Biochemical and Biophysical Research Communications 387 (2009) 778–783

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

The induction of trehalose and glycerol in Saccharomyces cerevisiae in response

to various stresses
LiLi Li, YanRui Ye, Li Pan, Yi Zhu, SuiPing Zheng, Ying Lin *
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts
Received 16 July 2009 during various stress conditions. We investigated the levels of glycerol and trehalose and the expression
Available online 25 July 2009 profiles of genes involved in their metabolism to determine their involvement in the response of Saccha-
romyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes
Keywords: involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that treha-
Saccharomyces cerevisiae lose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period)
Stress response
of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sor-
Trehalose
Glycerol
bitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interest-
Gene expression ingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable
conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glyc-
erol turnover are of general importance during cellular stress adaptation.
Ó 2009 Elsevier Inc. All rights reserved.

The budding yeast Saccharomyces cerevisiae is widely used in
the fermentation and brewing industries. During the fermentation
process, yeasts are subjected to a succession of stress conditions,
such as high temperature, high sugar and accumulation of ethanol,
which affect their viability and fermentation efficiency. When the
cells encounter such stresses, dynamic changes occur in the com-
plex biological networks that comprise genes, proteins, metabo-
lites, etc. and that underlie cellular function [1]. The investigation
of such molecular responses will help us to understand the molec-
ular mechanisms by which cells adapt to fermentation conditions.
Several studies have reported that trehalose is better as a pro-
tein stabilizer than any of a number of compatible solutes, because
of its unusual ability to alter the water environment surrounding a
protein, stabilizing the protein in its native conformation [2,3]. A
strong correlation between trehalose content and stress resistance
has been demonstrated for a variety of stresses [4–6], such as heat,
osmotic stress, and ethanol. Furthermore, the stress response is
mediated at the level of transcription, and a number of stress-in-
duced transduction pathways concerning trehalose [7,8].
Glycerol is clearly instrumental in adjusting the water potential
of yeast cells. It is well known that yeast cells accumulate glycerol
under conditions of high osmotic pressure [9]. Gene expression
profiling experiments [10,11] have also shown that genes related
to the formation of glycerol are highly expressed under conditions
of high osmotic pressure. In addition, glycerol can be used to in-
* Corresponding author. Fax: +86 020 39380698.
E-mail address: feylin@scut.edu.cn (Y. Lin).
crease the stability of thermolabile enzymes and also to prevent
the permanent inactivation of enzymes subjected to injurious con-
ditions [6]. Further research is required to understand the genetic
and metabolic changes in the production of this compatible solute
that occur under various stress conditions.
In this work, we examined the response with respect to treha-
lose and glycerol metabolism of S. cerevisiae to heat, sorbitol and
ethanol stresses. Although the genes involved in the synthesis
and degradation of trehalose and glycerol are stress induced in
S. cerevisiae, there were differences in the patterns of glycerol
and trehalose synthesis and related gene expression profiles. Our
objectives were to elucidate previously unclear connections be-
tween the gene expression profiles and the intracellular levels of
glycerol and trehalose, and to uncover the possible common pro-
tective mechanism in response to environmental stresses.
Materials and methods
Strains and cell culture. The yeast strain used in this study was
S. cerevisiae XQ1. S. cerevisiae was cultured in YPD medium (1%
yeast extract, 2% peptone, and 2% glucose). Yeast cells were grown
overnight at 30 °C in YPD medium, inoculated into fresh YPD med-
ium at an OD600 of 0.1, grown to an OD600 of 1, and then subjected
to stress treatments. Cell growth was monitored by measuring the
optical density at 600 nm. An increase of 1 OD600 unit was shown
to be equivalent to an increase of 0.36 g of dry cell weight per liter.
Heat treatment. Cells that had been grown continuously at 30 °C
were collected by centrifugation at 5000g and 30 °C for 2 min,

0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.07.113
 L. Li et al. / Biochemical and Biophysical Research Communications 387 (2009) 778–783
resuspended in an equal volume of YPD medium at 30 °C or 42 °C
(prewarmed to the corresponding temperature), and returned to
30 °C or 42 °C for growth, respectively.
Sorbitol treatment. The collected cells were resuspended in an
equal volume of fresh YPD medium with or without 1 M sorbitol,
and returned to 30 °C for growth.
Ethanol treatment. The experiment was carried out as described
for sorbitol treatment except that the collected cells were resus-
pended in an equal volume of fresh YPD medium with or without
6% (v/v) ethanol.
Sample preparation. Samples were collected at 0, 0.5, 1, 2, 4, and
6 h. For analysis of the intracellular and extracellular products, the
collected samples were centrifuged immediately at 10,000g and
4 °C for 5 min. The resulting supernatants were filtered through a
cellulose acetate filter (0.45 lm) and stored at 20 °C until analy-
sis. The harvested cells were washed with cold distilled water, and
then the samples were crashed in a microwave oven at 700 W by
five repeated treatments of 60 s each with a 30 s interval between
each treatment. The trehalose and glycerol were then extracted
with 1 ml of distilled water at room temperature for 1 h. The sam-
ples were centrifuged at 20,000g for 10 min, and then the trehalose
and glycerol in the supernatant were analyzed by HPLC [12].
Analysis of intracellular and extracellular metabolites. Glucose,
trehalose, glycerol, ethanol, and acetate in both the intracellular
and extracellular fractions were quantified using an Aminex
HPX-87H column (Bio-Rad) at 50 °C and a flow rate of 0.5 ml/min
with 5 mM H2SO4 as the eluent. A refractive index detector (Waters
2414) was used for all the chemicals mentioned.
RNA isolation and RT-PCR analysis. RNA isolation and RT-PCR
analysis was performed as reported in our previous work [13].
The specificity of the primer pairs (Supplementary Table 1) was
tested by amplification of genomic DNA before RT-PCR.
Fig. 1. Intracellular (A, B, and C) and extracellular (D, E, and F) levels of trehalose after heat (A, D), sorbitol (B, E) and ethanol (C, F) stress. Samples were collected after
cultivation in YPD medium for 0, 0.5, 1, 2, 4, and 6 h in the absence (open circle) or presence (filled circle) of the designated stress treatment. The values are the means and
standard deviations of three independent experiments.
779
Results
Cell growth of S. cerevisiae under different stress conditions
The growth of the cells subjected to thermal stress displayed no
significant difference (p > 0.05) compared to that of the control
cells (Supplementary Fig. S1A), which implied that either heat
treatment was not detrimental to the cells or the cells had devel-
oped a mechanism for rapid adaptation to high temperature during
long term evolution. Cell growth was inhibited strikingly during
the first 4 h in the presence of 1 M sorbitol, whereas after 4 h the
cells in the sorbitol-containing medium grew normally due to cel-
lular adaptation to osmotic stress (Supplementary Fig. S1B). The
inhibition of cell growth by 6% external ethanol was the most obvi-
ous among the three types of stress (Supplementary Fig. S1C).
Trehalose accumulation under different stress conditions
Intracellular trehalose levels increased rapidly and dramatically
in response to heat shock (Fig. 1A), in accordance with the results
of a previous study [14], which showed that intracellular trehalose
increased strikingly at the onset of heat shock and thus enabled
proteins to retain their native conformation at higher temperatures
and suppressed the aggregation of denatured proteins. However,
from 1 to 2 h after the initiation of heat stress, the levels of treha-
lose decreased markedly and approached the initial levels that
were observed. This degradation of trehalose is critical for recovery
from heat shock because very high levels of trehalose can interfere
with normal protein activity by stabilizing proteins in nonnative
conformations and inhibiting the refolding of these denatured pro-
teins by heat shock proteins [15,16]. From 2 to 4 h after the initia-
tion of heat shock, the amount of intracellular trehalose increased
780 L. Li et al. / Biochemical and Biophysical Research Communications 387 (2009) 778–783
once more, and reached its maximal levels (15.8% of dry cell
weight) at the 4 h time point. A significant decrease in trehalose
content occurred during the last 2 h of the experimental period
(as measured at the 6 h time point) because once glucose has been
depleted trehalose is used as a carbon source.
Under sorbitol and ethanol stresses, it appeared that trehalose
did not accumulate to levels that would have a major impact on
the adaptation of the cells. However, the concentration of intracel-
lular trehalose was higher than that in the control cells throughout
the stress treatments (Fig. 1B and C), which indicated that an in-
crease in trehalose may be a general response to physiological
stress. Interestingly, although the trehalose content under sorbitol
and ethanol stress was higher than that in the control cells, it did
decrease throughout the experimental period.
In addition, we found that the level of extracellular trehalose
was higher in the cultures that were subjected to stress than in
the control culture (Fig. 1D–F) throughout the entire experimental
period. This suggested that the mechanism of protection might
involve minimizing the damage caused to cells, which possibly re-
quire the presence of trehalose in vitro and in vivo of the cell; this
hypothesis is supported by the results of da Costa et al. [17], which
were obtained under oxidative conditions.
Glycerol accumulation under different stress conditions
Glycerol synthesis occurred mainly during the initial stages of
stress treatment, after which the concentration of glycerol de-
creased (Fig. 2A–C). However, the glycerol profile under sorbitol
stress was significantly different from that for the other two stres-
ses. Under sorbitol stress conditions, a rapid increase in glycerol
was observed during the first 2 h, and the highest concentration
Fig. 2. Intracellular (A, B, and C) and extracellular (D) levels of glycerol after heat (A), sorbitol (B, D) and ethanol (C) stress. The samples were collected after cultivation in YPD
medium for 0, 0.5, 1, 2, 4, and 6 h in the absence (open circle) or presence (filled circle) of the designated stress treatment. The values are the means and standard deviations of
three independent experiments.
of glycerol was measured at 2 h (32.9% dry cell weight). The intra-
cellular glycerol levels under sorbitol stress were far higher than
those in non-stressed cells throughout the entire experimental
course, whereas, under heat and ethanol stress, glycerol did not
accumulate to levels that would have a major impact on the adap-
tation of the cells. S. cerevisiae displayed the ability to cope with
both high temperature and sorbitol stress, which suggests that tre-
halose and glycerol make a remarkable contribution to the toler-
ance of heat and osmotic stress, respectively.
The trehalose metabolic pathway and the stress response
Trehalose is produced from glucose-6-phosphate and uridine
diphosphate glucose in a two-step process, and recycled back to
glucose by trehalases (Supplementary Fig. S2). Even though the
trehalose cycle consists of only a few metabolites and enzymatic
steps, its regulatory structure and operation are surprisingly com-
plex. To understand the mechanism by which yeast cells accumu-
late trehalose in response to stresses, the effects of stress on the
expression of the genes TPS1, TPS2, ATH1, NTH1, and GLK1, which
encode products involved in trehalase metabolism, were observed.
These five genes, which were induced immediately and strongly
after heat shock, shared similar expression profiles in response to
heat stress (Fig. 3). The highest level of expression occurred at
0.5 h, and was followed by an immediate decrease from 0.5 to
1 h. Under conditions of sorbitol stress, it was found that the
response profile was also similar for all five genes: they were in-
duced strongly during the first 0.5 h after the initiation of stress,
after which the expression levels dropped immediately to those
observed initially. This indicated a cellular adaptation to diverse
conditions. Under conditions of ethanol stress, TPS1, ATH1, GLK1,
 L. Li et al. / Biochemical and Biophysical Research Communications 387 (2009) 778–783 781

52

47

42

37

32

2
2 207 3 4 6 8 2 207 3 4 6 8

Fig. 3. Expression of TPS1 and TPS2 (responsible for trehalose synthesis, gray), and ATH1, NTH1 and GLK1 (responsible for trehalose degradation) was determined after heat
(striped), sorbitol (gray) or ethanol (dotted) stress treatment for 0, 0.5, 1, 2, 4, and 6 h in YPD medium. The samples collected at 0 h were adopted as controls (blank). Each
sample was analyzed in quadruplicate, and the values are the means and standard deviations of four replicates.

and TPS2 were induced. The first three showed a similar expression
pattern, whereas TPS2 was up-regulated the least among the four
ethanol-induced genes.
TPS1 and TPS2, which encode enzymes involved in trehalose
synthesis, were both induced under the experimental stress condi-
tions; this indicated that the synthesis of trehalose was stimulated
by stress. This rapid increase in trehalose has been attributed to an
increase in both the translation of the genes involved in the syn-
thesis of trehalose [18] and the substrates required for trehalose
synthesis [19]. It has been reported that the deletion of TPS1 or
TPS2 results in diminished accumulation of trehalose [4,20]. More-
over, strains that lack the TPS1 gene, which encodes Tps1p, an
essential component of the trehalose synthase complex, cannot
synthesize any trehalose [21]. Trehalose is degraded in the cyto-
plasm by neutral trehalase, which is encoded by the gene NTH1.
The transcription of NTH1 is stress responsive; however, the activ-
ity of Nth1p is greater during recovery from stress, which allows
Nth1p to compete successfully with the biosynthetic pathway
and reduce intracellular trehalose levels. The data also revealed
that heat shock resulted in the strongest induction of these genes,
as compared to osmotic or ethanol stress. This suggested that the
accumulation of trehalose was much more important for resistance
to heat shock than for the other two stress conditions, which was
consistent with the quantitative analysis of trehalose. Moreover,
the data showed that both the synthesis and degradation of treha-
lose were induced during stress conditions.
The glycerol metabolic pathway and the stress response
Glycerol is produced by the reduction of dihydroxyacetone
phosphate to glycerol-3-phosphate, which is then dephosphoryl-
ated to yield glycerol (Supplementary Fig. S2). The response of
the pathway of glycerol metabolism to the different stress condi-
tions was observed (Fig. 4). Firstly, induction of GPD2, GPD1,
GUT1, and GUT2 was observed during the first hour after the initi-
ation of heat shock. GPD1 and GUT2 shared similar expression pro-
files: the highest expression level was observed at the 0.5 h time
point, after which there was an immediate decrease from 0.5 to
1 h. Secondly, GPD1, HOR2, RHR2, and GUT2 were induced during
the first hour after sorbitol shock. The first three genes showed a
similar pattern of induction: the highest expression level was ob-
served at the 1 h time point, and was followed by an immediate de-
crease from 1 to 2 h. Thirdly, the six genes were all up-regulated at
the 0.5 h time point under ethanol shock, although the amount of
up-regulation of RHR2, GPD2 and GUT1 was low.
GPD1, which is involved in glycerol synthesis, was induced in
response to the stress conditions, whereas glycerol accumulated
markedly only during sorbitol shock. This indicates that other
mechanisms in addition to GPD1 must be involved in glycerol accu-
mulation under sorbitol stress. A previous study showed that intra-
cellular glycerol concentrations are not only controlled at the level
of production but also by regulated transmembrane transport
through the glycerol facilitator, Fps1p [22,23]. Under conditions
782 L. Li et al. / Biochemical and Biophysical Research Communications 387 (2009) 778–783

2 16 3
GPD2 GPD1 RHR2
14
2.5
1.6

Relative expression
Relative expression
Relative expression

12
2
10
1.2

8 1.5

0.8
6
1
4
0.4
0.5
2

0 0 0
0 0.5 1 2 4 6 0 0.5 1 2 4 6 0 0.5 1 2 4 6
Time(h) Time(h) Time(h)
7 20 45
HOR2 GUT1 GUT2
40
6
16
Relative expression

Relative expression

Relative expression
35
5
30
12
4 25

3 20
8
15
2
4 10
1
5

0 0 0
0 0.5 1 2 4 6 0 0.5 1 2 4 6 0 0.5 1 2 4 6
Time(h) Time(h) Time(h)

Fig. 4. Expression of GPD2, GPD1, RHR2, and HOR2 (responsible for glycerol synthesis, gray), and GUT1 and GUT2 (responsible for glycerol degradation) was determined after
heat (striped), sorbitol (gray), or ethanol (dotted) stress treatment for 0, 0.5, 1, 2, 4, and 6 h in YPD medium. The samples collected at 0 h were adopted as controls (blank).
Each sample was analyzed in quadruplicate, and the values are the means and standard deviations of four replicates.

of increased osmolarity, the Fps1p glycerol facilitator is closed,
which enables cells to accumulate intracellular glycerol [24]. In
our experiment the extracellular glycerol increased rapidly after
1 h (Fig. 2D), which is in accordance with the report that glycerol
leaks into the outside medium through Fps1p because the cells
had already adapted to the stress environment.
Discussion
A general stress response would involve a system that regulates
the coordinated induction of many stress genes through a common
element in their promoter (known as a stress response element,
STRE). The STREs would be induced by diverse stresses and the
transcriptional activation mediated through these elements would
enable cells to acquire tolerance towards stress conditions. The
Msn2p/4p transcription factors, which are found only in yeast, bind
to STREs found in the promoters of many heat shock genes [25].
STREs have been identified in the promoters of NTH1, TPS1, and
TPS2 [26], which suggests that the expression of most of the genes
that encode enzymes involved in trehalose metabolism is co-regu-
lated. This explains the fact that stress induced both the genes
involved in trehalose synthesis and those involved in degradation,
and why the genes responded in a similar pattern (Fig. 3). Simi-
larly, both GPD1 and HOR2 contain STRE sequences in their
promoters, which explain why GPD1 was induced by all three types
of stress (Fig. 4), although deletion of MSN2 and MSN4 has little
effect on the induction of GPD1 and HOR2 under conditions of
osmotic stress [7]. However, genes that contain an STRE in their
promoter will not necessarily show a uniform pattern of expres-
sion. The different patterns of expression among genes that contain
STREs could be due to the promoter context in which the STREs are
found and might depend on the severity of the stress condition [7].
The evolution of metabolic systems that allow adaptation to
sudden environmental changes could have occurred by the simul-
taneous selection and optimization of pathways that produce
important stress protectants in addition to providing glycolytic
safety valves. In this context both glycerol and trehalose appear
to be ideal candidates. One of the consequences of sudden stress
is a dramatic decrease in the ATP demand from macromolecular
biosynthesis. It has been proposed, by elegant theoretical consider-
ations from models of glycolysis, that under conditions of growth
arrest, with low ATP demand, cells face the threat of substrate-
accelerated death. However, the stress-induced metabolic ATP
imbalance could be counteracted by instigating a metabolic change
that would introduce futile ATP cycles and thus increase the de-
mand for ATP. This model, which was proposed by Blomberg [9],
has yet to be theoretically evaluated and experimentally verified.
However, the stimulation of the genes involved in trehalose and
glycerol synthesis and degradation (Figs. 3 and 4) under stress con-
ditions supports this hypothesis. Acetic acid can provide another
glycolytic safety valve under diverse stress conditions. In our
experiment, acetic acid accumulated in cells under sorbitol stress
(data not shown). This result was consistent with the latest work
by Hirasawa et al. [27], who showed that Ald6p, a protein related
 L. Li et al. / Biochemical and Biophysical Research Communications 387 (2009) 778–783
to acetate biosynthesis, is induced under conditions of high osmo-
tic pressure. However, the response of acetic acid metabolism to
detrimental stresses is not well understood, and thus the role that
acetic acid plays during unfavorable stress conditions needs fur-
ther investigation.
The stimulation of trehalose and glycerol synthesis (Figs. 1 and
2) and the up-regulation of related genes (Figs. 3 and 4) show that
the first hour after the initiation of stress treatment corresponds to
a sensitive response period. This is also supported by the hierarchi-
cal clustering of genes that are involved in trehalose and glycerol
metabolism (data not shown). The length of this responsive phase
can vary considerably, depending on a number of factors, which in-
clude the type and concentration of stress solute, the strain of
yeast, and the growth phase.
The cellular response to stress is also dependent on the physio-
logical state of cells. Gene expression shows complex changes as
glucose is depleted progressively from the growth media. In the
diauxic shift, Brauer [28] suggested that the cells, instead of arrest-
ing completely, initiate metabolic remodeling, cell cycle changes,
and accompanying stress responses. The changes in gene expres-
sion and metabolism that we observed during the last 2 h of the
experimental period result from the transition in growth phase
and heat treatment.
Acknowledgments
This work was supported by the National Natural Science Foun-
dation of China (Grant No. 20436020). We greatly thank Interna-
tional Science Editing Compuscript Ltd., Co. for the professional
editing of the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2009.07.113.
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