|
Plant Physiol. (1999) 119: 1473-1482
A Selaginella lepidophylla Trehalose-6-Phosphate
Synthase Complements Growth and Stress-Tolerance Defects in a Yeast
tps1 Mutant1
Rodolfo Zentella2,
José O. Mascorro-Gallardo2,
Patrick Van Dijck,
Jorge Folch-Mallol,
Beatriz Bonini,
Christophe Van Vaeck,
Roberto Gaxiola,
Alejandra A. Covarrubias,
Jorge Nieto-Sotelo,
Johan M. Thevelein, and
Gabriel Iturriaga*
Departamento de Biología Molecular de Plantas, Instituto de
Biotecnología-Universidad Nacional Autónoma de
México, Avenida Universidad 2001, Colonia Chamilpa, 62210 Cuernavaca Morelos, Mexico (R.Z., J.O.M.-G., J.F.-M., R.G., A.A.C.,
J.N.-S., G.I.); and Laboratorium voor Moleculaire Celbiologie and
Vlaams Interuniversitair Instituut voor Biotechnologie, Katholieke
Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee,
Flanders, Belgium (P.V.D., B.B., C.V.V., J.M.T.)
 |
ABSTRACT |
The
accumulation of the disaccharide trehalose in anhydrobiotic organisms
allows them to survive severe environmental stress. A plant cDNA,
SlTPS1, encoding a 109-kD protein, was isolated from the
resurrection plant Selaginella lepidophylla, which
accumulates high levels of trehalose. Protein-sequence comparison
showed that SlTPS1 shares high similarity to trehalose-6-phosphate
synthase genes from prokaryotes and eukaryotes. SlTPS1
mRNA was constitutively expressed in S. lepidophylla.
DNA gel-blot analysis indicated that SlTPS1 is present
as a single-copy gene. Transformation of a Saccharomyces
cerevisiae tps1 mutant disrupted in the
ScTPS1 gene with S. lepidophylla SlTPS1
restored growth on fermentable sugars and the synthesis of trehalose at
high levels. Moreover, the SlTPS1 gene introduced into
the tps1 mutant was able to complement both
deficiencies: sensitivity to sublethal heat treatment at 39°C and
induced thermotolerance at 50°C. The osmosensitive phenotype of the
yeast tps1 mutant grown in NaCl and sorbitol was also restored by the SlTPS1 gene. Thus, SlTPS1 protein is a
functional plant homolog capable of sustaining trehalose biosynthesis
and could play a major role in stress tolerance in S. lepidophylla.
| |
INTRODUCTION |
An amazing adaptation that allows survival under complete
dehydration is present in yeast cells, fungal spores, certain
invertebrate species, and resurrection plants that resume their vital
functions as soon as they resume contact with water (Clegg, 1965 ; Gaff, 1971; Thevelein, 1984). These anhydrobiotic organisms also withstand strong vacuum, high doses of ionizing radiation, and extreme
temperatures without suffering damage. In addition, many of these
organisms accumulate the nonreducing disaccharide trehalose (Weisburd,
1988; Crowe et al., 1992).
Among several well-characterized osmoprotectors (Yancey et al., 1982),
trehalose seems to be one of the most efficient at maintaining lipids
in a fluid phase in the absence of water, thus avoiding phase
separation, leakage, and membrane fusion (Crowe et al., 1984, 1987).
Trehalose
( -D-glucopyranosyl-1,1--D-glucopyranoside), like other polyols, plays a key role in the structural and functional stabilization of membranes and proteins in the anhydrous state, apparently by means of water replacement of osmolyte molecules (Clegg,
1985) or formation of a glassy state (Burke, 1985).
The biosynthesis of trehalose consists of two enzymatic steps catalyzed
by the oligomeric subunits TPS, which synthesizes trehalose-6-P from
Glc-6-P and UDP-Glc, and TPP, which forms trehalose (Cabib and Leloir,
1958; Vandercammen et al., 1989; Londesborough and Vuorio, 1993). The
genes encoding both enzymes from bacteria and yeast have already been
isolated and sequenced (Luyten et al., 1993; Kaasen et al., 1994).
Deletion mutants in the trehalose pathway in these microorganisms cause
a reduction in osmotolerance (Giaever et al., 1988; Mackenzie et al.,
1988) and thermotolerance (Hengge-Aronis et al., 1991; De Virgilio et
al., 1994).
The yeast tps1 mutant and its alleles are unable to grow
in Glc as the sole carbon source. There is strong evidence to suggest that this defect is attributable to the additional role of the TPS1
subunit in regulating the flow of Glc into the cell (Van Aelst et al.,
1993; Neves et al., 1995; Thevelein and Hohmann, 1995; Hohmann et al.,
1996).
Here we report the isolation and molecular characterization of a
full-length cDNA encoding the enzyme TPS (SlTPS1) from the resurrection plant Selaginella lepidophylla, which is one of
the organisms that accumulates trehalose at higher levels (Adams et al., 1990; Müller et al., 1995). We show that SlTPS1
cDNA encodes a functional TPS able to restore growth in fermentable
sugars of a yeast tps1 mutant. Furthermore,
SlTPS1 synthesizes high levels of trehalose and complements
osmotolerance and thermotolerance deficiencies in a tps1
mutant.
| |
MATERIALS AND METHODS |
Plant Material
Selaginella lepidophylla (Hook. & Grev. Spring.) plants
were collected from arid regions of Morelos state in Mexico. Plants were rehydrated and maintained in controlled conditions (24°C and
16 h of light with an average of 50% humidity) in growth chambers (Conviron, Asheville, NC). Subsequently, S. lepidophylla
microphyll fronds were air dried at the indicated times by placing them
on 3MM filter paper (Whatman).
Strains
The cDNA bank was plated in the Escherichia coli strain
XL-1-Blue MRF , and the strain SOLR was used to excise the
pBluescript from the  phage, following the manufacturer's
instructions (Stratagene). The E. coli DH5 strain was
used to subclone and make constructs. The yeast strains were wild type,
W303-1A (Mat a leu2-3, 112 ura3-1, trp1-1, his3-11, 15 ade2-1,
can1-100, GAL, SUC2) (Thomas and
Rothstein, 1989); tps1, YSH290 (W303-1A,
ggs1/tps1::TRP1)
(Hohmann et al., 1993); tps2, YSH450 (W303-1A,
tps2::LEU2) (Neves et al., 1995); and tps1tps2, YSH652 (W303-1A,
ggs1/tps1::TRP1,
tps2::LEU2) (Neves et al., 1995).
Construction of the cDNA Bank of S. lepidophylla
An expression bank was prepared with mRNA isolated from S. lepidophylla microphylls dehydrated for 2.5 h using the ZAP
cDNA synthesis kit, the Uni-ZAP XR vector, and the Gigapack II Gold packaging extracts following the manufacturer's instructions
(Stratagene). The initial titer of the bank was 2 × 106 plaques of bacteriophage/mL and 1.5 × 1011 plaques of bacteriophage/mL after bank
amplification. After 4 × 105 recombinant
bacteriophages from the amplified cDNA bank of S. lepidophylla were plated, 13 plaques were obtained that hybridized with a mixture of the five oligonucleotides TPS5-1, TPS5-2, TPS5-3,
TPS3-1, and TPS3-2 (see Fig. 1A). In a second screening round, only 6 of the initial 13 plaques hybridized with a mixture of the
oligonucleotides TPS5-1 and TPS5-2. In a third step, pIBT6 was the
only clone that hybridized with the oligonucleotide TPS5-1,
corresponding to the most 5 end of the selected region for isolation
of the cDNA. Plaques were converted into plasmid by in vivo excision
according to the manufacturer's instructions (Stratagene). Plasmid DNA
was digested with EcoRI and XhoI to excise the
corresponding insert, transferred to a Hybond N+
nylon membrane (Amersham), and hybridized to oligonucleotides P labeled with T4 polynucleotide kinase.
View larger version (104K):
[in this window]
[in a new window]
| Figure 1.
Comparison of S. lepidophylla
SlTPS1 with other TPS protein sequences. A, Alignment of an internal
region of plant TPS amino acid sequences with bacteria, yeast, and
animal sequences. Gaps that were introduced to optimize the alignment
are indicated by dashes; identical residues are shaded. B, Dendrogram
of TPS sequences. Amino acid sequences of TPS from (top to bottom)
M. thermoautotrophicum (MtTPS1; accession no. AE000931);
E. coli (EcOtsA; accession no. X69160); D. melanogaster (DmTPS1; accession no. AC004373); A. thaliana (AtTPS1; accession no. Y08568); S. lepidophylla (SlTPS1; accession no. U96736); S. cerevisiae (ScTPS1; accession no. X68214); and A. thaliana (AtTPS4; accession no. Z97344). DmTPS1 and AtTPS4
expressed sequence tags were found in databases from the respective
systematic genome-sequencing programs. C, Diagram of TPS and TPP
protein sequences. A comparison of protein size and structure is shown
based on sequence-alignment data presented in A and compared with
E. coli (EcotsB) and S. cerevisiae
(ScTPS2) sequences. Protein size is shown in amino acid (aa) length. D,
The 5 leader sequence of the SlTPS1 gene is shown.
Three ATG triplets are present (boldface), but only the one close to
the 3 end is in frame with the open reading frame sequence; thus, it
is considered to be the putative initiation codon. The
NcoI restriction site is underlined.
|
|
The following degenerated oligonucleotides were synthesized for
screening of the cDNA bank: TPS5-1,
5-YTNTGGCCNBCNTTYCAYTAY-3; TPS5-2,
5-GGNTKBTTYYTNCAYAYNCCNTTYCC-3; TPS5-3,
5-MGNY-TNGAYTAYWBNAARGGNBTNCC-3; TPS3-1,
5-SWN-ACNARRTTCATNCCRTCNCK-3; and TPS3-2,
5-CCRW-ANTKNCCRTTDATNCKNCC-3 (single-letter abbreviations
for wobble nucleotides are B, C, G, or T; K, G or T; M, A or C; N, A or
C or G or T; R, A or G; S, C or G; W, A or T; D, A, G, or T; and Y, C
or T). These oligonucleotides were designed based on conserved domains
of TPS sequences (depicted in Fig. 1A) using the following
accession numbers: E. coli, X69160; Schizosaccharomyces pombe, Z29971; Aspergillus
niger, U07184; Saccharomyces cerevisiae, X68214; and
Kluyveromyces lactis, X72499.
Hybridization of Nucleic Acids
To screen the cDNA bank, the bacteriophage plaques were
transferred to a nylon membrane and the filter was hybridized with oligonucleotides and labeled with the 32P isotope
by means of T4 polynucleotide kinase using 6× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate) at 37°C.
The filter was washed three times at the same temperature for 10 min
each under the following conditions: 6× SSC, 4× SSC, and 2× SSC.
Southern- and northern-blot techniques were used according to standard
protocols (Sambrook et al., 1989) with the following modifications. For
the genomic Southern-blot analysis, the DNA was fractionated on a 0.8%
agarose gel in TBE (0.09 M Tris-borate and 0.002 M EDTA) buffer and transferred to a nylon membrane. The
filter was hybridized using SlTPS1 cDNA labeled with
32P isotope as a probe, using 2× SSC at 65°C.
The filter was washed three times at the same temperature for 20 min
each time under the following conditions: 2× SSC, 1× SSC, and 0.5×
SSC. For the northern-blot analysis, a 1.2% agarose gel was used in a
Mops-formaldehyde buffer and a Hybond N+ nylon
membrane was also used for the transfer. Hybridization conditions were
in 50% formamide and 2× SSC at 42°C. The three successive filter
washings were performed with 2× SSC, 2× SSC, and 1× SSC at 55°C.
DNA Sequencing
Nested deletions of the insert were created with the enzymes
exonuclease III and nuclease S1 from the selected clone to subsequently determine the nucleotide sequence of both strands using Sequenase version 2.0 (United States Biochemical). Protein sequence alignments were analyzed using the CLUSTAL W software program (Thompson et al.,
1994).
DNA Manipulation and Constructs
Recombinant DNA techniques such as bacterial transformation,
isolation of DNA from plasmid, bacteriophage, and labeling of
radioactive fragments were carried out according to standard procedures
(Sambrook et al., 1989). For expression in yeast, two new
shuttle-expression vectors named pSAL4 and pRS6 were constructed. pSAL4
is similar to pSAL1 (Mascorro-Gallardo et al., 1996), but it has the
pRS426 backbone (Christianson et al., 1992) with the 2 µ origin of
replication and the URA3 marker. The pRS6 vector was
constructed using the PMA1 gene promoter from the pRS699
vector (Serrano and Villalba, 1995), a polylinker, and the
CYC1 terminator. This cassette was cloned in the pRS423
backbone (Christianson et al., 1992) with the 2 µ origin of
replication and the HIS3 marker.
Two out-of-frame ATG triplets in SlTPS1 cDNA leader (see
Fig. 1D) were removed by digesting the pIBT6 clone with NcoI
and treatment with nuclease S1. The intact leader precludes expression of SlTPS1 in yeast (data not shown). The 3.2-kb SlTPS1 cDNA
was subcloned into pSAL4 or pRS6, and the resulting constructions were
designated pSTS1 and pRTS1, respectively. The
ScTPS1 and ScTPS2 yeast genes were isolated from
genomic yeast DNA by PCR using primers ScTPS1-5
(5-CCGCTCGAGGGTACTCACATACAGAC-3), ScTPS1-3 (5-ATAGTTTTGCGGCCGCATCGGGTTCATCAG-3), ScTPS2-5
(5-CCGCTCGAGCACTATTTCTGTGCCG-3), and ScTPS2-3
(5-CGGG-GTACCATGGTGGGTTGAGAC-3). Amplified fragments were cloned
into pSAL4 to give plasmids pSTS2 and pSTS3 or into pRS6 to give
plasmids pRTS2 and pRTS3. A truncated SlTPS1
(SlTPS1C) was constructed by deleting the DNA
sequence coding for the 431-amino acid C terminus. The
SlTPS1 region coding for the resting 563-amino acid N
terminus was amplified by PCR with primers SlDC400-5
(5-CATGCCATGGCTATGCCTCAGCCTTACC-3) and SlDC400-3
(5-CGGGGTACCTCACTTTGACTCCGAGTACTTTGC-3) with TAG termination codon. A
deletion of SlTPS1 (SlTPS1N)
comprising the 396-amino acid C terminus was constructed by PCR with
primer SlDN600-5 (5-CCGCTCGAGCCATGGTGCATATTCCGCCTCAATTGCC-3) and
universal primer (5-GTAAACGACGGCCAGT-3). Both
SlTPS1C and SlTPS1N
were amplified using pIBT6 as a template. The PCR products were
subcloned into pSAL4 vector to give plasmid pSTS4 or into pRS6 vector
to give plasmid pRTS4. PCR was conducted using the Expand High Fidelity PCR System (Boehringer Mannheim), and reaction conditions were 1 cycle
at 94°C for 3 min; 30 cycles at 94°C for 1 min, at 55°C for 1 min, and at 72°C for 2 min; and 1 cycle at 72°C for 10 min.
Transformation, Complementation, and Stress Assays in Yeast
Yeast was grown at 30°C in minimal medium (0.7% Bacto-yeast
nitrogen base without amino acids, pH 6.0, supplemented with 0.002% adenine, 0.002% His, 0.003% Leu, 0.003% Trp, and 0.002% uracil) plus 2% Gal. Transformation was performed as described previously (Elble, 1992), and transformants were selected in medium without uracil
for pSAL4 or in medium without His for pRS6. For each construct, at
least three independent transformants were chosen to test their ability
to restore the growth defect on minimal medium plus 2% Glc. The
candidates were streaked on agar plates with minimal medium with 2%
Gal, Glc, or Fru. As a control, the same strains were transformed with
the pSAL4 or the pRS6 vector alone. After 3 d, growth was visible
in positive control and complemented mutants.
For the thermotolerance assays, liquid cultures were grown at 25°C in
minimal medium plus 2% Gal up to the mid-log phase (4 × 106 colony-forming units/mL, corresponding
to 0.4 A600), then shifted to 39°C for
1 h for thermoinduction, and further incubated at 50°C for
different times to evaluate induced thermotolerance. Decimal dilutions
were plated in solid YPGal (2% Bacto-peptone, 1% yeast extract, 2%
Gal, and 2% agar) and grown at 25°C for 3 d before colony
counting. The level of thermotolerance was expressed as the ratio
(percentage) of viability after the 50°C treatment and immediately
before the 50°C treatment.
For the osmotolerance assays, exponential cultures were first
diluted until A600 was 0.1 and then
serially diluted 5-fold at each step. Spots were made with 4 µL of
each dilution and plated in solid YPGal supplemented with 0.9 M NaCl or 1.6 M sorbitol. Growth was recorded after 3 d for the control plates and after 4 d for the osmotic stress treatments.
Trehalose Determination
Essentially, yeast cells (25-50 mg fresh weight) were collected
by vacuum filtration through 0.22- to 0.45-µm membranes (Gelman Sciences, Ann Arbor, MI) and washed several times with ice-cold water
to remove external Glc. Yeast cells were scraped from the filter
membrane before being frozen in liquid nitrogen and stored at  80°C
or immediately transferred to a screw-capped tube containing 1 mL of
0.25 M Na2CO3
and boiled in a water bath for 20 min. After cooling, samples were
centrifuged in a microfuge and 0.2 mL of supernatant was mixed with 0.1 mL of 1 M acetic acid to neutralize before the addition of
0.1 mL of buffer T (0.3 M sodium acetate plus 0.03 M CaCl2, pH 5.5). For trehalose
quantification, 0.05 mL of samples or trehalose standards and 0.05 mL
of Humicola grisea trehalase (or water to determine Glc not
derived from trehalose) were incubated for 45 min at 40°C. One
milliliter of Tris-HCl buffer, pH 8.0, containing 100 units each of Glc
oxidase and peroxidase (Sigma) plus 0.1 mg of o-dianisidine
(Sigma) was added. Incubation was for 1 h at 30°C, and the
reaction was stopped with 0.5 mL of 56% (v/v) sulfuric acid. The
A546 was determined before 1 h. H. grisea trehalase was purified according to the
method of Neves et al. (1994).
| |
RESULTS |
Cloning and Sequence Analysis of S. lepidophylla SlTPS1
To isolate a gene involved in trehalose synthesis, a comparison of
the deduced amino acid sequences of TPS was made from the reported
nucleotide sequences from bacteria and yeast (see ``Materials and Methods''). Highly conserved regions were selected to synthesize
degenerated oligonucleotides (Fig. 1A),
which were used to screen a cDNA library from S. lepidophylla (see ``Materials and Methods''). The largest clone
isolated, pIBT6 (3.2 kb), contains a 110-nucleotide 5 leader sequence,
a 96-nucleotide 3-untranslated sequence, and an open reading frame
coding for a protein of 994 amino acids with a calculated molecular
mass of 109 kD; it was designated SlTPS1. The alignment of deduced amino acid sequences of TPS from bacteria, yeast, plants, and animals
showed several regions of homology (Fig. 1A). Only the most conserved
region in all of the compared TPS sequences is shown. These deduced
amino acid sequences were plotted in a dendrogram (Fig. 1B). The most
distant sequence from SlTPS1 was the archaebacterium Methanobacterium thermoautotrophicum MtTPS1, which is only
27% identical to S. lepidophylla SlTPS1. The E. coli EcOtsA and the Drosophila melanogaster DmTPS1
sequences shared 35% and 40% identity to SlTPS1, respectively. Plant
and yeast TPS sequences were found closely related to each other.
SlTPS1 is the most related to the recently reported AtTPS1 sequence
from Arabidopsis (Blázquez et al., 1998), and both sequences
share an identity of 67%, whereas the S. cerevisiae ScTPS1
is 50% identical to both SlTPS1 and AtTPS1. It is interesting that
another Arabidopsis TPS homolog, AtTPS4, is more closely related to
ScTPS1 (41% identity) than to SlTPS1 and AtTPS1 (37% identity).
AtTPS4 may represent a divergent TPS gene that probably
arose by duplication of the ScTPS1 ancestor gene.
An additional feature of plant TPS sequences is the presence of two
putative domains (Fig. 1C). Only the N-terminal sequence of SlTPS1,
which contains 563 amino acids, has similarity to TPS sequences (Fig.
1, A and B), whereas the remaining 431-amino acid sequence of SlTPS1,
which constitutes its C-terminal region, is also present in AtTPS1 and
AtTPS4 but is absent in yeast and E. coli TPS sequences
(Fig. 1C). This C-terminal region of SlTPS1 shows a certain degree of
identity to sequences encoding TPP: 29% to the yeast ScTPS2 subunit
and 22% to the E. coli EcOtsB enzyme.
Expression Pattern and Copy Number of
SlTPS1
RNA-blot analysis was performed to investigate the expression
pattern of the SlTPS1 gene. Poly(A+)
RNA was isolated from S. lepidophylla microphylls (lycophyte leaves) that were fully turgid or desiccated for different lengths of
time. SlTPS1 mRNA is expressed as a single band
corresponding to a 3.2-kb transcript present in fully hydrated and
dehydrated S. lepidophylla microphylls at similar levels
(Fig. 2). This constitutive expression of
the S1TPS1 gene is in agreement with the comparable levels
of trehalose in both nonstressed and desiccated S. lepidophylla plants (Adams et al., 1990).
View larger version (39K):
[in this window]
[in a new window]
| Figure 2.
Expression of SlTPS1 mRNA in
S. lepidophylla. Poly(A+) RNA (2 µg) was
extracted from fully hydrated (lane C) S. lepidophylla
microphylls or dehydrated for 2.5 h (lane 2.5), 5 h (lane 5),
or 1 year (lane Y). The RNA blot was hybridized with
32P-labeled SlTPS1 cDNA (top row). An rRNA
gene fragment was hybridized to the same filter as a loading control
(bottom row).
|
|
The copy number of the SlTPS1 gene was determined by DNA
gel-blot analysis. S. lepidophylla genomic DNA was digested
with EcoRI, which cuts internally, and BamHI and
XbaI, which do not cut the cDNA. After probing with the
full-length SlTPS1 insert, two expected bands were obtained
with EcoRI (Fig. 3). Digestion with XbaI corresponded to a single band, but two bands were
obtained using BamHI, suggesting either that there are two
SlTPS1 genes or that the BamHI site is present in
an intron. To test this latter possibility, we used specific
oligonucleotides matching the coding 5 and 3 ends of
SlTPS1 to amplify genomic homologs by PCR. A single fragment
was obtained that led to two bands after digestion with
BamHI, thus suggesting the possibility of a BamHI
site in an intron. Partial nucleotide sequence of these PCR fragments matched exactly the cDNA sequence (data not shown). Therefore, according to these data, SlTPS1 seems to be a single-copy
gene.
View larger version (18K):
[in this window]
[in a new window]
| Figure 3.
DNA gel-blot analysis of the SlTPS1
gene. Genomic DNA (20 µg) of S. lepidophylla was
digested with EcoRI (lane E), XbaI (lane
X), or BamHI (lane B). The filter was probed with
32P-labeled SlTPS1 cDNA. The positions of
the DNA markers are indicated on the left.
|
|
Functional Analysis of SlTPS1 in Yeast
To determine whether the SlTPS1 gene can complement the
physiological defects of mutant yeast cells devoid of trehalose
biosynthesis genes, the corresponding 3.2-kb cDNA was subcloned into
the yeast pSAL4 vector behind a CUP1 promoter that is
inducible by copper ions (Mascorro-Gallardo et al., 1996). The
resulting plasmid pSTS1 was used to transform the yeast
tps1 and tps1tps2 mutants. These mutants are unable to grow with Glc or Fru as the carbon source,
apparently because of the role of TPS1 in regulating the flow of Glc in
glycolysis (Van Aelst et al., 1993; Thevelein and Hohmann, 1995). After
transformation with pSTS1 containing the SlTPS1 gene, the
tps1tps2 mutant was able to grow in Glc in the presence of copper ions (Fig. 4),
whereas no growth was observed for the tps1 mutant. All
strains grew well in Gal, as expected. Transformation of the
tps1 mutant with plasmid pSTS2, which harbors the
homologous ScTPS1 gene, restored growth for both the
tps1 and tps1tps2 mutants
(Fig. 4). Trehalose levels were measured in the tps1 and
tps1tps2 mutants transformed with pSTS1
plasmid (containing SlTPS1) grown in different carbon
sources (Table I). Neither the
tps1 nor the tps1tps2 mutant
transformed with SlTPS1 accumulated trehalose above the
level detected for the same mutants transformed with the pSAL4 vector
alone. We decided to express SlTPS1 cDNA under the control
of a stronger promoter to observe a possible accumulation of trehalose.
Thus, the promoter of the PMA1 gene that encodes the
H+-ATPase (Serrano and Villalba, 1995) was
subcloned in a multicopy vector to give plasmid pRS6 (see ``Materials and Methods''). After SlTPS1 was subcloned under the
control of the PMA1 promoter (plasmid pRTS1), this
construction was used to transform the yeast tps1 mutant.
It was observed that the tps1 mutant transformed with
pRTS1 plasmid was able to grow in Glc (Fig.
5).
View larger version (95K):
[in this window]
[in a new window]
| Figure 4.
Complementation of the
tps1tps2 mutant by
SlTPS1 under the control of the CUP1
promoter. Yeast mutants lacking the TPS1 protein
(tps1) or both TPS1 and TPS2
(tps1tps2) were transformed with
pSTS1 (pSAL4 containing SlTPS1), pSTS2 (pSAL4 containing
ScTPS1), or pSAL4 vector alone and spread on 2% agar
plates in minimal medium without uracil (ura) supplemented with 2%
Gal (SGal), 2% Glc (SGlc), or 2% Glc with copper sulfate (SGlc +100
µM CuSO4). The wild-type control strain
W303-1A (WT) was also transformed with pSAL4.
|
|
View this table:
[in this window]
[in a new window]
|
Table I.
Trehalose content of the transformed
tps1 and tps1tps2 mutants
with pSAL4-derived vectors
Three independent transformants of wild-type yeast, the
tps1 mutant, or the tps1tps2
mutant transformed with pSAL4 vector alone or harboring the
SlTPS1, SlTPS1C, or ScTPS1 genes
were grown in minimal medium with the indicated carbon source plus 100 µM CuSO4. Trehalose content was determined in
the stationary phase (7.0 A600). Values are
means ± SD.
|
|
View larger version (50K):
[in this window]
[in a new window]
| Figure 5.
Complementation of the tps1
mutant by SlTPS1 under the control of the
PMA1 promoter. The yeast mutant lacking TPS1 protein
(tps1) was transformed with pRTS1 (pRS6 containing
SlTPS1), pRTS2 (pRS6 containing ScTPS1),
or pRS6 vector alone and spread on 2% agar plates in minimal medium
without His (his) supplemented with 2% Gal (SGal) or 2% Glc (SGlc).
The wild-type control strain W303-1A (WT) was also transformed with
pRS6.
|
|
The trehalose content was determined for tps1 mutant
strains transformed with the pRTS1 (containing SlTPS1) and
pRTS2 (harboring ScTPS1) plasmids. Trehalose was detected in
the tps1 mutant complemented with SlTPS1 grown
in different carbon sources (Table II).
The trehalose levels were higher when the tps1 mutant was
complemented with the S. cerevisiae ScTPS1 gene than when
SlTPS1 was used. These results indicate that heterologous
S. lepidophylla SlTPS1 is not completely fulfilling the
function of the yeast ScTPS1 protein, considering the sequence
divergence and the fact that the SlTPS1 polypeptide is larger than
ScTPS1. Therefore, we constructed a deletion mutant of
SlTPS1, named SlTPS1C, to remove
the sequence encoding its C-terminal domain of 431 amino acids, which
lacks similarity to ScTPS1 or EcOtsA (Fig. 1C). Plasmid pRTS4
containing SlTPS1C was used to transform the
tps1 and tps1tps2 mutants. Growth in Glc was complemented in both mutants by the C-terminal deletion of SlTPS1. When SlTPS1C
was cloned under control of the CUP1 promoter (plasmid
pSTS4), only partial complementation was observed in the
tps1tps2 mutant: Growth in Glc was slow compared with that in the same mutant transformed with the full-length SlTPS1 gene in pSAL4 (data not shown). Trehalose levels in
the tps1 mutant transformed with pRTS4 were 2.5 times
lower than the levels with the full-length SlTPS1 gene but
significantly higher than in the tps1 mutant transformed
with vector alone (Table II). These results show that the C-terminal
region of SlTPS1 is required for full activity.
View this table:
[in this window]
[in a new window]
|
Table II.
Trehalose content of the transformed
tps1 mutant with pRS6-derived vectors
Three independent transformants of wild-type yeast or the
tps1 mutant transformed with pRS6 vector alone or
harboring the SlTPS1, SlTPS1C, or
ScTPS1 genes were grown in minimal medium with the indicated
carbon source.
|
|
Given that the C-terminal region of SlTPS1 shares relative similarity
to TPPs (Fig. 1C), we tested the ability of full-length SlTPS1 and its C-terminal domain
(SlTPS1N) to complement the defect to grow at
37.5°C associated with the yeast tps2 and
tps1tps2 mutants. pRTS1 (containing
SlTPS1) and pRTS5 (harboring
SlTPS1N) were used to transform these mutants.
It is known that the tps2 mutant grows normally in Glc
but is unable to grow continuously at 37.5°C (De Virgilio et al.,
1993). The tps1tps2 mutant has both growth
defects. SlTPS1 was not able to restore the growth of the
tps2 and tps1tps2 mutants at
37.5°C. Transformation with pRTS3, which harbors the homologous
ScTPS2 gene, complemented both mutants (data not shown).
Trehalose is required for the acquisition of thermotolerance in yeast
and E. coli (Hengge-Aronis et al., 1991; De Virgilio et al.,
1994). The yeast tps1, tps2, and
tps1tps2 deletion mutants are deficient in
both induced and noninduced thermotolerance. The phenotypes are
restored by complementation with the corresponding homologous gene. To
determine whether the SlTPS1 gene can also complement these
deficient stress responses, the yeast tps1 mutant was
transformed with plasmid pRTS1 (harboring SlTPS1) or pRTS2 (containing ScTPS1). We assayed the ability to survive both
a sublethal heat shock of 39°C for 1 h (thermoinduction) and a
lethal heat shock of 50°C for 20 min after an acclimation treatment
of 39°C for 1 h (induced thermotolerance). The viability of the
tps1 mutant after thermoinduction decreased to 30%, but
almost complete viability was recovered after transformation with the
SlTPS1 or the ScTPS1 gene (Table
III). After lethal heat shock, S. lepidophylla SlTPS1 restored the induced
thermotolerance as effectively as yeast ScTPS1 in
tps1 mutant cells compared with the level in the
wild-type cells (Fig. 6). Both
SlTPS1- and ScTPS1-transformed cells displayed a
10-fold higher induced thermotolerance than the tps1
mutant cells transformed with the pRS6 vector alone after 20 min at
50°C.
View this table:
[in this window]
[in a new window]
|
Table III.
Viability of tps1 mutant cells
transformed with SlTPS1 gene after thermoinduction
Thermoinduction was performed by incubating yeast cells in liquid SGal
(-His) medium up to approximately 0.4 A600 at
25°C, and then shifted for 1 h at 39°C. Aliquots for colony
counting were taken just before and after thermoinduction. Data
represent the mean of three independent transformants and their
SD.
|
|
View larger version (19K):
[in this window]
[in a new window]
| Figure 6.
Complementation of thermotolerance deficiency of
the tps1 deletion mutant transformed with the
SlTPS1 gene. Transformed strains were grown in liquid
culture and assayed for induced thermotolerance (see ``Materials and Methods''). Values shown for each construct are the averages of three
independent transformants. WT, Wild-type cells transformed with the
pRS6 vector alone; tps1, mutant transformed with the
pRS6 vector alone; tps1+SlTPS1, mutant
transformed with plasmid pRTS1;
tps1+ScTPS1, mutant transformed with
plasmid pRTS2. Error bars denote ±SD.
|
|
Finally, it has been shown that the tps1 mutant is
osmosensitive (Hounsa et al., 1998). To determine whether
SlTPS1 could restore the osmotolerance defect of the
tps1 mutant, this strain was transformed with the
plasmids pRTS1 and pRTS2 and growth was evaluated under osmotic stress
conditions. The tps1 mutant transformed either with the
SlTPS1 or the ScTPS1 gene was able to grow in medium containing 1.6 M sorbitol or 0.9 M NaCl (Fig. 7). As
a positive control, we used the wild-type strain, which also grew normally. In contrast, the tps1 mutant transformed with
the vector alone was unable to grow in high-osmoticum conditions. All
of these results suggest that the levels of trehalose accumulated in
the tps1 mutant transformed with SlTPS1 are
sufficient to restore growth under osmotic and heat stress.
View larger version (27K):
[in this window]
[in a new window]
| Figure 7.
SlTPS1 restores the osmotolerance
defect of a yeast tps1 deletion mutant. Osmotolerance
assay of a tps1 deletion mutant transformed with the
SlTPS1 gene. Yeast mutants lacking the protein TPS1
(tps1) were transformed with pRTS1 (containing
SlTPS1), pRTS2 (containing ScTPS1), or
pRS6 vector alone and spotted on 2% agar plates in rich YPGal medium
supplemented with 0.9 M NaCl or 1.6 M sorbitol.
The wild-type control strain W303-1A (WT) was also transformed with
pRS6.
|
|
| |
DISCUSSION |
Trehalose is one of the most efficient osmoprotectors and
thermoprotectors in nature (Colaço et al., 1992; Crowe et al., 1992). Although much experimental work concerning trehalose has been
done in bacteria and yeast, little information has been reported from
animals or plants. Here we report the molecular and functional characterization of a full-length cDNA encoding a novel TPS (SlTPS1) from the resurrection plant S. lepidophylla. This plant is
known to accumulate trehalose at levels comparable to yeast and other fungi and at levels much higher than those found in other plants (Adams
et al., 1990; Müller et al., 1995). It is likely that a major
factor determining the anhydrobiotic ability of resurrection plants is
their capacity to accumulate high levels of osmoprotectors such as
trehalose. Until a few years ago, it was thought that only resurrection
plants had the capacity to synthesize trehalose (Müller et al.,
1995). Recently, the presence of trehalose was reported in tobacco,
potato (Goddijn et al., 1997), and rice (Garcia et al., 1997), but at
levels about 3000-fold less than in S. lepidophylla (Adams
et al., 1990). Although the actual level of trehalose in Arabidopsis
has not yet been reported, presumably it is not significant because
AtTPS1 mRNA is expressed at very low levels (Blázquez et al., 1998). Additionally, it is possible that the low levels of
trehalose in most higher plants could be the result of very active
trehalase. Nevertheless, when Goddijn et al. (1997) incubated tobacco
and potato plants in the presence of the trehalase inhibitor validamycin A, trehalose did not reach concentrations comparable to
those in a resurrection plant.
Another possibility is that substitutions in the amino acid sequence
that occurred during evolution in different plant TPS have an important
influence on trehalose synthesis among plant species.
In this work, we present evidence that the expression of the
SlTPS1 gene in the yeast tps1 mutant under
control of the strong PMA1 promoter leads to an
accumulation of trehalose in the stationary phase equivalent to 64% to
100% of the levels reached by wild-type yeast, depending on the carbon
source. Similarly, Blázquez et al. (1998) used the
PGK1 promoter to express AtTPS1, which resulted in 25% of the trehalose found in the wild-type yeast grown in Gal.
These results might be attributable to either a difference between
PGK1 and PMA1 promoter strength or an increased
capacity of SlTPS1 to synthesize trehalose. Therefore, it remains to be shown if the difference in the trehalose content between S. lepidophylla and nonresurrection plants is the result of
transcriptional control and/or balance between trehalase and TPS enzyme
activities.
Transformation of the yeast tps1 mutant with the
SlTPS1 gene under the control of a moderate promoter such as
CUP1 did not restore the ability of the wild-type phenotype
to grow in Glc, whereas the growth of the
tps1tps2 mutant was complemented. Nevertheless, trehalose levels in both the tps1 and
tps1tps2 mutants transformed with
SlTPS1 were negligible, thus suggesting that restoration of
growth in Glc of the tps1tps2 mutant by SlTPS1 is independent of the presence of trehalose. This
observation is interesting because SlTPS1 expressed in a
moderate promoter, such as CUP1, allows separate analyses of
both TPS functions, i.e. trehalose synthesis capacity and regulation of
Glc influx in glycolysis (Van Aelst et al., 1993; Thevelein and
Hohmann, 1995).
Moreover, the fact that tps1 was not complemented by the
SlTPS1 gene expressed under the control of the
CUP1 promoter led us to suggest that maybe SlTPS1 was
somehow inhibited by ScTPS2. One possible explanation for these results
is a negative interaction or sequestration of SlTPS1 by ScTPS2. In
yeast, ScTPS1, ScTPS2, ScTPS3, and ScTSL1 interact with each other to
form the holoenzyme complex (Reinders et al., 1997). Thus, given the
sequence similarity between yeast and plant TPS1, ScTPS2 and SlTPS1 may
interact with each other, resulting in a structural constraint of
SlTPS1 enzyme activity.
The SlTPS1 cDNA encodes a 109-kD protein that shares strong
similarity to TPS sequences and has a C-terminal extension with some
similarity to TPP. We tested whether SlTPS1 had TPP activity by transforming the yeast tps2 and
tps1tps2 mutants with the full-length
SlTPS1 gene or just its C-terminal region under the control
of the strong PMA1 promoter. However, the lack of
complementation for growth at 37.5°C suggested that SlTPS1 does not
have TPP activity. It has been shown that all TPP enzymes from bacteria
to plants have two short and well-conserved regions of homology (Vogel
et al., 1998). These sequences are absent in SlTPS1 and AtTPS1,
providing further evidence for the absence of TPP activity in SlTPS1.
In yeast, it is well established that trehalose is involved in acquired
thermotolerance and tolerance to continuous growth at sublethal
temperatures (De Virgilio et al., 1994; Elliot et al., 1996). It has
been shown that the tps1 mutant is deficient in induced
thermotolerance, and this phenotype can be restored after
complementation with the homologous ScTPS1 gene (De Virgilio et al., 1994). Here we show that SlTPS1 is able to restore
the yeast capacity for both thermoinduction and induced
thermotolerance. Another aspect of SlTPS1 that we addressed
was its capacity to confer osmotolerance. A recent work (Hounsa et al.,
1998) analyzed the osmosensitive phenotype tps1 mutant
under moderate and severe osmotic stress, revealing the strong
correlation between the presence of trehalose in yeast and survival
under osmotic stress conditions. In the present study, we showed that
both SlTPS1 and ScTPS1 are able to complement and
restore growth of yeast cells under osmotic stress. These data suggest
that trehalose may play a similar role in S. lepidophylla as
a stress protectant.
A few TPS homologs have been cloned from bacteria, fungi, and higher
eukaryotes (Fig. 1). It is possible that TPS1 is present in most
organisms, although not necessarily involved in the synthesis of
significant amounts of trehalose. That plant or animal TPS might have a
role similar to that of yeast ScTPS1 as a controller of the influx of
sugar into glycolysis is an intriguing possibility. The fact that both
SlTPS1 and AtTPS1 are able to complement the growth defect on fermentable sugars of the yeast tps1
mutant suggests this possibility and opens a new perspective on the
regulation of glycolysis in plants and animals that should be explored.
| |
FOOTNOTES |
1
This work was supported by grant no. IN202795 to
G.I. and J.N.-S. from Dirección General de Asuntos del Personal
Académico-Universidad Nacional Autónoma de México,
Mexico; by grants to J.M.T. from the Fund for Scientific
Research-Flanders and the Research Fund of the Katholieke Universiteit
Leuven, Concerted Research Actions, Belgium; and by grant no. 938032MX
to R.G. from the European Economic Community, Belgium.
2
These authors contributed equally to the paper.
*
Corresponding author; e-mail iturri{at}ibt.unam.mx; fax
52-73-172388.
Received August 21, 1998;
accepted December 21, 1998.
| |
ABBREVIATIONS |
Abbreviations:
TPP, trehalose-6-P phosphatase.
TPS, trehalose-6-P synthase.
| |
ACKNOWLEDGMENTS |
We thank Paul Gaytán and Eugenio López for
oligonucleotide synthesis and Mario Trejo, Elena Arriaga, Guadalupe
Ochoa, Martine De Jonge, and Willy Verheyden for technical assistance.
We acknowledge Dr. G.J. Ruijter (Wageningen Agricultural University,
The Netherlands) for providing purified A. niger hexokinase.
J.O.M.-G. thanks Consejo Nacional de Ciencia y Tecnología,
Mexico, and B.B. thanks Conselho Nacional de Desenvolvimento Cientifico
e Tecnológico, Brazil, for Ph.D. fellowships.
| |
LITERATURE CITED |
Adams RP,
Kendall E,
Kartha KK
(1990)
Comparison of free sugars in growing and desiccated plants of Selaginella lepidophylla.
Biochem Syst Ecol
18:
107-110
[CrossRef]
Blázquez MA,
Santos E,
Flores C-L,
Martínez-Zapater JM,
Salinas J,
Gancedo C
(1998)
Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase.
Plant J
13:
685-689
[CrossRef][Web of Science][Medline]
Burke MJ (1985) The glassy state and survival of anhydrous
biological systems. In AC Leopold, ed, Membranes, Metabolism
and Dry Organisms. Cornell University Press, Ithaca, NY, pp 358-363
Cabib E,
Leloir LF
(1958)
The biosynthesis of trehalose phosphate.
J Biol Chem
231:
259-275
[Free Full Text]
Christianson TW,
Sikorski RS,
Dante M,
Shero JH,
Hieter P
(1992)
Multifunctional yeast high-copy number shuttle vectors.
Gene
110:
119-122
[CrossRef][Web of Science][Medline]
Clegg JS
(1965)
The origin of trehalose and its significance during the formation of encysted embryos of Artemia salina.
Comp Biochem Physiol
14:
135-143
[Medline]
Clegg JS (1985) The physical properties and metabolic status of
Artemia cysts at low water contents: "the water
replacement hypothesis." In AC Leopold, ed, Membranes,
Metabolism and Dry Organisms. Cornell University Press, Ithaca,
NY, pp 169-187
Colaço C,
Sen S,
Thangavelu M,
Pinder S,
Roser B
(1992)
Extraordinary stability of enzymes dried in trehalose: simplified molecular biology.
Biotechnology
10:
1007-1011
[CrossRef][Medline]
Crowe JH,
Crowe LM,
Carpenter JF,
Aurell Wistrom C
(1987)
Stabilization of dry phospholipid bilayers and proteins by sugars.
Biochem J
242:
1-10
[Web of Science][Medline]
Crowe JH,
Crowe LM,
Chapman D
(1984)
Preservation of membranes in anhydrobiotic organisms: the role of trehalose.
Science
223:
701-703
[Abstract/Free Full Text]
Crowe JH,
Hoekstra FA,
Crowe LM
(1992)
Anhydrobiosis.
Annu Rev Physiol
54:
579-599
[CrossRef][Web of Science][Medline]
De Virgilio C,
Bürckert N,
Bell W,
Jenö P,
Boller T,
Weimken A
(1993)
Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae, causes accumulation of trehalose6-phosphate and loss of trehalose-6-phosphate phosphatase activity.
Eur J Biochem
212:
315-323
[Web of Science][Medline]
De Virgilio C,
Hottiger T,
Domínguez J,
Boller T,
Wiemken A
(1994)
The role of trehalose synthesis for the acquisition of thermotolerance in yeast. I. Genetic evidence that trehalose is a thermoprotectant.
Eur J Biochem
219:
179-186
[Web of Science][Medline]
Elble R
(1992)
A simple and efficient procedure for transformation of yeasts.
BioTechniques
13:
18-20
[Web of Science][Medline]
Elliot B,
Haltiwanger RS,
Futcher B
(1996)
Synergy between trehalose and Hsp104 for thermotolerance in Saccharomyces cerevisiae.
Genetics
144:
923-933
[Abstract]
Gaff DF
(1971)
Desiccation-tolerant flowering plants in southern Africa.
Science
174:
1033-1034
[Abstract/Free Full Text]
Garcia AB,
de Almeida Engler J,
Iyer S,
Gerats T,
Van Montagu M,
Caplan AB
(1997)
Effects of osmoprotectants upon NaCl stress in rice.
Plant Physiol
115:
159-169
[Abstract]
Giaever HM,
Styrvold OB,
Kaasen I,
Strom AR
(1988)
Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli.
J Bacteriol
170:
2841-2849
[Abstract/Free Full Text]
Goddijn OJM,
Verwoerd TC,
Voogd E,
Krutwagen RWHH,
de Graaf PTHM,
Poels J,
van Dun K,
Ponstein AS,
Damm B,
Pen J
(1997)
Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants.
Plant Physiol
113:
181-190
[Abstract]
Hengge-Aronis R,
Klein W,
Lange R,
Rimmele M,
Boos W
(1991)
Trehalose synthesis genes are controlled by the putative sigma factor encoded by rpoS and are involved in stationary-phase thermotolerance in Escherichia coli.
J Bacteriol
173:
7918-7924
[Abstract/Free Full Text]
Hohmann S,
Bell W,
Neves MJ,
Valckx D,
Thevelein JM
(1996)
Evidence for trehalose-6-phosphate-dependent and -independent mechanisms in the control of sugar influx into yeast glycolysis.
Mol Microbiol
20:
981-991
[Web of Science][Medline]
Hohmann S,
Neves MJ,
de Koning W,
Alijo R,
Ramos J,
Thevelein JM
(1993)
The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII.
Curr Genet
23:
281-289
[CrossRef][Web of Science][Medline]
Hounsa C-G,
Brandt EV,
Thevelein J,
Hohmann S,
Prior BA
(1998)
Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress.
Microbiology
144:
671-680
[Abstract/Free Full Text]
Kaasen I,
McDougall J,
Strom AR
(1994)
Analysis of the otsBA operon for osmoregulatory trehalose synthesis in Escherichia coli and homology of the OtsA and OtsB proteins to the test trehalose-6-phosphate synthase/phosphatase complex.
Gene
145:
9-15
[CrossRef][Web of Science][Medline]
Londesborough J,
Vuorio OE
(1993)
Purification of trehalose synthase from baker's yeast: its temperature-dependent activation by fructose 6-phosphate and inhibition by phosphate.
Eur J Biochem
216:
841-848
[Web of Science][Medline]
Luyten K,
de Koning W,
Tesseur I,
Ruiz MC,
Ramos J,
Cobbaert P,
Thevelein JM,
Hohmann S
(1993)
Disruption of the Kluyveromyces GGS1 gene causes inability to grow on glucose and fructose and is suppressed by mutations that reduce sugar uptake.
Eur J Biochem
217:
701-713
[Medline]
Mackenzie KF,
Singh KK,
Brown AD
(1988)
Water stress plating hypersensitivity of yeasts: protective role of trehalose in Saccharomyces cerevisiae.
J Gen Microbiol
134:
1661-1666
[Abstract/Free Full Text]
Mascorro-Gallardo JO,
Covarrubias AA,
Gaxiola R
(1996)
Construction of a CUP1 promoter-based vector to modulate gene expression in Saccharomyces cerevisiae.
Gene
172:
169-170
[CrossRef][Web of Science][Medline]
Müller J,
Boller T,
Wiemken A
(1995)
Trehalose and trehalase in plants: recent developments.
Plant Sci
112:
1-9
Neves MJ,
Hohmann S,
Bell W,
Dumortier F,
Luyten K,
Ramos J,
Cobbaert P,
de Koning W,
Kaneva Z,
Thevelein JM
(1995)
Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis.
Curr Genet
27:
110-122
[CrossRef][Web of Science][Medline]
Neves MJ,
Terenzi HF,
Leone FA,
Jorge JA
(1994)
Quantification of trehalose in biological samples with a conidial trehalase from the thermophilic fungus Humicola grisea var. thermoidea.
World J Microbiol Biotechnol
10:
17-19
Reinders A,
Bürckert N,
Hohmann S,
Thevelein JM,
Boller T,
Wiemken A,
De Virgilio C
(1997)
Structural analysis of the subunits of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock.
Mol Microbiol
24:
687-695
[CrossRef][Web of Science][Medline]
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual, Ed 2.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Serrano R,
Villalba JM
(1995)
Expression and localization of plant membrane proteins in Saccharomyces.
Methods Cell Biol
50:
481-496
[Web of Science][Medline]
Thevelein JM
(1984)
Regulation of trehalose mobilization in fungi.
Microbiol Rev
48:
42-59
[Free Full Text]
Thevelein JM,
Hohmann S
(1995)
Trehalose synthase: guard to the gate of glycolysis in yeast?
Trends Biochem Sci
20:
3-10
[CrossRef][Web of Science][Medline]
Thomas BJ,
Rothstein RJ
(1989)
Elevated recombination rates in transcriptionally active DNA.
Cell
56:
619-630
[CrossRef][Web of Science][Medline]
Thompson JD,
Higgins DG,
Gibson TJ
(1994)
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res
22:
4673-4680
[Abstract/Free Full Text]
Van Aelst L,
Hohmann S,
Bulaya B,
de Koning W,
Sierkstra L,
Neves MJ,
Luyten K,
Alijo R,
Ramos J,
Coccetti P,
and others
(1993)
Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae.
Mol Microbiol
8:
927-943
[Web of Science][Medline]
Vandercammen A,
François J,
Hers H-G
(1989)
Characterization of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Saccharomyces cerevisiae.
Eur J Biochem
182:
613-620
[Web of Science][Medline]
Vogel G,
Aeschbacher RA,
Müller J,
Boller T,
Wiemken A
(1998)
Trehalose-6-phosphate phosphatase from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant.
Plant J
13:
673-683
[CrossRef][Web of Science][Medline]
Weisburd S
(1988)
Death-defying dehydration.
Sci News
133:
107-110
Yancey PH,
Clark ME,
Hand SC,
Bowlus RD,
Somero GN
(1982)
Living with water stress: evolution of osmolyte systems.
Science
217:
1214-1222
[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
N. Avonce, J. Wuyts, K. Verschooten, L. Vandesteene, and P. Van Dijck
The Cytophaga hutchinsonii ChTPSP: First Characterized Bifunctional TPS-TPP Protein as Putative Ancestor of All Eukaryotic Trehalose Biosynthesis Proteins
Mol. Biol. Evol.,
February 1, 2010;
27(2):
359 - 369.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Vandesteene, M. Ramon, K. Le Roy, P. Van Dijck, and F. Rolland
A Single Active Trehalose-6-P Synthase (TPS) and a Family of Putative Regulatory TPS-Like Proteins in Arabidopsis
Mol Plant,
January 28, 2010;
(2010):
ssp114v2 - ssp114.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. C. F. Proctor, R. Ligrone, J. G. Duckett, M. C. F. Proctor, R. Ligrone, and J. G. Duckett
Desiccation Tolerance in the Moss Polytrichum formosum: Physiological and Fine-structural Changes during Desiccation and Recovery
Ann. Bot.,
June 1, 2007;
99(6):
1243 - 1243.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. C. F. Proctor, R. Ligrone, and J. G. Duckett
Desiccation Tolerance in the Moss Polytrichum formosum: Physiological and Fine-structural Changes during Desiccation and Recovery
Ann. Bot.,
January 1, 2007;
99(1):
75 - 93.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. PRESSEL, R. LIGRONE, and J. G. DUCKETT
Effects of De- and Rehydration on Food-conducting Cells in the Moss Polytrichum formosum: A Cytological Study
Ann. Bot.,
July 1, 2006;
98(1):
67 - 76.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Avonce, B. Leyman, J. O. Mascorro-Gallardo, P. Van Dijck, J. M. Thevelein, and G. Iturriaga
The Arabidopsis Trehalose-6-P Synthase AtTPS1 Gene Is a Regulator of Glucose, Abscisic Acid, and Stress Signaling
Plant Physiology,
November 1, 2004;
136(3):
3649 - 3659.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. D. Elbein, Y.T. Pan, I. Pastuszak, and D. Carroll
New insights on trehalose: a multifunctional molecule
Glycobiology,
April 1, 2003;
13(4):
17R - 27R.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. J. Eastmond, Y. Li, and I. A. Graham
Is trehalose-6-phosphate a regulator of sugar metabolism in plants?
J. Exp. Bot.,
January 3, 2003;
54(382):
533 - 537.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. K. Garg, J.-K. Kim, T. G. Owens, A. P. Ranwala, Y. D. Choi, L. V. Kochian, and R. J. Wu
Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses
PNAS,
December 10, 2002;
99(25):
15898 - 15903.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Van Dijck, L. De Rop, K. Szlufcik, E. Van Ael, and J. M. Thevelein
Disruption of the Candida albicans TPS2 Gene Encoding Trehalose-6-Phosphate Phosphatase Decreases Infectivity without Affecting Hypha Formation
Infect. Immun.,
April 1, 2002;
70(4):
1772 - 1782.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Vogel, O. Fiehn, L. Jean-Richard-dit-Bressel, T. Boller, A. Wiemken, R. A. Aeschbacher, and A. Wingler
Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues
J. Exp. Bot.,
September 1, 2001;
52(362):
1817 - 1826.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Wingler, T. Fritzius, A. Wiemken, T. Boller, and R. A. Aeschbacher
Trehalose Induces the ADP-Glucose Pyrophosphorylase Gene, ApL3, and Starch Synthesis in Arabidopsis
Plant Physiology,
September 1, 2000;
124(1):
105 - 114.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
B. Shen, S. Hohmann, R. G. Jensen, and a. H. J. Bohnert
Roles of Sugar Alcohols in Osmotic Stress Adaptation. Replacement of Glycerol by Mannitol and Sorbitol in Yeast
Plant Physiology,
September 1, 1999;
121(1):
45 - 52.
[Abstract]
[Full Text]
|
|
|
|
|
Top
Abstract
Introduction