From the Department of Biochemistry, University of Arizona, Biosciences West, Tucson, Arizona 856721-0088
Received for publication, September 7, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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Transcript expression of Saccharomyces
cerevisiae at high salinity was determined by microarray analysis
of 6144 open reading frames (ORFs). From cells grown in 1 M
NaCl for 10, 30, and 90 min, changes in transcript abundance >2-fold
were classified. Salinity-induced ORFs increased over time: 107 (10 min), 243 (30 min), and 354 (90 min). Up-regulated, functionally
unknown ORFs increased from 17 to 149 over this period. Expression
patterns were similar early, with 67% of up-regulated transcripts
after 10 min identical to those at 30 min. The expression profile after 90 min revealed different up-regulated transcripts (identities of 13%
and 22%, respectively). Nucleotide and amino acid metabolism exemplified the earliest responses to salinity, followed by ORFs related to intracellular transport, protein synthesis, and destination. Transcripts related to energy production were up-regulated throughout the time course with respiration-associated transcripts strongly induced at 30 min. Highly expressed at 90 min were known salinity stress-induced genes, detoxification-related responses, transporters of
the major facilitator superfamily, metabolism of energy reserves, nitrogen and sulfur compounds, and lipid, fatty acid/isoprenoid biosynthesis. We chose severe stress conditions to monitor responses in
essential biochemical mechanisms. In the mutant, High salinity represents a stress for organisms, because the
excess of sodium or other monovalent cations imbalances the osmotic potential and generates water deficit, and the influx of sodium may
lead to metabolic toxicity (1, 2). Protective biochemical reactions
range from the synthesis of osmolytes, to increased chaperone activity,
enhanced radical oxygen scavenging, changes in redox control, increased
proton pumping activity, adjustments in carbon/nitrogen balance, and
altered ion and water uptake (2-7). These biochemical activities have
been documented in a wide range of organisms, from bacteria to
specialized vertebrate tissues, and suggest that the responses, with
species-specific adjustments, utilize common cellular defense programs
that balance water deficit and ion excess.
In yeast, many components underlying the signaling pathways that
control these biochemical entities are known (4, 8-12). Most
information is available about signal transduction-altering carbohydrate metabolism, where
MAPK1 phosphorelays,
exemplified by protein kinase Hog1p (High
Osmolarity Glycerol), transmit osmotic changes
(13, 14) leading to the induction of transcription that activates
downstream biochemical functions (10, 15-18). Activation of Hog1p
constitutes an early phase of the salinity stress response, which then
seems to diverge into different pathways (18-20). One pathway is
mediated by the transcription factors Msn2p/Msn4p binding to stress
response elements (STREs) and acting in signal amplification (21-23).
Hot1p, another transcriptional activator, has recently been identified
as a Hog1p partner in this signaling (12, 20). In addition, the
derepression of genes, for example through the regulated action of
Sko1p, seems to add another level of complexity controlling the
salinity stress response machinery (24, 25). Partially interacting with
HOG-based signal transduction is a pathway associated with the action
of the protein phosphatase calcineurin, a mediator for many cellular responses to calcium signals (8, 11, 26). The calcineurin pathway
responds predominantly to challenges in the ionic environment.
The rationale for focusing on transcriptional reactions of yeast to
salt stress through a genome-wide expression analysis is based on our
interest in plant salinity stress responses (2, 6, 27). Similar abiotic
stress-induced gene expression programs seem to exist in yeasts and
plants, including conserved signaling pathways and biochemical defense
determinants (28, 29). Comparative studies promise to reveal the
similarities and distinctions between cell-specific and organismal
components involved in tolerance acquisition. An experimental outline
that describes the portion of the yeast genome required for osmotic
stress tolerance will aid in delineating the conserved cellular
functions of homologous elements in multicellular organisms.
Yeast microarrays provide information about the transcription of all
genes. Genome-wide monitoring of transcript changes in yeast could show
previously unrecognized cellular aspects of stress protection and
reveal genes that represent a yeast-specific solution to survival in
high sodium. Such studies with yeast have recently become available
(25, 30). The three times replicated time-course experiments reported
here add to these analyses. One novel aspect is the description of a
succession of biochemical categories that are progressively
up-regulated. Early stress responses, affecting mainly nucleotide and
protein biosynthetic pathways, are different from later responses,
which included intracellular protein and metabolite transport
activities and increased energy consumption for metabolic and ion
homeostasis. Transcription after prolonged stress also exemplifies
ascending functions in cell rescue, in aging (cell death) and
defense-related roles, and reveals a large number of functionally
unknown ORFs.
A yeast genome array, 6144 coding regions deposited on nylon filters,
was used for a complete analysis of changes in transcript expression
following hyper-osmotic stress. The results confirm many of the ORFs
and proteins previously reported as stress-regulated (3, 10, 15, 23)
and adds a number of stress-regulated transcripts that had not been
recognized before. We describe early response transcripts distinguished
from those that act at later times in different functional categories
that seem to maintain cell integrity. We identify a set of ~200 salt
stress-regulated functionally unknown ORFs. Some of the unknown ORFs
have orthologs in cDNA libraries from salt-stressed plants.
Our results complement and extend through the use of a salt
stress-sensitive mutant, which is unable to synthesize glycerol, recent
reports that have targeted yeast salinity stress responses though the
analysis of arrayed ORFs (25, 30).
Yeast Strains, Growth, and Stress
Conditions--
Saccharomyces cerevisiae strain S150-2B
(MAT Yeast Gene Filters--
Yeast Gene Filters (Research
Genetics Inc.) contain 6144 PCR products bound to nylon filters
(available at the Research Genetics web site). The size of the
PCR products ranged from 300 bp to 4 kb. The filters are missing ~300
PCR products from chromosome 16. Average changes in transcript
abundance for each time point were calculated relative to the "no
salt" control using "Pathways" software (also available at the
Research Genetics web site). Approximately 30% of the ORFs whose
hybridization signal varied at background levels under all experimental
conditions were eliminated from the final analysis. The filters were
used only once to avoid variations caused by unequal stripping of probe
or DNA from the membranes. ORFs with (partially) overlapping reading
frames are identified in the supplemental material. Averaging reduced
the number for absolute fold induction but the spread over all
hybridizations indicated that a 2-fold induction was significant.
Preparation of mRNA and Hybridizations--
Total RNA was
isolated from frozen cell pellets by extraction with hot acidic phenol
(32). Complementary DNA was prepared using oligo(dT) primers and 5 µg
of total RNA labeled with [ Microarray Analysis--
Phosphorimages of the yeast microarray
filters were captured with a resolution of 50 µm on a Storm
PhosphorImager (Molecular Dynamics Inc.) and analyzed using Pathways
software, version 2.01, which provided a 16-bit imaging capability
(Research Genetics Inc.). Normalization between sets of filters was
based on the average of the signal intensities of all the data points
on the individual filters. Comparisons for each of the experimental
conditions (10, 30, and 90 min of NaCl stress) were calculated relative
to the no-stress control. The relative fold changes in transcript abundance for 10, 30, and 90 min of salt stress represent the average
changes in gene expression for three experiments each. We considered
expression levels >2.0-fold as induced, <2.0-fold as repressed, and
between Northern Blots and Densitometry--
Total RNA was isolated from
mid-log phase (A600 = 1.0) yeast cultures after
0, 10, 30, and 90 min of exposure to 1 M NaCl. Probes were
made by PCR amplification of specific ORFs from genomic DNA and random
primer-labeled with [ Microarray Evaluation--
Global gene expression during salinity
stress was determined using Yeast Gene Filters (Research Genetics
Inc.), assembled from PCR-amplified open reading frames (ORFs) for 95%
of the yeast putative and confirmed genes. Yeast cultures were grown to
mid-log phase in complete media supplied with 1 M NaCl for
0, 10, 30, and 90 min. Exploratory experiments at different time
points, which have been performed only once, are not included. Compared with previous microarray experiments (25, 30), the lag phase for
recovery of growth in our experiments was ~2 times longer. Each
hybridization was repeated three times. The [
High salinity affected expression levels of ~10% of the yeast ORFs.
During salt stress the number of ORFs induced increased more than
2-fold from 107 (10 min) to 354 (90 min) (Fig. 1). After 10 and 30 min,
27 and 78 ORFs, respectively, were induced more than 3-fold, and an
additional 87 (10 min) and 165 (30 min) ORFs had average changes in
transcript levels between 2- and 3-fold. After 90 min of salt stress,
170 ORFs (>3-fold) and 185 ORFs (2- to 3-fold) showed increased
average changes in transcript abundance. The up-regulated ORFs in major
MIPS categories are shown (Fig. 2). The
nature of regulated transcripts over time changed, suggesting that
different functions needed to be activated at different time points. As
a control, 39 ORFs were examined (Fig. 3,
not all data included) by RNA blot analysis, to independently verify
changes for transcripts in different abundance categories. Open reading frames were chosen with varying levels of transcript abundance in
microarrays; 11 ORFs > 3.0 fold, 13 ORFs > 2.0- to
3.0-fold, 7 ORFs with no change in message levels, and 8 with decreases of more than 2-fold. ImageQuaNT software was used to determine changes
in transcript levels in Northern hybridizations, which were compared
with the average changes in gene expression from the microarrays. Among
the selected ORFs, 36 of 39 agreed with the microarray data (3 ORFs
predicted to be up-regulated by a factor of less than 2 showed no
change in RNA blot hybridizations) (Fig. 3). Overall, low and moderate
changes in transcript abundance in the comparison between the RNA blots
and the averaged microarray data differed by less than 2-fold, but
large changes in abundance can differ by >10-fold, mainly attributable
to low basal transcript levels (e.g. YGL037C, YGR243W, and
YHR087W).
Correlation with known Yeast Stress Responses--
The behavior of
many transcripts in the analyses correlated with known biochemical
hyper-osmotic stress responses. Glycerol, for example, an
osmoprotectant known to accumulate rapidly in response to stress in
yeast (31), accumulated as documented by high pressure liquid
chromatography analyses (data not shown), and transcripts in the
glycerol biosynthetic pathway increased. Indeed, dehydrogenases and
phosphatases leading to glycerol production, GPD1/2, GPP1/2, were
up-regulated at all time points during salinity stress, most strongly
as time progressed (Fig. 4).
Similarly, ORFs for enzymes involved in trehalose metabolism,
GLK1, PGM2, HXK1, YKL035W,
TPS1, TPS2, and NTH1, were
up-regulated at 90 min, but not at 10 and 30 min of salt stress.
Transcripts for all enzymes of the pathway were among those most highly
induced (Fig. 4). Trehalose, like glycerol, is implicated in yeast
stress responses as an osmoprotectant, although trehalose does not
accumulate to osmotically significant concentrations in salt-stressed
bakers' yeast (34). PGM2, UGP1, TPS1, TPS2, and the regulatory factor encoded by TSL1, catalyze trehalose biosynthesis, whereas
Nth1p and Ath1p (trehalases) lead to trehalose
degradation and the formation of glucose (35). Completion of this cycle
seems to be indicated by the up-regulated transcripts for the
kinases HXK1 and GLK1 (Fig. 4). The presence of high transcript amounts
for Nth1p, Hxk1p, and Glk1p may explain why the osmoprotectant
trehalose does not accumulate during salt stress. A circular flux of
carbon, based on the induction of all ORFs in this pathway, seems to
indicate a function for trehalose in a regulatory role, for example in redox control, similar to what has been documented for the functions of
the two GPD enzymes (36, 58). Trehalose synthesis and degradation, in
combination with glycerol production, plays a key metabolic role in the
protection against high salinity. Such a conclusion, also based on gene
expression changes, has recently been put forward (58). The 1,4-glucan
branching enzyme involved in glycogen biosynthesis was only
moderately up-regulated under our conditions. These results are similar
to recently published data with the exception that the high NaCl
concentration, 1 M, tended to delay up-regulation compared
with what has been reported for the yeast transcriptome response in 0.4 M (for 10 and 20 min) or 0.7 M NaCl (45 min)
and 0.95 M sorbitol (30 min) (25, 30). At the lower sodium
concentration (0.4 M) nearly 1400 ORFs increased, most of
them transiently (30). The up-regulated ORFs shown in the study
by Posas et al. (30) (0.4 M NaCl, 10 and 20 min)
tended to be early-induced ORFs in our studies (see the supplemental
material). Induced ORFs reported by Rep et al. (25) at a
concentration of 0.7 M NaCl (45 min) are mostly found among
those ORFs up-regulated after 90 min in our experiments.
The extent to which transcript increases correlate with protein amount
has been verified in some studies. Apart from increases in the activity
of enzymes and the phenotype of knockout mutants, two-dimensional
electrophoresis of proteins and partial sequencing of up-regulated
peptides indicated general proportionality between RNA and protein
amounts for metabolic enzymes, and this also extended to the
down-regulation of, for example, enolases (ENO1/2) (Refs. 60-62; see
Supplemental Table sIII).
Induced Gene Expression during Hyper-osmotic Stress--
Global
gene expression patterns were determined for ORFs induced more than
2-fold after 10, 30, and 90 min of exposure to 1 M NaCl
(Fig. 2). Expression patterns for early-induced transcripts, at 10 and
30 min following stress, were similar, with 67% of the ORFs induced
after 10 min being identical to those induced after 30 min. The
profiles are characterized by rapid transcript increases for ORFs in
protein metabolism, mainly attributable to transcripts for components
of protein synthesis, protein destination, and the regulation of
protein fate. Nearly half of all up-regulated transcripts (42%; 44 ORFs) originated from the categories "protein destination" (ORFs
related to protein modification, transport, and targeting),
"intracellular transport" (cellular import, protein trafficking,
and vesicular transport), and "protein synthesis" (ribosomal
proteins). These three categories represented 13% (53 ORFs) of the
ORFs up-regulated after 90 min. Based on a relative scale, the
difference seems to indicate the significance of these ORFs for the
adjustment of metabolism early during the salt stress.
ORFs for Ribosomal Proteins--
Ribosomal proteins (RPO) account
for the majority of induced ORFs after 10 min (17 ORFs) and 30 min (45 ORFs), and the 17 ORFs found after 10 min are included in the group of
45 ORFs found at 30 min (Table I). This
is in contrast to the expression patterns of all ORFs for ribosomal
proteins at 90 min of salt stress when 93 of the 176 (55%) strongly
down-regulated ORFs encoded ribosomal proteins. At the late time point,
only three RPO were up-regulated, all of them encoding
mitochondrial RPO (MRP8, MRPS18, MET13). The number of transcripts
strongly repressed after 90 min included 31 of the early-induced
RPO (Table I). The up-regulated transcripts (30 min)
represented 22 subunit proteins of small and 22 subunit proteins of
large subunits of cytoplasmic ribosomes and, in addition, one subunit
protein for mitochondrial ribosomes (MRP8). In only six cases
(RPL1A/B, RPL19A/B, RPL34A/B,
RPS25A/B, RPS26/A/B, and RPS27A/B)
were both isoforms of a ribosomal protein up-regulated (possibly
because of cross-hybridization), whereas only one of two isoforms
showed increased transcript levels for the other RPO. Of the 137 yeast
cytoplasmic ribosomal proteins of which 59 are duplicated (37), the 44 up-regulated cytosolic RPO suggest specific promoter and
possibly also signaling events. Their up-regulation may also indicate a
requirement for the presence of specific RPO under stress
conditions. We have found a similar theme in the salt stress response
of rice (Oryza sativa), recorded by microarrays of 1728 transcripts. The early time points of salinity stress in rice lead
within 1 h to the up-regulation of a large number of rice
transcripts encoding plant cytosolic ribosomal proteins (38). Our
hybridizations with yeast distinguished isoforms at identity scores of
~90% at the 2-fold increase threshold that was chosen. Relaxing the
stringency to, for example, 1.6-fold increase would have included an
even larger number of Rpo transcripts. RPO
up-regulation has also been observed by Posas et al.
(30).
Other Up-regulated Functions--
Other differences in gene
expression between 10 and 30 min of salt stress were related to amino
acid and nucleotide biosynthesis. At 10 min, induced ORFs in the
categories amino acid (11.2%) and nucleotide metabolism (9.3%) are
higher relative to 30 min (6.2% and 3.3%, respectively). Strongly
up-regulated are 10 of 12 ORFs associated with amino acid biosynthesis
(YAL004w, THR4, HOM2, YER081w, PRS3, THR1, YIL074c, MET25,
GLN1, and LYS21). Five of 11 are related to nucleotide
metabolism: URA1, ADE1, PRS3, SHM2, and ADE13.
The earliest metabolic activities, it seems, in need of up-regulation
include amino acid biosynthesis and ribonucleotide metabolism. Between
10 and 30 min the transcriptional and translational machineries
generate, modify, and transport proteins within the cell, and this
process seems to be completed by 90 min. At that time, ORFs involved in
protein synthesis have declined precipitously, and the majority of gene
expression changes now target different functions, such as chaperone
and detoxification increases and intracellular transport.
The category "energy supply" showed a peak of induction in the
number of regulated transcripts, specifically in the subcategories respiration and the metabolism of energy reserves (Fig. 2 and supplemental material). After 30 min of salt stress, 9.1% of the induced ORFs are components of the electron transport chain, such as
cytochromes, or subunits of the ATP synthase complex, with the
comparable percentages of induced ORFs, 5.6% and 4.0% after 10 and 90 min, respectively.
Signal Transduction Components--
Transcriptional regulation
under hyper-osmotic conditions by the HOG1 protein kinase and the CNB1
protein phosphatase (calcineurin) has been well documented (10, 15, 17,
39, 40). Especially well studied is the signaling pathway terminating
at HOG1, with the sensors Sln1p and Sho1p converging in the MAPK
cascade from Ssk2/22p through Pbs2p to Hog1p (10, 18, 24, 41-43).
Additional evidence for genes regulated downstream of HOG1
and the transcriptional activators MSN2/MSN4 has
recently been provided in microarray experiments comparing wild type
and deletion mutants of HOG1 and MSN2/MSN4 (25,
30). Our results indicated that calcineurin was induced during the 10- and 30-min time points, but HOG1 was not found among the
significantly up-regulated transcripts. Several other protein kinases,
phosphatases, and several transcription factors were consistently
up-regulated. These included ORFs with established functions in cell
signaling (MFA1, SRA3, and HAC1), cell
cycle control and mitosis (PPH22, PHO85, PPH21, and
CIN5), and mRNA transcription (PHO4, PHO85, GCN4,
CUP2, TIS11, YHR056C, and YER130C).
Functions in Cell Defense--
Not surprisingly, the number of
ORFs in the categories cell rescue, defense, cell death, and aging
increased during the stress (Table II).
These included the ORFs for glycerol and trehalose production, and five
genes involved in cellular detoxification (GRX1 and
TTR1 (glutaredoxin), the duplicated CUP1A/CUP1B
(metallothionein), and CCP1 (cytochrome-c
peroxidase precursor)). In the case of CUP1A/B, their high
sequence homology led to cross-hybridization and only one of the genes
may be up-regulated. YGP1 (gp37, glycoprotein secreted in
response to nutrient limitation) is also induced, although its function
during salt stress is not clear. It may be associated with cell wall
biogenesis, because it is 47% homologous to SPS100, which
is involved in cell wall formation (44).
The percentages of ORFs related to the defense response (Fig. 2) are
10% (10 min), 5% (30 min), and 7% (90 min). The lower percentage at
30 min relative to other times can be attributed to heat shock proteins
(HSPs) (Table II), which have been reported to be up-regulated in a
variety of stresses such as oxidative stress
(H2O2), methyl methanesulfonate, and heat shock
(45-47). Four HSPs (SSA1, HSC82, YGL128C, and
YDR033W) are induced within 10 min; one HSP
(HSP12) after 30 min, and nine HSPs after 90 min of exposure
to salt (HSP12, YRO2, HSP26, SSE2, HSP78, SSA4, MDJ1, HSP104, and DDR48). Only HSP12 was induced during two
time points (30 and 90 min). The groups of HSPs induced during 10 and
90 min are distinct, indicating that different sets of HSPs could have different functional targets in the early and later responses to
salinity stress. Also, the majority (5 of 6) of induced genes involved
in DNA repair (Table II) showed increased transcript amounts only
during the later stress response (90 min). These included HSP12,
FUN30, HEL1, THI4, and RNR4.
Transport Functions--
During the early response to
salinity stress, few induced ORFs were observed in the categories of
the major facilitator superfamily (MFS) (three ORFs), nitrogen and
sulfur metabolism (one ORF), and in lipid, fatty acid, and isoprenoid
metabolism (six ORFs) (Fig. 2). This changed during the 90-min time
point: 17 up-regulated ORFs encode MFS transport proteins, and 9 and 16 induced ORFs were involved in nitrogen and sulfur metabolism and
in lipid, fatty acid, and isoprenoid metabolism, respectively (Table
III). Members of the MFS are
characterized as permeases with 12 transmembrane domains. Many proteins
in this class act as sugar, amino acid, or multidrug transporters (49).
Five of the 17 induced MFS genes have been characterized: the iron
transporter, FET3; the amino acid transporters
FUR4, PUT4, and BAP2; and the low
affinity hexose transporter, HXT1. It remains to be seen
whether the 12 unknown MFS transcripts are translated into
membrane-associated proteins, and if their induction is
salt-specific.
Among the ion transporters known to play a role in sodium
detoxification (3, 48, 50, 51), several transcripts were up-regulated.
The Na-ATPases, ENA1-4, were induced after 30 min of salt stress.
Vacuolar H+-ATPase subunits were induced during all time
points, and the H+-ATPase PMA1 was not induced (Fig. 2, and
supplemental information). It is difficult to determine metabolic
functions in stressed cells for the induced ORFs relative to nitrogen,
sulfur, lipid, fatty acid, and isoprenoid metabolism. However, 12 ORFs
related to nitrogen and sulfur metabolism were found to be up-regulated
following a different stress, MMS (47), and four of these
are also found during 90 min of salt stress (Table IV): NPR1
(serine/threonine kinase), and three
unknown ORFs, YFL030W (similar
to transaminases) and YPL135W and YOR226C
(both similar to proteins involved in nitrogen fixation).
We also analyzed the regulation of stress response genes in a group of
84 ORFs, which include at least two STREs in their 5'-upstream regions
(23). A subset of these genes is affected by the
Hog1p-dependent signal transduction pathway (10, 25, 30).
Transcripts for 34 ORFs containing multiple STREs were up-regulated
with the majority induced after 90 min of salt stress: 3 (10 min), 8 (30 min), and 30 ORFs (90 min) (see the supplemental material).
Magnification of the Stress Response in a
In addition, another 34 ORFs were induced for which no
function is known (or where the putative function has not
experimentally been verified). At least 11 of the ORFs strongly
up-regulated in the mutant are functionally associated with
mitochondria, possibly indicating a function in aging or cell death.
Also, Functionally Unknown Salinity-regulated ORFs--
Unclassified,
putative, and unconfirmed ORFs represent ~40% of the total yeast
genome. Many of these were induced by the stress: 16% (17 ORFs) of all
induced transcripts at 10 min,
24%2 at 30 min, and 42%
(149) at 90 min of salt stress (Fig. 2). Reasons for a gradual increase
of functionally ORFs not being studied are that long-term stress
experiments have been few and that these ORFs may only be expressed
under severe stress conditions. The program for the systematic
elimination and analysis of ORFs (53) has already produced data (a data
base of protein-protein interactions is available on the Web from the
University of Washington Departments Web Server). None of these
knockout mutants show a clear phenotype under salt stress conditions
(streaking and analysis for survival after long-term growth)
(59).3 However, several of
the KO strains show phenotypes that are related to stresses, for
example, the addition of 0.03% SDS or hygromycin, indicating that
these ORFs could have a function in cell wall synthesis or integrity
(Ref. 59; see also the Yale Genome Analysis Center Web site).
A Comparison of Yeast and Plant Salinity Stress
Responses--
Comparisons of unclassified yeast ORF expression
profiles to gene expression changes among unknown genes from other
organisms such as bacteria and plants will provide clues to the
functions of the unknown yeast ORFs. Table
V lists ORFs with unknown function in
yeast for which homologous plant ESTs have been found. As indicated, several of the plant homologs are significantly up-regulated at different time points during stress. A comparison is made with ESTs
from a salt-tolerant plant, Mesembryanthemum crystallinum (ice plant), including ESTs that show significant homology to functionally unknown yeast ORFs or questionable ORFs.2 In
addition, more than 50 yeast genes for which functions are known are
also up-regulated in salt-stressed plants where a similar progression
of different up-regulated functions has been observed (38).3 Several of the plant homologs identified ORFs termed
hypothetical in yeast and exhibited similarities to putative
serine/threonine-type protein kinases. Yeast knockout strains that
eliminated these ORFs did, however, not show a clear phenotype under
salt stress conditions.3
Salinity Tolerance: A Progression of Programs?--
We have
catalogued changes in gene expression in yeast over time following a
severe salt shock, which affects growth significantly. Determining
number and nature of induced transcripts and the changes in time of
transcript categories allowed for several conclusions. The profile of
the genes expressed is similar at the early times (10 and 30 min), but
changes later (90 min) are considerable. The evidence points toward the
initiation of protein synthesis and restructuring of protein
composition as a major task for the initial period (ribosomal proteins,
transcription machinery, amino acid synthesis, and utilization).
Another part of the early response is the induction of chaperone-type
proteins. Only early on are a few components of signaling pathways
found up-regulated; we think that more of these components could be
induced at even earlier time points or under less severe stress.
To place the information into context, we indicate four response types
that allow for clear statements. 1) A few transcripts are rapidly and
strongly up-regulated early. The most dramatically early up-regulated
ORF (YDR276c) encodes PMP3/SNA1. Deletion of PMP3 confers hyper-sensitivity to sodium in mutants that
lack sodium efflux systems (e.g. Pmr2p/Enap and Nha1p). A
function for Pmp3p has recently been documented in the control of
membrane polarization, such that its deletion leads to membrane
hyper-polarization and increased influx of monovalent cations (56). A
plant (Arabidopsis thaliana) PMP3 homolog complements this
phenotype. PMP3 homologues, which are salt
stress-dependently up-regulated in microarray analyses (57), have also been found in the halophytic plant
Mesembryanthemum crystallinum (27, 57) and in salt-stressed
rice (38). PMP3 transcript induction in our experiments changed from an
early induction (17-fold at 10 min, 14-fold at 30 min) to 2-fold
induction at 90 min. 2) Ribosomal proteins and several other functions
exemplify the second response type, gradual up-regulation to a peak at
30 min followed by the later repression of all RPOs with the exception of a few mitochondrial proteins. Among those up-regulated, some if them
transiently, in the second phase are also components for mitochondrial
functioning, glycolysis, transcription, inter-compartmental protein
transport, and protein turnover. 3) The third type comprises ORFs
unaffected or down-regulated early and gradually induced later. These
included many known yeast salinity stress responses. ORFs in this
category have also been reported as up-regulated in two previous array
analyses (25, 30) with few cases where the induction was close to the
cutoff value of 2-fold (see supplemental materials). The increases
reported in previous studies are generally higher than the fold
increases found in our experiments, but all three data sets show the
same trend (25, 30). What might be up-regulated 50-fold or 10-fold in
previous reports is shown in our analyses to have an averaged induction
of 20- to 5-fold (see Supplementary Table sI). In addition to these
ORFs (25, 30), our analyses detected other strongly up-regulated
transcripts. A secreted glycoprotein (YGP1;
YNL160w) for example, reported as 3-fold up-regulated (25),
was 26-fold up-regulated in our experiments. ARE2 (acyl-CoA sterol
acyltransferase) is strongly up-regulated as are a putative resistance
protein (YOR273c), PEP4 (aspartyl protease), ERG5 (C-22 sterol
desaturase), YLL028c (similarity to multidrug resistance proteins),
GYP6 (GTPase-activating protein), or the CUP1A/1B metallothioneins.
These have not been reported before (25, 30), indicating strain
differences or reflecting the different stress conditions. 4) The
fourth response type, consistently up-regulated transcripts, falls in
many different functional categories. Each of the four response
categories includes up-regulated functionally unknown, putative, and/or
questionable ORFs with the absolute numbers increasing at the 90-min
time point (Fig. 2).
Among the late-induced ORFs, highly expressed transcripts identify
membrane transporters, cell detoxification functions, and a number of
unclassified ORFs. Plant ESTs exist with homology not only to
functionally identified transcripts but also to ORFs whose functions
remain to be determined. Resources that will aid in deciphering the
roles of these unknown ORFs include the generation and availability of
yeast deletion strains (e.g. Ref. 53), programs for protein
function determination by protein-protein interactions (54) or in
computation and the microarray expression data from related
organisms such as other fungi, bacteria, and plants. The advantage
provided by the three time points chosen in connection with a strong
salt shock seems to lie in a drawn-out stress response that allowed for
a separation of successively engaged response pathways. This succession
identifies early, strongly up-regulated functions and intermediate and
late functions. The analysis presented here does not, however, include
the equally numerous down-regulated functions, which provide additional
information about the effects of stress and the responses by which
tolerance can be achieved. Further in-depth analyses, including
additional time points, different salt stress treatments or other
abiotic stress factors, and additional mutant strains will be necessary
for a complete understanding of the yeast stress response. Our results
extend the reported data (25, 30) through the inclusion of several time
points under severe stress conditions and integrates immediate cellular responses with long term metabolic and physiological events that seem
to assure survival. The kinetics of this response may serve as a
paradigm against which plant salinity stress responses could be scaled.
gpd1/gpd2, lacking
glycerol biosynthesis, the stress response was magnified with a
partially different set of up-regulated ORFs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
ura3-52 his3
leu2-3,112
trp1-289) was grown at 30 °C to mid-log phase
(A600 = 1.0) in standard rich media, YPD
(1% yeast extract/2% peptone/2% glucose, pH 5.5). Control (no salt)
cultures were harvested immediately, and salt-stressed yeast cultures
were harvested after 10, 30, and 90 min. To salt stress the cells, an
equal volume of YPD containing 2 M NaCl was added directly
to the yeast cultures. Cell count, optical density measurements, and
streaking of cells on non-selective media during the stress experiments
indicated a decline in cell numbers by ~25% during the 10-min time
point but cell number remained constant thereafter and increased after
~4 h of stress. Cells were collected by centrifugation at 3000 rpm
for 5 min and rapidly washed once in sterile water, and the cell pellet
was frozen and stored at
70 °C until RNA extraction. The
experiment was repeated three times with independently grown cultures.
As an additional control, yeast strain W3031A (MAT
leu2-3,112 ura 3-1 trp1-1 his3-11,15 ade2,1 can1-100 SUC2 GAL
mal0 GPD1::URA3
GPD2::TRP1), which is defective in glycerol
biosynthesis (kindly provided by Dr. S. Hohmann, Gøeteborg, Sweden),
was grown to mid-log phase in rich medium and stressed with 0.5 M NaCl for 60 min (31).
-33P]dCTP and purified
through a QIAquick column (Qiagen Inc.). The cDNAs (three for each
time point) were hybridized to 12 individual sets of gene filters.
Detailed experimental procedures for treatment of the filters can be
found at the Research Genetics web site.
1.9- and +1.9-fold as unchanged. Induced ORFs were
categorized based on the MIPS classification of yeast ORFs (available
on the Web). Approximately 30% of the 6144 signal intensities were
removed from the analysis, because they were equal to background
intensities under all experimental conditions. The complete data sets
can be found at the Research Genetics Web site.
-32P]dCTP. Probes were chosen
based on the microarray data. We chose seven ORFs with no change in
gene expression and 32 ORFs with induced gene expression (see the
Stress Genomics Web site for a list). Standard procedures for RNA blot
hybridizations were followed (33). Phosphorimaging analyses of the RNA
hybridizations were created on the Storm PhosphorImager, and
densitometry was calculated using ImageQuaNT software (Molecular
Dynamics Inc.). Statistical analyses used standard commercial software programs.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-33P]dCTP
signal intensities for all hybridizations ranged from background levels
(3-65 dpm) to high intensity (~72,000 dpm). The most highly expressed transcripts for both stressed and unstressed yeast
represented ~10% of the yeast genome with an average signal
intensity value of 4500 dpm, whereas 60% of the transcripts had an
average signal intensity value of 169 dpm (Fig.
1). Standard deviations, which included
all hybridization were within ± 1.5 S.D. for the most highly
expressed transcripts (614 ORFs) and slightly lower for transcripts
expressed in the medium to low range (~1000 ORFs).
View larger version (41K):
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Fig. 1.
Intensity of 33P incorporation
for 6144 S. cerevisiae ORFs. The Pathways
software "single filter analysis application" was used to obtain
intensity values for each ORF. Intensity values in the range from 3 to
72,000 dpm are representative of 12 individual filters used in the
experiments.
View larger version (40K):
[in a new window]
Fig. 2.
Gene expression profiles in functional
categories. The classification is based on the MIPS data base
available on the Web.
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[in a new window]
Fig. 3.
Comparison of RNA blot hybridizations and
microarray expression data. RNA blots (12 are shown) for 39 ORFs
were done in triplicate using RNA isolated from control ( ) and
salt-stressed yeast (+) exposed for 90 min to 1 M NaCl. The
RNA blot data (N) were generated using ImageQuaNT software
and represent average changes in signals comparing stress to control.
The probes were generated by PCR amplification of entire ORFs from
genomic DNA and by random primer labeling with
[
-33P]dCTP. The average microarray data (M)
represent the average of three experiments (no salt versus
90-min salt stress), and standard deviation (S) was
calculated using the "nonbiased" or "n
1"
method. The results for 3 of 39 ORFs did not agree (YDR387c
(ITR1-like), YHR048w (unknown), YFR017c (unknown)), because expression
at one of the time points was at background level and could not be
measured accurately.
View larger version (22K):
[in a new window]
Fig. 4.
Regulation of transcripts in trehalose and
glycerol biosynthesis, acetate production, and the trichloroacetic acid
cycle. Gene names are presented in boxes with fold
up-regulation indicated. For three of the four up-regulated coding
regions for trichloroacetic acid cycle enzymes the fold regulation is
shown for all three time points.
Yeast ribosomal protein genes induced at 10 and 30 min (>2.0-fold) and
repressed at 90 min (<2.0-fold)
Listing of upregulated ORFs in the stress response categories "Cell
Rescue, Defense, Cell Death and Aging"
ORFs induced more than 2-fold during 90 min of salinity stress in the
categories "MFS, Nitrogen/Sulfur Metabolism and in Lipid, fatty Acid,
and Isoprenoid Biosynthesis"
Transcripts in yeast gpd1/gpd2 induced higher than in wild type
gdp1/2 after 60 min in 0.5 M
NaCl, including the ~200 most highly induced ORFs, in comparison to
transcripts upregulated at 10, 30, and 90 min in wild type.
gdp1,
gdp2
Strain--
As a control to test filter reliability, hybridizations
used RNA from a mutant strain, gpd1
gpd2
(31), which is deficient in glycerol production. The GPD1 and GPD2
transcripts were not detectable (not shown). The inability of these
cells to osmotically adjust to high salinity also affected a number of
other transcripts. The
gpd1/2 strain, when
grown for 60 min in 0.5 M NaCl, showed a significantly
different transcript induction profile compared with wild type grown in
1 M NaCl (Table IV), a concentration that is lethal for
this mutant. Up-regulated transcripts were mostly comparable to the
90-min time point in wild type in 108 of highly induced transcripts
(see the supplemental material), including most of the metabolic
functions. Up-regulated (>2-fold) in the deletion mutant, but not in
wild type, were another 87 transcripts (Table IV). Most conspicuous
were transcripts for components of retrotransposons. Although one gene
related to retrotransposons (YOR344c) was among the
up-regulated ORFs in wild type, the number increased to 17 (including
YOR344c) in
gpd1/2. Cellular stress has been implicated as an agent that increases transposition
(e.g. Ref. 63). In yeast, FUS3, a mitogen-activated protein
kinase inhibits Ty1 retrotransposition (64). This transcript is
strongly down-regulated in
gpd1/2 (
3.0) but
unaffected by 1 M NaCl in wild type (see Supplemental Table sIV).
-factor receptor, mating pheromone
-2 factor, mating factor
, agglutinin, and transcripts for surface proteins increased.
Finally, an ORF with strong similarity to a protein kinase (Stl2p), and
ORFs for two transcription factor-like proteins (YLR256w, YKL109w
[HAP4]) were also newly up-regulated in the glycerol-deficient
mutant. The transcriptional behavior in this mutant supports a
connection between osmoregulatory and pheromone response pathways: The
cross talk in signaling to regulate growth and osmotic stress defense pathways reported before (e.g. Ref. 52). We can only
speculate that, in the absence of glycerol synthesis and accumulation
in this strain, the hyper-osmotic condition leads to a severe stress that affects cell integrity to a much larger extent than in the wild
type at even higher osmolarity.
Salinity stress-upregulated yeast ORFs with homology to
Mesembryanthemum cDNAs
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ACKNOWLEDGEMENTS |
---|
We thank Ariana Call, Chris Borchert, and Robert Hershoff for help and the people from Research Genetics for advice. We are indebted to Drs. Tracie Matsumoto, Mike Hasegawa (Purdue University), Carol Dieckmann (University of Arizona), and Rolf Prade and Patricia Ayoubi (Oklahoma State University) for discussions or help with annotations. Drs. Stefan Hohmann (University of Gøeteborg, Sweden) and Carol Dieckmann generously provided yeast strains and advice.
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FOOTNOTES |
---|
* This work was supported by Grant DBI-9813360 from the National Science Foundation, Plant Genome Initiative.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Tables sI-sIV.
To whom correspondence should be addressed: Dept. of Biochemistry,
University of Arizona, 1041 E. Lowell St. Tucson, AZ 85721-0088, USA.
Tel.: 520-621-7961; Fax: 520-621-1697; E-mail:
bohnerth@u.arizona.edu.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M008209200
2 C. B. Michaloski, S. Kawasaki, M. Deyholos, C. Borchert, S. Brazille, D. W. Galbraith, and H. J. Bohnert, unpublished data.
3 J. Yale and H. J. Bohnert, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; STRE, stress response element; EST, expressed sequence tag; HOG, high osmolarity glycerol; ORF, open reading frame; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase(s); GPD, glycerol-3-phosphate dehydrogerase; RPO, ribosomal protein; HSP, heat shock protein; MES, 4-morpholineethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.
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