubious
ORF, dubious
3-phosphoglycerate kinase
Dubious ORF
DNA polymerase IV
ORF uncharacterized. Renamed as CTO1 (this
work)
H/ACA box small nucleolar RNA (snoRNA).
(Samarsky and Fournier 1999)
Putative protein of unknown function
YML122c
GTR1
YCT2 1.10
1
III
191 688–202 606
10 918
NDI1
RRP43
RBK1
PHO87
BUD5
YCR038w-A
MATALPHA2
MATALPHA1
YCT2 1.19
1
X
58 443–66 673
8230
YCT2 1.23
2
III
137 841—145 054
7213
YCR041w
PHO90
MBB1
YJL197c-A
PGK1
YCR013c
POL4
YCR015c
SNR33
YCR016w
8
FEMS Yeast Research, 2015, Vol. 15, No. 3
CTO1/YCR015c function is related to phosphate uptake
Figure 4. Phosphate and tryptophan uptake of strains overexpressing CTO1. (A)
Statistical data of the initial rate of phosphate uptake (pmol × 106 cells−1 ×
min−1 ) of W303 strains overexpressing the empty episomal plasmid (Yep24) or
the plasmid containing the CTO1 or PHO90 genes and grown at 8◦ C and starved
for phosphate for 24 h. The averages of three determinations with the standard deviations are shown. (B) Cultures of the W303 strain transformed with the
empty episomal plasmid (YEp24) (squares) or with the episomal plasmid containing CTO1 (circles) were grown at 28◦ C (filled) or 10◦ C (empty), and uptake
of radiolabeled tryptophan monitored using a scintillation counter. Results of a
typical experiment are shown.
previously uncharacterized ORF YCR015c was responsible for
the phenotype. We named it as CTO1 for Cold TOlerance 1
(Fig. 3A).
CTO1 was discovered in our screening in tryptophan-rich
medium. This suggests that the phenotype is related to phosphate uptake, which is in agreement with all the genes isolated
in the same screening. We confirmed this observation by measuring the uptake of radiolabeled tryptophan in strains overexpressing CTO1 (see above). We could not observe any significant change relative to the wild type neither at 28◦ C nor at 10◦ C
(Fig. 4B). Database mining gave more evidence supporting the
role of CTO1 in regulating phosphate uptake. SPELL transcriptomic data analysis (Hibbs et al. 2007) showed that expression
is constant under most conditions, but even though this apparent stability is repressed by heat shock (Causton et al. 2001)
and expression is upregulated by phosphate starvation (Saldanha, Brauer and Botstein 2004). Moreover, two independent
proteomic analysis have shown its physical interaction with
Gtr2p (Gavin et al. 2002; Sekiguchi et al. 2008). Gtr2p (Nakashima,
Noguchi and Nishimoto 1999) is a regulator of Gap1p amino-acid
permease (Jauniaux and Grenson 1990), and also controls autophagy depending on the TOR and the EGO signaling pathways
(Dubouloz et al. 2005) indicating that CTO1 function could be related to the regulation of membrane proteins. Standard blast
analysis confirmed that CTO1 is in single copy in the genome
of all the strains of S. cerevisiae included in the databases. CTO1
also has orthologs in different yeasts, being the most conserved
the homologs found in Zygosaccharomyces rouxii, Kazachstania
africana, Torulaspora delbrueckii and Candida glabrata. Moreover,
blast analysis against a general database found that there are
two uncharacterized proteins in soybean (Glycine max) that share
25% of identity with CTO1. This protein homology can be found
also in other plants such as grapevine, tomato and barrel medic,
suggesting an evolutionary conserved role for the newly discovered gene (data not shown). Yeast proteome wide analysis
showed that CTO1 encodes a predicted protein of 317 amino
acids, 36278.8 Da and an isoelectric point of 7.41. Structure prediction indicates that it is a soluble globular protein. No molecular function can be predicted from its structure. Data from the
LoQaTe project indicate that Cto1p localization is cytoplasmic,
and this localization remains unchanged upon stress caused by
H2 O2 , DTT or starvation (Breker et al. 2014).
Quantification of phosphate uptake under cold
conditions
Relation among phosphate and tryptophan uptake
We investigated if the results obtained in solid media could
be extrapolated to liquid media and the growth improvement
quantified. For this purpose, we performed growth curves at
8◦ C in rich YPD media supplemented with excess tryptophan (2
mg ml−1 ). All genes confirmed the phenotype in liquid media.
On average, the lag period was reduced by about 25% (from 161
to 121 h) and the generation time by about 8% (from 8.6 to 7.9
h) (Fig. 3B). Therefore, most of the effect of the cold-tolerance
genes under these conditions is based on shortening of the lag
time.
To quantify the effect exerted by the overexpression of some
representative genes isolated in our screening, we quantified the
initial uptake rate of inorganic phosphate (Pi) at 8◦ C in cells overexpressing either the empty plasmid or CTO1 or PHO90 genes.
Overexpressing CTO1 or PHO90 increases by more than one order of magnitude the rate of inorganic phosphate uptake at 8◦ C
(Fig. 4A).
The results of our different screenings indicate that tryptophan
becomes limiting under cold conditions, with Pi acting as the
second bottleneck for yeast growth. As our screening was performed in a strain which is auxotrophic for tryptophan (trp1), in
a strain prototrophic for tryptophan (TRP1) the uptake or availability of this amino acid should not be limiting and the main
restricting factor would be phosphate uptake. To prove this, we
studied the effect of exogenous tryptophan or phosphate in TRP1
or trp1 strains. We used the W303 strain as trp1 and strain RS452
as TRP1. It is important to indicate that strain RS452 behaviors
in all assays in a similar way than strain W303 transformed
with the YEp24-TRP1 plasmid. The addition of 0.2% w/v tryptophan to the medium increased growth at 10◦ C in the W303
trp1 strain. The effect was much less evident in the RS452 TRP1
strain. In agreement with our hypothesis, addition of 10 mM inorganic phosphate to the growth medium only had effect in the
RS452 (TRP1) strain. Addition of phosphate had no effect in a trp1
Vicent et al.
9
Identification of additional limiting factors,
independent of tryptophan or inorganic phosphate
uptake
To further characterize the mechanism of cold tolerance determined by the isolated genes, we overexpressed the positive
genes in the RS452 strain, prototroph for tryptophan. TAT1, TAT2
and TRP1 could not improve the growth of the strain at 8◦ C. NSG2
also did not conferred cold tolerance, indicating that its phenotype depends on the tryptophan transport, but PCK1 and PRO2
conferred tolerance, even upon addition of exogenous tryptophan indicating that the mechanism of tolerance is independent of tryptophan availability (Fig. 5B). PHO84 + GTR1, PHO87
and PHO90 also conferred tolerance in tryptophan-rich medium
as compared with control strains, but this effect diminished
upon the addition of exogenous phosphate (Fig. 5C). The coldtolerance phenotype of CTO1 could be reproduced under high
exogenous tryptophan concentration, but was almost lost upon
the addition of exogenous phosphate, similar to the phenotype exhibited by the overexpression of phosphate transporters
(Fig. 5C).
Transcriptional regulation by cold of the most
representative genes identified in our screening
Figure 5. Cold tolerance conferred by the addition of exogenous tryptophan or
phosphate. (A) Comparison of the cold tolerance of the W303 trp1 strain (W303)
transformed with the empty episomal plasmid (W303 + YEp24) and the RS452
TRP1 strain (RS452) transformed with the empty episomal plasmid (RS452 +
YEp24). (B) Phenotype in cold conditions conferred by the identified genes in
the RS452 TRP1 strain. RS452, RS452 transformed with the empty episomal plasmid (RS452 + YEp24) or with the plasmid containing the genes identified in the
screening in YPD medium (indicated in each lane). (C) Phenotype in cold conditions of the identified genes in the RS452 TRP1 strain. RS452, RS452 transformed
with the empty episomal plasmid (RS452 + YEp24) or with the plasmid containing the genes identified in the screening in tryptophan-rich medium (indicated
in each lane). In all cases (A, B and C), the strains transformed with the empty
episomal plasmid (YEp24) and the different plasmids with the genomic fragments identified in the screening (indicated in each lane) were grown in selective (SD) medium until saturation. Serial dilutions of each strain (1/10, 1/100 and
1/1000) were spotted onto YPD medium (YPD), or YPD medium containing 0.2%
w/v of tryptophan (YPD + W), 10 mM of inorganic phosphate (YPD + Pi) or both
(YPD + W + Pi) and maintained at 10 or 8◦ C (as indicated). Growth was recorded
after 8 days.
strain without exogenous tryptophan, but upon addition of tryptophan, exogenous phosphate could increase the tolerance in
the trp1 strain, indicating a synergistic and hierarchical effect
between phosphate and tryptophan (Fig. 5A).
There are several reports in the literature on the transcriptional
response of yeast at low temperature. The results of these reports referring to the genes identified by us are compiled in
Fig. 6A. None of the published evidence can be directly extrapolated to our results as is made in other genetic strains, and
the experimental conditions are very different to the growth at
10 or 8◦ C that we have used in our screenings. To investigate a
possible transcriptional regulation, we performed an RT-PCR of
the TAT1, TAT2 and PHO90 genes, and compared its expression
when grown at 28 or 10◦ C. The constitutive ACT1 gene was used
to normalize the level of gene expression and minimize the effect of the general decrease of transcription caused by cold. In
our conditions, TAT1 and TAT2 are induced while PHO90 is repressed (Fig. 6B). This is in contradiction with previously published results showing that both TAT2 (three different reports)
and TAT1 (two separate papers) were repressed under a variety
of low-temperature conditions (Fig. 6A). PHO90 expression was
not previously found to be regulated by cold, but we have found
that is repressed in our experimental conditions. For the other
positive genes, a literature search on cold shock transcriptional
regulation showed that PCK1 was found to be induced in three of
five reports and PHO87 in one. PHO84 was repressed in two papers and induced in one confirming the high variability depending on the design of the experiment (Fig. 6A). We also recover
the genome wide transcription data available in databases (Hibbs et al. 2007). These uncovered a transcriptional joint regulation of these three genes. PHO90 and TAT1 are repressed under
oxidative stress (Shapira, Segal and Botstein 2004), heat shock
(Segal et al. 2003) or by the activation of the Hog1 MAPK pathway
(O’Rourke et al. 2004). The adjusted correlation score for PHO90
in relation to TAT1 is 2.2 (Hibbs et al. 2007). This indicates a strong
correlation in the transcriptional regulation, given that it is the
13th in the score ranking, and the higher value in the list is 2.4.
On the other way, TAT2 expression remains unchanged in many
genome wide transcription experiments, but it has an adjusted
correlation score of 2 relative to TAT1, ranking the seventh (data
not shown).
10
FEMS Yeast Research, 2015, Vol. 15, No. 3
Figure 6. Transcriptional regulation of TAT1, TAT2 and PHO90. (A) Comparison of the published data from genome wide transcriptome analysis for the genes identified
in the screening on YPD medium. (B) Relative expression of TAT1, TAT2 and PHO90 in relation to the constitutive ACT1 gene in the W303 strain at 28◦ C (filled bars) or
10o C (empty bars) measured by quantitative RT-PCR. Results are the average of three independent experiments and error bars represent standard deviation.
DISCUSSION
Yeast gene overexpression approach has proven to be a powerful technique to identify limiting factors under stress conditions. That was the strategy utilized with the HAL genes identified for its ability to confer salt resistance (Serrano et al. 2003). We
have adapted the same strategy to identify genes able to confer
yeast the ability to grow at suboptimal temperatures (10 or 8◦ C).
A similar strategy has been used by other authors (HernandezLopez et al. 2011) but with different outcomes. The divergence
could be explained because in the latter screening the drug phytosphingosine was included to promote cellular stress responses
including ubiquitination and degradation of amino-acid transporters. Therefore, only genes related to ubiquitination were
found (Hernandez-Lopez et al. 2011).
When a gene confers tolerance to a stress by overexpression,
this can be due to the fact that the gene is part of the stress response or that it is part of a physiological process that becomes
limiting under the stress condition. It is known that cold alters the fluidity of membranes and therefore, affects membrane
transporters. The first transport that becomes limiting during
growth at low temperatures is tryptophan uptake, and can be
suppressed by overexpressing high- (TAT2) or low-affinity (TAT1)
tryptophan transporters, by using strains prototroph for tryptophan or by supplementation of media with excess tryptophan.
This is in agreement with previous results showing that under
high pressure tryptophan uptake also becomes limiting (Abe and
Horikoshi 2000) as high pressure resembles cold by also triggering a decrease in membrane fluidity. TAT2 has also been shown
previously to confer tolerance to 10◦ C (confirming the validity
of our screening), quinidine (Khozoie, Pleass and Avery 2009),
zaragozic acid (Daicho et al. 2007) and weak organic acids (Bauer
et al. 2003) indicating that TAT2 is limiting under most conditions
that affects fluidity of the plasma membrane and/or tryptophan
uptake. Our results also indicate that TAT1 and TRP1 can also
rescue this growth defect under cold conditions. The growth advantage at low temperature conferred by increased tryptophan
transport (overexpression of TAT1 or TAT2) or internal biosynthesis (transformation with TRP1) is mostly reflected by a reduced
lag (Fig. 1C and D). This suggests that starting cell growth requires a certain level of tryptophan inside cells.
A recent report suggests that tryptophan availability is not
the only limiting factor for wine yeast when grown at low temperatures (López-Malo et al. 2014). In our screening, we also
identified NSG2 which participates in the biosynthesis of ergosterol (Flury et al. 2005). There are previous references indicating that fluidization of membrane lipids could confer cold tolerance (Rodriguez-Vargas et al. 2007) and that yeast strains with
deletion or overexpression of lipid metabolism genes show phenotypes related to growth at low temperature (López-Malo et al.
2013). Overexpression of NSG2 did not confer tolerance to cold
stress in tryptophan-rich medium or in an RS452 (TRP1) strain,
pointing out that its mechanism of tolerance is dependent on
tryptophan availability. It has been shown that ergosterol affects
the sorting of Tat2p to the membrane (Umebayashi and Nakano
2003). Here, we show that NSG2 is a likely limiting factor in that
process. The other genes identified in our screening at 10◦ C under tryptophan-limiting conditions were the gamma-glutamyl
phosphate reductase Pro2p, which participates in the proline
biosynthesis pathway, and the gluconeogenic enzyme Pck1p,
a phosphoenolpyruvate carboxykinase. A mutant allele of another gene in the proline biosynthesis pathway (PRO1) was previously characterized for conferring freezing tolerance (Morita,
Nakamori and Takagi 2003). Pro2p is probably the limiting factor for proline biosynthesis, not Pro1p. We have also identified
Pck1p, a key enzyme in gluconeogenesis. It is known that under cold temperatures there are several changes affecting carbohydrate metabolism. Specifically, it has been described that
cold can induce the accumulation of trehalose or glycerol (Aguilera, Randez-Gil and Prieto 2007). At low temperature, the rate of
metabolism is severely reduced, as denoted by the drastic increase in generation and the lag times. Thus, at low temperatures probably gluconeogenesis becomes more important than
at 28◦ C and the step catalyzed by Pck1p is rate limiting. Surprisingly, the kinetics of growth at low temperature of cells transformed with the PCK1 and PRO2 genes is similar to those transformed with genes related to tryptophan uptake (about 100 h
lag). This could be explained if Pck1 and Pro2 are needed to
Vicent et al.
synthesize some metabolites (trehalose and proline, respectively) that need to be accumulated to partially bypass, by some
unknown mechanism, the requirements of intracellular tryptophan.
In tryptophan-rich medium, at 8◦ C, our screening revealed
that the second limiting factor is the uptake of inorganic phosphate, given that all positive clones were related to this physiological process. This screening has allowed the characterization
of a novel gene encoded by the ORF YCR015c, which we named
CTO1 for Cold TOlerance 1. The genomic fragment containing
this gene was isolated twice in our screening. Subcloning confirmed that the phenotype was dependent on this ORF (Fig. 3).
Overexpression of CTO1 could also confer tolerance upon addition of exogenous tryptophan, and uptake of radiolabeled tryptophan in W303 strain overexpressing CTO1 was similar to the
wild type, both at 28 and 10◦ C (data not shown), so we completely ruled out that the phenotype was related to tryptophan
uptake. Furthermore, the phenotype disappeared upon addition
of exogenous phosphate (Fig. 4C) confirming the relationship of
this gene with phosphate uptake. Likewise, the initial rate of inorganic phosphate uptake at 8◦ C was higher than in the control
strain and similar to the strain overexpressing PHO90. Database
mining provided additional evidence for the role of CTO1 in the
regulation of phosphate uptake. Genome wide transcriptome
analysis revealed that this gene is upregulated by phosphate
starvation and interacts physically with a protein involved in
membrane protein sorting (Gtr2p). Blast analysis showed that
CTO1 is in a single copy in the yeast genome, but is conserved
in yeasts and plants, so this report is the first evidence for a
novel and conserved gene related to inorganic phosphate uptake whose function becomes limiting under cold stress. Deep
characterization of this gene is currently being undertaken in
our laboratory.
Taken together, all our results point to a joint regulation of
growth at low temperatures of laboratory strains by phosphate
and tryptophan uptake. Phosphate uptake only becomes limiting in tryptophan-rich media, and there is a strong correlation
between the transcriptional regulation of PHO90 and TAT1. Under cold conditions, Tat2p is targeted to the membrane, in a way
similar to starvation conditions (Beck, Schmidt and Hall 1999).
TOR pathway has been reported to be a negative regulator of
the stress response dependent on calcineurin (Mulet et al. 2006),
and the calcineurin-dependent transcription factor CRZ1/HAL8
is able to confer freeze tolerance by overexpression (Panadero
et al. 2007). The mechanism of tolerance to near freezing temperatures depends on the stress-induced transcription factors
Msn2p and Msn4p, which are also inhibited by the TOR pathway (Kandror et al. 2004; Schmelzle et al. 2004). The TOR pathway senses nutrients, so an interesting hypothesis that deserves
further investigation is that the TOR pathway is also regulating
inorganic phosphate uptake.
We have also obtained expression data for TAT1, TAT2 and
PHO90, finding that TAT1 and TAT2 expression increases at low
temperatures while PHO90 expression decreases. There is a lack
of agreement between our expression data and the results of
previous genome-wide transcriptome analysis. One of the reasons could be that none of the published reports reflect our
experimental design. The fact that one gene is upregulated by
cold does not necessarily mean that this gene is limiting under
stress conditions. None of the isolated genes in our screening are
among those described as early-induced or late-induced genes
upon cold stress. Moreover Tip1p, the major cell wall mannoprotein, described as an early induced gene, was present in one of
the isolated plasmids (YCT19.9), but its overexpression did not
11
confer tolerance to cold stress in our conditions, confirming that
Tip1p is not a limiting factor regardless its increase of expression
upon cold stress (Fig. 1A).
We also investigated if overexpression of the positive genes
could as well confer tolerance to freeze-thaw stress. We performed experiments with a W303 strain overexpressing the positive genes, but in most cases tolerance to this type of stress
was diminished in relation to the wild type (data not shown)
indicating that the underlying survival mechanisms operating
at low temperatures are very different from those involved in
resistance to freeze-thaw stress.
SUPPLEMENTARY DATA
Supplementary data is available at FEMSYR online.
FUNDING
I. Vicent was a recipient of a FPI fellowship from the Generalitat Valenciana. This work was supported by Grant AGL200303757 from the Spanish Ministry of Science and Technology
and by Grant ACOMP06/66 from Generalitat Valenciana (both
awarded to A.N.), and funded by Universidad Politécnica de Valencia (Grants PPI2742/2002 and PPI5621-05-04) awarded to A.N.
and R.S.
Conflict of interest. None declared.
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