Download Comparative Analysis of Short- and long-Term

Plant Physiol. (1994) 106: 703-712
Comparative Analysis of Short- and long-Term Changes in
Gene Expression Caused by l o w Water Potential in Potato
( S o h u m tuberosum) Cell-Suspension Cultures’
Antonella Leone*, Antonello Costa, Marina Tucci, and Stefania Crillo
Research Centre for Vegetable Breeding, National Research Council, (A.L., A.C., S.G.), and Department of
Agronomy and Plant Genetics (M.T.), University of Naples, 80055 Portici, Italy
changes in gene expression in response to extemal stimuli
occur in a coordinated manner. As suggested by Sachs and
Ho (1986) for hsps, the synthesis of polypeptides in response
to variations in the environment is biphasic: synthesis of
‘early” proteins, which are implicated in the perception and
transduction of the stress signal, and synthesis of ‘late”
proteins involved in the assumption of a new homeostatic
cellular condition and the recovery of a normal cellular
metabolism. Without polypeptides induced both early and
late, acquisition of tolerance is improbable and productivity
under stress conditions is unachievable. For water stress, it
has frequently been observed that there is simultaneous
activation of specific protein synthesis, a general inhibition
of plant and cell growth (Trewavas and Jones, 1991), and
suppression of the synthesis of some constitutive cellular
proteins (Bartholomewet al., 1991). The final result of these
two mechanisms (Le. activation and inhibition of the synthesis of specific proteins) is that protein synthesis capacity may
be unchanged but preferentially directed to the synthesis of
the so-called ‘stress proteins.”
When studying plant-stress response, an aspect that has to
be carefully considered is that some of the observed alterations in protein synthesis may be due to cellular injury, which
is a function of the stress intensity, duration, and mode of
imposition. Shock conditions (long exposure to and/or intense water stress) lead to irreversible damage of the cellular
constituents, such as membranes (Stewart, 1989), whereas a
gradual imposition may not lead to significant cellular injuries
and may enable plants to tolerate an even more intense
stress. Therefore, appropriate and accurate investigations
have to be designed to distinguish polypeptides whose synthesis is associated with the ability of plants to cope with
water stress from those merely caused as a result of cellular
damage.
Regardless of the time necessary for their induction, waterstress proteins can be divided broadly into two categories:
those that are induced by ABA and those that are not. A
large number of the drought-induced proteins identified so
far come under the former category and are generally better
characterized than the other water-stress proteins (for a review, see Skriver and Mundy, 1990). It is well known that
mesophytic plants have the capacity to synthesize rapidly
To dissect the cellular response to water stress and compare
changes induced as a generalized response with those involved in
tolerance/acclimation mechanisms, we analyzed changes in twodimensional electrophoretic patterns of in vivo [35S]methioninelabeled polypeptides of cultured potato (Solanum tuberosum) cells
after gradual and long exposure to polyethylene glycol (PEG)mediatedlow water potential versus those induced i n cells abruptly
exposed to the same stress intensity. Protein synthesis was not
inhibited by gradual stress imposition, and the expression of 17
proteins was induced in adapted cells. Some polypeptides were
inducible under mild stress conditions (5% PEG) and accumulated
further when cells were exposed to a higher stress intensity (10
and 20% PEG). The synthesis of another set of polypeptides was
up-regulated only when more severe water-stress conditions were
applied, suggesting that plant cells were able to monitor different
levels of stress intensity and modulate gene expression accordingly.
In contrast, in potato cells abruptly exposed to 20% PEG, protein
synthesis was strongly inhibited. Nevertheless, a large set of polypeptides was identified whose expression was increased. Most of
these polypeptides were not induced in adapted cells, but many of
them were common to those observed in abscisic acid (ABA)treated cells. These data, along with the finding that cellular ABA
content increased in PEG-shocked cells but not in PEG-adapted
cells, suggested that this hormone is mainly involved in the rapid
response to stress rather than long-term adaptation. A further
group of proteins included those induced after long exposure to
both water stress and shock. Western blot analysis revealed that
osmotin was one protein belonging to this common group. This
class may represent induced proteins that accumulate specifically
in response to low water potential and that are putatively involved
in the maintenance of cellular homeostasis under prolonged stress.
In recent years, great attention has been focused on
changes in plant gene expression caused by responses to
environmental stresses. Quantitative and qualitative variations in the electrophoretic pattems of in vivo-labeled and in
vitro-translated proteins have been identified, but the function of these proteins has been elucidated in only a few cases
(for a review, see Leone et al., 1993). Many of the observed
This work was supported by a grant from the Italian Ministry of
Agriculture and Forestry in the framework of the project ’Resistenze
genetiche delle piante agrarie agli stress biotici e abiotici.”Conhibution No. 113 of Research Centre for Vegetable Breeding, Consiglio
Nazionale delle Richerche, Portici, Italy.
* Corresponding author; fax 39-81-775-35-79.
Abbreviations: ZD,two-dimensional; HS, heat shock; hsp, heatshock Drotein.
703
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Leone et al.
704
(within 30 min) large quantities of ABA in response to
drought (Quarrie, 1983; Cohen and Bray, 1990). The rapidity
of ABA induction suggests that it is involved in short-term
response to stress and in transduction of the stress signal
(Hetherington and Quatrano, 1991). When applied, ABA
triggers the synthesis of polypeptides that are also induced
as a rapid response to water stress (Skriver and Mundy,
1990). Application of the hormone also reproduces some of
the inhibitory and detrimental effects on growth components
visible after long exposure to drought (Trewavas and Jones,
1991). Therefore, it would be interesting to ascertain whether
or not ABA, in addition to being involved in the rapid
responses, is also involved in long-term responses to water
stress and whether the hormone is responsible for the downregulation of protein synthesis observed when plants are
exposed to drought.
In an attempt to answer these questions, we compared the
changes in the synthesis of in vivo-labeled polypeptides of
cultured potato (Solanum tuberosum) cells that were gradually
adapted to grow in a medium supplemented with high mol
wt PEG (20%) versus the modifications induced in cells
abruptly transferred to the same medium. PEG is a molecule
commonly used to mimic water stress because it reduces
water availability without penetrating the cell (Rains, 1989).
Although suspension cultures may be considered an oversimplification of the complex mechanisms that might be
involved in the response of plants to drought, they represent
a highly controllable and homogeneous experimental system
that allows the study of long-term adaptation to environmental stresses at a cellular level without interference of different
plant tissues and developmental stages. The comparison also
permitted speculation concerning the relationships between
some physiological and biochemical cellular modifications
after short- and long-term exposure to low water potential
and the changes in gene expression we observed.
MATERIALS AND METHODS
Cell Cultures and Stress Treatments
Cell-suspension cultures obtained from leaf callus of the
potato (Solanum tuberosum) dihaploid clone SVPl1 were
kindly provided by Dr. T. Cardi (Portici, Italy) and were
maintained in modified Murashige-Skoog medium (Tavazza
et al., 1988) in a rotary shaker at 28OC in the dark, subcultured
every 7 d when growth reached the stationary phase. These
cells will be referred to as “unadapted cells.” Gradual adaptation to low water potentials was obtained by subcultures in
media with increasing concentrations of PEG 8000 (Sigma)
from O to 5, 10, and 20% (w/v), corresponding to an osmotic
potential of about -0.5, -1.0, -1.4, and -2.3 MPa, respectively (deduced from Lee-Stadelmann and Stadelmann,
1989). Cells were maintained at 5 and 10% PEG for two
subculture passages and then at 20% PEG for 45 culture
cycles before further analysis (“adapted cells”).Osmotic shock
was imposed by abrupt transfer of unadapted cells to a
medium containing 20% PEG for 24 h (“PEG-shockedcells”).
In other experiments, unadapted cells were either treated
with 100 PM ABA for 24 h (“ABA-treated cells”) or exposed
to 37OC for 3 h (“heat-shocked cells”). Before any determi-
Plant Physiol. Vol. 106, 1994
nations were made, cells were thoroughly ,ind rapidly
washed with an isotonic salt solution for each treatment to
eliminate PEG.
Cell Growth and Viability
For cell growth determination, 20 mL of cell-suspension
culture were filtered through filter paper and dried to a
constant weight at 7OOC. Cell growth was expxessed as mg
mL-’ of cell dry weight at d 7 of subculture. Cell viability
(stainable cells divided by the total cell number analyzed)
was determined by fluorescein diacetate staining (Widholm,
1972) during the whole growth cycle and expressed as mean
percentage of the value found in unadapted control cells.
ABA Measurement
ABA content of cells at each day of the growing cycle was
measured in an aqueous extract using a compi2titive radioimmunoassay, as described by Quame et al. (1988). The
monoclonal antibody Mac62 was kindly provided by Dr. S .
Quarrie (Norwich, UK).
Free Pro Content
Total free amino acids were extracted with 5% (w/v) sulfosalicylic acid from lyophilized cells (500 mg) harvested at
d 3 of culture, and Pro was detected by HPLC.
In Vivo Protein labeling and 2D-PAGE
In vivo labeling of unadapted, PEG-adapted, and PEGshocked cells and of cells during the adaptation cycle (5 and
10% PEG) was performed at d 3 from subcultuxe, during the
exponential growth phase, with 1.85 MBq of [35S]Met (37
PBq mol-’) for 3 h. Cells were treated with lOCl PM ABA for
24 h and labeled in the last 3 h of the treatment. HS was
imposed for 3 h, during which cells were radicilabeled. Following in vivo labeling, cells were harvested by centrifugation, thoroughly rinsed with an isotonic salt solution, and
immediately ground in liquid nitrogen. Proteins were extracted with TCA-acetone according to the method of Granier
(1988). The acetone-precipitated proteins were :suspended in
lysis buffer containing 9.5 M urea, 3% (w/v) CHAPS (Sigma),
50 m K2C03, 50 m DTT, and 2% ampholite; (4 parts pH
5-8 and 1 part pH 3.5-10, Pharmacia). Equal amounts of
TCA-precipitableradioactive proteins (250 or 500 X lo3 cpm
on each gel) were separated by 2D-PAGE according to the
method of O’Farrell (1975) with the modifications described
gels
by Hochstrasser et al. (1988). After the electro~~horesis,
were fixed, processed for fluorography (Bonnei. and Laskey,
1974), dried, and exposed to x-ray film for a time, proportional
to the amount of radioactivity loaded on each gel. All experiments were replicated at least once, and only reproducible
changes in labeled proteins are reported.
RNA Analysis
Total RNA was prepared by the acid guanidmium thiocyanate phenol chloroform method (Chomeczynsld and Sacchi,
1987) and separated on 1.5% agarose denaturing (2.2 M
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Water Stress and Protein Synthesis in Potato Cells
formaldehyde) gels. Gels were blotted in 20X SSC onto nylon
membranes and RNA fixed by UV cross-linking. Filters were
hybridized with a [cY-~'P]~CTP
random-primed cDNA clone
encoding tobacco osmotin (Singh et al., 1989). Prehybridization and hybridization were performed at 42OC in 6X SSC,
5X Denhardt's solution (1% gelatin, 1% Ficoll, 1% BSA),
0.1% SDS, 50% formamide, and 100 r g mL-' salmon sperm
DNA. Filters were washed twice for 20 min at room temperature in 2X SSC, 0.1% SDS and once in 0.1% SSC, 0.1%
SDS at 65OC and placed at -8OOC with Kodak X-Omat AR
films and intensifying screens. Fold increase in osmotin transcript was estimated by scanning the film with a laser densitometer (LKB, Uppsala, Sweden). Transcript size was determined by comparison with mol wt standards (0.24- to
9.5-kb RNA ladder; BRL).
Protein lmmunoblotting
Proteins were extracted in sample buffer (62.5 lll~Tris [pH
6.81, 5% glycerol, 2% SDS, and 40 lll~DTT) and quantified
according to a modified Bradford procedure (LaRosa et al.,
1989). Equal amounts of proteins (30 Pg) were separated by
SDS-PAGE on a 12.5% polyacrylamide gel according to the
method of Laemmli (1970) and electroblotted onto nitrocellulose membrane. Osmotin protein was detected according
to the procedure of LaRosa et al, (1989) on western blots
with anti-tobacco osmotin antibodies, obtained from Dr. R.
Bressan (West Lafayette, IN).
RESULTS
Cellular Adaptation to PEG-Mediated Low Water Potential
In Table I the main characteristics of our cellular system
are summarized. At d 7 of the cellular growth cycle (stationary
phase), the dry weight of cells gradually adapted was not
significantly different from the value attained by potato cells
grown in the standard medium. In contrast, cellular growth
was strongly inhibited (by about 50%) in cells directly transferred to a medium containing 20% PEG. Adapted and
705
Table II. Comparison of [35S]Met incorporation ability during shock
or gradual adaptation of potato cells to PEG-mediated low water
potential conditions
Potato cells, after 3 d of subculture, were subjected to different
treatments and incubated with [35S]Metfor 3 h. Unadapted cells
were PEG shocked for 24 h and labeled in the last 3 h of t h e
treatment. Cells were kept in medium containing 5 and 10% PEG
for at least two generations each before transfer to the final PEG
concentration (20%). Cells were maintained in the medium supplemented with 20% PEG for at least 45 generations. The radioactivity
incorporated in TCA-insoluble proteins was determined and expressed as cpm mg-' dry wt. Values are means of two or three
experiments.
Incorporation of
13WMet
Medium
Treatment
OsmoIarit v
OSM
%
Control
PEG shock
0.225
0.925
5% PEG
10% PEG
0.400
0.575
20% PEG
0.925
1O0
70
81
114
120
shocked cells also differed in their ABA and Pro content. The
maximum ABA content, corresponding to a 5-fold increase,
was found in PEG-shocked cells 5 d after transfer to a
medium with 20% PEG, whereas no significant increment
over the basal level was observed in adapted cells. Moreover,
compared to unadapted cells, a substantially higher free Pro
content was detected in adapted and shocked cells, which
represented about 42 and 21.7% of total cellular free amino
acids, respectively.
Effect of Gradual Adaptation or Shock on
[35S]MetIncorporation
Cellular incorporation of [35S]Met (cpm mg-' cell dry
weight) was determined in unadapted, PEG-adapted, and
PEG-shocked cells and expressed as a percentage of the value
Growth characteristicsand ABA and Pro content of unadapted, PEG-adapted, and
PEG-shocked potato cells
Dry weight was determined at d 7 of culture when cell growth reached the stationary phase. Cell
viability (fluorescein diacetate-stainable cells divided by the total cell number) is expressed as a
percentage of the value of t h e unadapted cells. ABA content was measured by radioimmunoassay.
Data reported refer to t h e maximum ABA accumulation detected during t h e cell growth cycle (d 3
for unadapted and adapted cells and d 5 for shocked cells). Fold increase was calculated as the ratio
between maximum ABA cellular content and content at d 1. Pro, expressed as a percentage of total
free amino acids, was measured at d 3 of culture by HPLC. Means of three replicates were analyzed
by Duncan's multiple range test. Values followed by the same letter are not significantly different at
P 5 0.05.
Table 1.
ABA
Dry Wt
Viability
Pro
Max content
mg mL-'
Unadapted
PEG adapted
PEG shocked
14.2a
13.3a
7.5b
O h of
control
1OOa
102a
87b
Fold increase
ng g-' dry wt
179.4a
198.8a
624.1 b
%
1.4
1.3
5.2
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
3.3a
41.9~
21.7b
706
Leone et al.
8.0
Plant Physiol. Vol. 106, 1994
IEF
- S.O
5.5
ri Mr
£ 80.0
a
49.5
32.5
27.5
18.5
Unadopted
80.0
49.5
32.5
27.5
18.5
»
- •
5H
PEG-Shocked
ABA 100 uM
Figure 1. Fluorographs of in vivo-labeled polypeptides of unadapted (A), PEG-adapted (B), PEC-shocked (C), and ABAtreated (D) potato cells. Unadapted cells were maintained in modified Murashige-Skoog medium. PEC-adapted cells
were gradually adapted in a medium supplemented with 20% PEG for at least 45 generations. PEG-shocked cells
represent unadapted cells abruptly transferred to a medium with 20% PEG. ABA-treated cells were exposed to 100 /JM
ABA for 24 h. For each treatment cells were labeled with [ 35 S]Met for 3 h. Equal amounts of radiolabeled proteins were
electrophoresed by IEF followed by an 8 to 13% gradient SDS-PAGE. In each treatment prominent polypeptides are
indicated as follows: A, up regulated; V, down regulated. Arrows in B and C indicate up-regulated polypeptides common
to the two treatments. Regions enclosed in boxes (I, II, and III) include polypeptides that varied during gradual adaptation
(see Fig. 2). Lowercase letters indicate major reference proteins. Molecular mass markers are shown in kD.
PEG-induced water stress, 2D electrophoretic patterns of [35S]Met in vivo-labeled polypeptides of unadapted and adapted
cells were compared (Fig. 1 and Table III). Protein profiles
were very complex, but only the most significant and reproducible differences were considered (indicated with open
triangles). To facilitate the localization of the polypeptides in
the fluorographs, eight reference proteins were identified
whose relative position in the gel was highly replicable (indicated with lowercase letters).
The overall pattern of in vivo-labeled polypeptides of
Protein Expression and Kinetics of Induction during
adapted cells was similar to the protein profile observed in
Gradual Adaptation to Low Water Potential
unadapted control cells. As already estimated in terms of [35S]Met incorporation, the synthesis of most of the constitutive
To identify changes in gene expression that may account
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was
inhibited
for the ability of adapted cells to Copyright
sustain active
growth
under
© 1994 American Society ofpolypeptides
Plant Biologists.
All not
rights
reserved.by gradual stress imposition
measured in unadapted cells (Table II). As expected, abrupt
transfer to a medium supplemented with 20% PEG caused a
30% reduction in the amount of [35S]Met incorporated into
potato cells. Incorporation was inhibited (19%) in cells maintained in a medium with 5% PEG but fully resumed when
cells were transferred to 10% PEG. Interestingly, incorporation capacity of cells kept for more than 45 generations in a
medium with 20% PEG was 20% higher than that observed
in unadapted cells.
Water Stress and Protein Synthesis in Potato Cells
707
Table 111. Summary of the major in vivo-labeled polypeptides up or down regulated in PEC-adapted cells at different stages of adaptation to
PEG-mediated low water potential
indicate different intensities of t h e proteins on the fluorographs; - indicates absence of the protein. Regulation of
the same polypeptides by ABA is also indicated. Polypeptides are numbered as in Figure 1 .
+, ++, +++, ++++
Polypeptide
Unadapted
No.
5% PEC
10% PEC
20% PEC
ABA
M.
U p regulated
2
3
4
6
11
12
19
20
21
22
26
27
28
29
35
48
49
82,500
82,500
80,700
67,400
60,600
58,700
54,500
53,900
50,600
47,500
41,400
3 7,600
36,000
33,l O0
26,800
14,800
13,500
+
+
+
+
++
+
+
+
+++
+
+
++
+
++
+
+
+
+
+
++
++
+
+
++
++++
+
+
++
+
++
++
++
+
+
+
++
++++
++
+
+++
+
+++
-
++
+
+
-
+
-
+
++
++
++
+++
+++
+++
++
+++
++++
++
+++
+++
+++
+++
+++
+++
+++
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
Down regulated
30
31
50
29,100
28,000
13,000
+++
++
++
and only three polypeptides (nos. 30, 31, and 50) were
significantly down regulated. Nevertheless, it was possible to
identify a specific set of proteins whose synthesis was up
regulated in adapted cells. This group included 17 polypeptides with an M, range of 13,500 to 82,500, among which the
most interesting was a polypeptide of 14.8 kD (no. 48),which
was de novo synthesized in adapted cells and undetectable
in unadapted control cells (Fig. 1, A and B).
Adaptation is a gradual phenomenon, and, therefore, we
followed the kinetics of induction of the major polypeptides
during the different steps of the adaptive process (5, 10, and
20% PEG). The main results are summarized in Table 111, and
some of the most representative examples are reported in
Figure 2. The synthesis of many polypeptides (nos. 3, 11, 12,
19, 26, 28, 35, and 49) was up regulated only after exposure
to the highest PEG concentration, whereas the synthesis of
others (nos. 2, 4, 6, 20, 21, 22, 27, 29, and 48) was found to
be enhanced when cells were treated with 5 and 10% PEG.
Despite the strong inhibition of protein synthesis that we
observed at 5% PEG (Table 111), three polypeptides (nos. 6,
20, and 21) were already induced in cells at this concentration. Although a few polypeptides (eg. nos. 30, 31, and 50)
were immediately and permanently down regulated, the
syntheses of polypeptides 11 and 35 were only transiently
repressed and subsequently fully recovered (see Fig. 2 for
details). It should be noted that transient repression of protein
synthesis and subsequent recovery during the adaptive process was a generalized response involving many more polypeptides than the group reported in Table I11 (data not
shown).
+
+
+
+
+
+
+
+
+
Yes
Yes
Yes
We checked whether any of the changes in protein synthesis detected during prolonged exposure to water stress might
)
of unadapted cells
be reproduced by ABA (100 p ~ treatment
(Fig. 1D and Table 111). Except for polypeptide 35, most of
the proteins induced in adapted cells were not up regulated
in ABA-treated cells, whereas the few down-regulated polypeptides identified in adapted cells were repressed by ABA
treatment .
Protein Expression in PEG-Shocked Cells
Comparison of in vivo-labeled protein pattems of PEGadapted and PEG-shocked cells (Fig. 1, B and C) demonstrated that abrupt transfer of unadapted cells to low water
potential had a substantially different effect on protein synthesis than gradual adaptation to the same conditions. A
drastic down regulation of many major polypeptides (21 in
total) was observed, which was consistent with the data on
[35S]Metincorporation. However, inhibition of the general
protein synthesis was associated with the increased synthesis ’
of 19 polypeptides (see Table I V for a summary). Among
them, the most prominent [35S]Met-labeledpolypeptides had
molecular masses of approximately 63 to 70 kD (nos. 7-10)
and 17 to 20 kD (nos. 40-46) (Fig. 1C). Despite the overall
major differences, it is worth noting that four polypeptides
(nos. 4, 27, 28, and 35), indicated by arrows in Figure 1, B
and C, were up regulated in both PEG-shocked and PEGadapted cells. Finally, we found that the synthesis of 14
of the 19 induced proteins and a11 of the proteins repressed
by shock conditions was regulated in a similar fashion by
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708
Leone et al.
Figure 2. Kinetics of induction or repression of
some polypeptides during gradual adaptation
to increasing PEC concentrations (5, 10, and
20%). Cells were kept in medium containing 5
and 10% PEC for at least two generations each
before transfer to the final PEC concentration
(20%). Cells were maintained in the medium
supplemented with 20% PEC for at least 45
generations. See Figure 1 for identification of
gel regions.
5% PEG
Plant Physiol. Vol. 106, 1994
10% PEG
20% PEG
H
O48
» -y to.
in
V35
exogenous ABA treatment (Fig. 1, C and D, and Table IV).
ABA also induced the synthesis of a group of M, 18,000 to
24,000 proteins (indicated by open squares without numbers
in Fig. ID), which were detected neither in PEG-adapted
cells nor in PEG-shocked cells.
In Vivo-Synthesized Polypeptides Induced in Unadapted
Cells during HS
The next question we addressed was whether or not the
HS response shared any similarities with changes in protein
synthesis caused by prolonged stress. When unadapted cells
were exposed to HS at 37°C for 3 h, protein synthesis was
inhibited to such an extent that most of the normal polypeptides were not detectable (Fig. 3). However, HS induced the
synthesis of a group of several hsps with a low molecular
mass in the range of 18 to 24 kD in potato cells. Another
group of hsps was found in the range of 65 to 80 kD. Several
hsps (nos. 1, 37,42,43,44, and 46) appeared to be in common
with PEG-shocked cells rather than PEG-adapted cells, at
least according to their electrophoretic mobilities. HS also
induced unique polypeptides (indicated by open triangles
without numbers), which are not discussed further in this
paper.
Northern blot hybridization with a cDNA clone encoding
tobacco osmotin revealed that osmotin mRNAs (1.2 kb) were
constitutively expressed in unadapted cells (Fig. 4A). As
estimated by densitometry, a 2-fold increase of osmotin
mRNAs over the basal level was found in potato cells adapted
to both 10 and 20% PEG. The abrupt transfer to a medium
containing 20% PEG caused a 3-fold increase, quantitatively
comparable with the signal detected in ABA-treated cells (Fig.
4B). It is interesting to note that osmotin transcripts accumulated in PEG-adapted cells without any apparent increase
in ABA cellular content, whereas the two events were coincident in PEG-shocked cells (Fig. 4D). To ascertain whether
the observed accumulation of osmotin mRNAs upon adaptation or shock resulted in the expression of the corresponding protein, we used antibodies prepared against tobacco
osmotin for western blot analysis. A single polypeptide of Mr
24,000 was immunodetected in potato cells. Osmotin accumulated above the constitutive level in PEG-adapted, PEGshocked, and ABA-treated cells. However, although we do
not have quantitative data, the estimated change in osmotin
content for each treatment did not consistently relate to the
corresponding change in osmotin transcript level (Fig. 4C).
DISCUSSION
Accumulation of Osmotin Protein and mRNAs in
PEG-Adapted and PEG-Shocked Cells
Osmotin is a protein that has been demonstrated to accumulate in tobacco cells adapted to NaCl (Singh et al., 1987).
An understanding of the complex events that follow the
onset of water stress requires a detailed description of gene
expression patterns at various stages of the stress response.
Many studies have identified polypeptides that appeared
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709
Water Stress and Protein Synthesis in Potato Cells
Table IV. Changes of in vivo-labeled polypeptides observed in
PEC-shocked potato cells as compared with unadapted cells
+, ++, +++, ++++ indicate different intensities of the proteins
on the fluorographs; — indicates absence of the protein. Regulation
of the same polypeptides by ABA is also indicated. Polypeptides
are numbered as in Figure 1.
Polypeptide
Unadapted
No.
Shocked
ABA
M,
Up regulated
1
4
5
7
8
9
10
27
28
35
36
37
39
40
42
43
44
45
46
83,300
80,700
71,100
67,400
66,000
65,300
65,300
37,600
36,000
26,800
26,200
23,800
23,500
19,900
18,700
18,700
18,700
1 7,900
1 7,500
+
+
+
+
+++
++
+++
++
Yes
No
No
Yes
+
++
+++
+++
Yes
Yes
++
+
+
+
++
+
++
+
+
++
+++
+++
+++
+++
++++
+++
++
+++
++++
++++
++++
Yes
No
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
+
+++
Yes
+
+++
Yes
Down regulated
6
13
14
15
16
17
18
23
24
25
26
30
31
32
33
34
38
41
47
50
51
67,400
56,000
55,000
55,000
55,000
54,700
54,700
46,000
46,000
42,700
41,400
29,100
28,000
27,200
27,200
27,000
23,600
19,900
17,000
13,000
12,500
+
-
Yes
+++
+++
+
+
Yes
Yes
+++
+++
++
++
++
+
+
-
Yes
Yes
Yes
Yes
Yes
++
+++
+
Yes
Yes
+
+++
++
+
+
Yes
Yes
Yes
+++
+++
+++
-
Yes
Yes
Yes
+++
+
Yes
+++
+++
++
+++
+
+
+
Yes
Yes
Yes
Yes
best characterized stress proteins, which accumulates in tobacco cells (Singh et al., 1987) as well as in tomato plants
(King et al., 1988) exposed to NaCl. However, so far, little
attention has been directed to establish the relationship, if
any, between the time-dependent regulation of the stress
proteins and the acquisition of tolerance to drought, defined
as plant capacity to maintain a normal cellular metabolism.
We compared short-term versus long-term changes in gene
expression in response to water stress. We demonstrated that
gradual imposition of water stress enabled potato cells to
sustain active growth at water potentials (about —2.0 MPa)
that inhibited the growth of cells exposed abruptly to the
same stress intensity. When water stress was imposed progressively, [35S]Met incorporation was not inhibited but was
activated, and the protein pattern was very similar to that
observed in unstressed control cells. The synthesis of only a
few polypeptides was repressed, whereas a new 14.8-kD
protein and at least 17 preexisting proteins were identified
whose synthesis was enhanced. Closer analysis of the kinetics
of induction demonstrated that a general initial repression of
protein synthesis was observed in potato cells when exposed
to a mild water stress (5% PEG, equivalent to -1.0 MPa).
However, the reduction was only transient and was followed
by recovery of almost the whole protein synthesis in the
subsequent steps (10 and 20% PEG). Some of the upregulated polypeptides were induced already at mild water
stress and accumulated proportionally to the increase in stress
intensity. Another set of polypeptides appeared only when
more severe water-stress conditions were applied. Altogether
these data suggest that plant cells seem to be able to monitor
different levels of stress intensity and to modulate gene
expression accordingly.
IEF
5.5
g
Mr
Q.
'&
V)
80.0
49.5
32.5
27.5
within minutes or during the first hours after the imposition
of the stress (Bray, 1988; Guerrero and Mullet, 1988; Guerrero
et al., 1990; Ho and Mishkind, 1991). Alternatively, research
has been focused on the identification of polypeptides induced in plant cells after long exposure to water stress or
NaCl (Singh et al., 1987; Borkird et al., 1991a, 1991b) and
associated with the ability to adapt to low water potential
conditions. To this last group belongs osmotin, one of the
18.5
HS 37°C
Figure 3. Fluorograph of in vivo-labeled polypeptides of cultured
potato cells during HS. Unadapted cells were labeled with [35S]Met
at 37°C for 3 h. Molecular mass markers are indicated in kD.
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Leone et al.
710
•o
IQ.
1
2
i
a
a.
«
•o
0,
'10% 20%'
28 S
18 S
1.2kb
1.0 1.9
1.8
3.3
3.0
24 kD
0.4
Figure 4. Expression of osmotin transcripts and protein during
gradual adaptation to increasing PEC concentrations, PEC shock,
and ABA (100 ^M) treatments. Equal amounts of total RNA (25 ng)
were separated on a 1.5% agarose denaturing gel (A). After transfer
onto a nylon membrane, filters were hybridized with a cDNA clone
encoding tobacco osmotin. Fold increase of osmotin transcripts as
determined by densitometry is indicated under each lane (B). For
western blot analysis (C), 30 /ig of total cellular protein were
separated on SDS-PACE, and osmotin content was immunodetected with antibodies prepared against tobacco osmotin. The
endogenous ABA content of potato cells after each treatment is
also reported (D).
The pattern of gene expression observed in potato cells
abruptly exposed to high PEG concentration was quite different. Protein synthesis, estimated as [35S]Met incorporation
and number of labeled proteins visualized on 2D gels, was
strongly inhibited. Nevertheless, a large set of polypeptides
was identified whose expression was increased or induced de
novo. The molecular mass of most of these proteins was in
the 18- to 24-kD range, which resembled ABA-induced proteins. The characterization of this group as ABA induced was
confirmed by two associated events: cellular accumulation of
ABA in PEG-shocked cells and appearance of polypeptides
with the same electrophoretic mobility in ABA-treated potato
cells. We do not know whether any of the proteins found to
be ABA induced was a lea-type protein (Dure et al., 1989),
since we did not detect, even using low-stringency conditions
in northern blots of potato cells, transcripts hybridizing to
several heterologous lea genes (D-ll, rab!7, and rab28, Em)
(data not shown). It is interesting to note that the synthesis
of the majority of proteins found to be down regulated in
Plant Physiol. Vol. 106, 1994
PEG-shocked cells was also repressed upon ABA treatment.
This finding supports the opinion that the hormone, in addition to promoting activation of genes useful to cope with
water stress, is also involved in the general process of growth
inhibition observed in water-stressed plants (Trewavas and
Jones, 1991) and in the down regulation of other genes.
Transcription of the rbsS and cab genes encoding two key
plant enzymes, the small subunit of Rubisco and Chl a/bbinding proteins, respectively, has been demonstrated to be
reduced by ABA (Bartholomew et al., 1991). Except for
polypeptide 35, none of the major polypeptides induced in
ABA-treated potato cells was detected in cells during gradual
water stress. This is not surprising, since ABA content did
not change significantly during the cellular growth cycle in
PEG-adapted cells. Genes encoding polypeptides that are
drought induced but not regulated by ABA have already been
characterized in other species, such as rice (Borkird et al.,
1991a, 1991b), pea (Guerrero et al., 1990), and common ice
plant (Thomas et al., 1992). Moreover, the presence of three
polypeptides induced by both long exposure and shock conditions, which are not ABA regulated, strongly supported
previous evidence that ABA does not account for all of the
differences in gene expression caused by water stress. The
proteins of this class may represent the best candidates
for the maintenance of cellular homeostasis under stress
conditions.
Since a high cellular content of free Pro was detected in
both PEG-adapted and PEG-shocked cells, we could speculate that one of the proteins belonging to this common group
could be an enzyme of the biosynthetic pathway of this
amino acid. Recently, it was shown that in Arabidopsis plants,
which accumulated high Pro content during salt treatment,
the transcription of a gene encoding a reductase that catalyzes
the reduction of pyrroline-5-carboxylic acid to Pro was activated (Verbruggen et al., 1993). We also demonstrated that
osmotin was another induced protein common to PEGshocked and PEG-adapted cells. Although the gene encoding
this protein is known to be ABA regulated (Singh et al.,
1987), osmotin transcripts and protein accumulated in PEGadapted cells without any appreciable increase of cellular
ABA content. As already suggested by Singh et al.
(1987), under prolonged water stress osmotin-induced
mRNAs might be stabilized and a high cellular protein
amount maintained without new gene transcription. This was
further supported by the finding that the osmotin protein
amount in PEG-adapted cells was comparable to the level
immunodetected in PEG-shocked and ABA-treated cells, although the steady-state level of osmotin transcript was lower
in PEG-adapted cells than in PEG-shocked or ABA-treated
cells.
The last question we addressed was whether or not waterstress response had similarities to HS response. Although a
comparison based only on polypeptide electrophoretic properties is not sufficient for an unequivocal result, some of the
hsps in heat-shocked cells were common to proteins synthesized in cells abruptly exposed to water stress but were not
visible in the fluorographs of in vivo-labeled proteins of
gradually adapted cells. Hsps and HS genes have been found
to be expressed in different plant systems in response to
drought (Czarnecka et al., 1984; Heikkila et al., 1984; Chen
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Water Stress and Protein Synthesis in Potato Cells
a n d Tabaeizadeh, 1992; Almoguera e t al., 1993). By using
anti-hsp antibodies, it h a s been recently documented that
low mol w t hsps are induced by water stress in sunflower
plants (Almoguera et al., 1993) and potato plants (Rossouw
et al., 1993). Moreover, Borkird e t al. (1991a, 1991b) have
found that two of the genes cloned by differential screening
from a cDNA library of rice cells adapted to PEG encoded
a n hsp 70 and ubiquitin, respectively. Incorrect protein folding and assembly h a v e been hypothesized a s possible effects
of water stress. Hsps, because of their proposed role a s
molecular chaperones, may help to recover native protein
conformation and/or attenuate t h e effect of stress. Failure to
detect hsps in cells exposed gradually to water stress may
indicate that such a function might not be required i n adapted
cells i n which normal protein synthesis w a s found to be fully
recovered.
The results presented here, based on the comparison of
changes i n gene expression caused by short-term or shock
and long-term exposure to water stress, contribute to the
analysis of stress responses of plant cells, in particular by
distinguishingbetween transient and long-term changes.
Received March 17, 1994; accepted June 13, 1994.
Copyright Clearance Center: 0032-0889/94/106/0703/10.
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