Drought- and ABA-Induced Changes in Polypeptide and mRNA Accumulation in Tomato Leaves.

Abstract

Drought stress triggers abscisic acid (ABA) biosynthesis resulting in ABA accumulation. The ABA-deficient tomato mutant, flacca (Lycopersicon esculentum Mill. cv Ailsa Craig), does not synthesize ABA in response to drought stress. This mutant has been used to distinguish polypeptides and in vitro translation products that are synthesized during drought stress in response to elevated ABA levels from those that are induced directly by altered water relations. A set of polypeptides and in vitro translation products was synthesized during drought stress in the wild type. These polypeptides and in vitro translation products were synthesized to a lesser extent in the drought-stressed ABA-deficient mutant. Treatment of flacca with ABA resulted in the synthesis of the drought-stress-induced polypeptides and in vitro translation products. These results support the hypothesis that many of the polypeptides that are synthesized during drought are regulated by alterations in ABA concentration. Similarly, the mRNA population was altered by ABA during drought stress.

Full text

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Plant Physiol. (1988) 88, 1210-1214
0032-0889/88/88/1210/05/$01.00/0
Drought- and ABA-Induced Changes in Polypeptide and mRNA
Accumulation in Tomato Leaves'
Received for publication May 16, 1988 and in revised form July 16, 1988
ELIZABETH A. BRAY
Department ofBotany and Plant Sciences, University ofCalifornia, Riverside, California
ABSTRACr
Drought stress triggers abscisic acid (ABA) biosynthesis resulting in
ABA accumulation. The ABA-deficient tomato mutant, flacca (Lycoper-
sicon esculentum Mill. cv Ailsa Craig), does not synthesize ABA in
response to drought stress. This mutant has been used to distinguish
polypeptides and in vitro translation products that are synthesized during
drought stress in response to elevated ABA levels from those that are
induced directly by altered water relations. A set of polypeptides and in
vitro translation products was synthesized during drought stress in the
wild type. These polypeptides and in vitro translation products were
synthesized to a lesser extent in the drought-stressed ABA-deficient
mutant. Treatment offlacca with ABA resulted in the synthesis of the
drought-stress-induced polypeptides and in vitro translation products.
These results support the hypothesis that many of the polypeptides that
are synthesized during drought are regulated by alterations in ABA
concentration. Similarly, the mRNA population was altered by ABA
during drought stress.
During drought stress, leaf ABA concentration increases dra-
matically. This increased ABA serves to close stomata, but other
rolesofthis large increase inABA are unknown. The experiments
reported here address the possibility that ABA induces specific
genes during periods of stress. During drought and salt stress,
changes in gene expression have been documented (1, 4-7, 9,
10, 17-21). Induction and repression of genes during drought
stress may be a direct response to environmental conditionsand/
or a response to changes in hormone concentration. Alterations
in ABA concentration during drought stress may serve to coor-
dinate drought responses and the adaptation of the plant to the
environment.
The tomato mutant, flacca, is wilty due to a lack of stomatal
closure (2, 22). This is correlated with decreased ABA content
(13) and can be reversed with ABA application (23). Therefore,
this mutant has been used to implicate ABA's role in stomatal
closure. Flacca has 26% of the ABA of the wild type when it is
well watered (13), and leaves do not produce additional ABA
upon drought stress (13). Therefore, ABA does not accumulate
during drought stress in the mutant as it does in the wild type.
In this study, the pattern ofpolypeptide synthesis and mRNA
accumulation in tomato was determined in response to drought
stress. The ABA-deficient mutant,flacca, was used to determine
if specific proteins and mRNAs accumulate in response to stress
and ifany ofthese proteins are synthesized in response to elevated
ABA concentrations.
'Research supported by a University ofCalifornia, Riverside, Faculty
Development Award to E. A. B.
MATERIALS AND METHODS
Plant Material. Plants of Lycopersicon esculentum cv Ailsa
Craig and the isogenic ABA-deficient mutant flacca (obtained
from Dr. J. W. Maxon Smith at the Glasshouse Crops Research
Institute in Littlehampton, West Sussex, England), were grown
in a growth chamber for 2 to 3 months, and plants were well
watered.
Experimental Treatments. All treatments were completed on
detached tomato leaves. Leaves were removed from the plant
and immediately placed in water. Leaves were kept in water for
nonstress treatments or were wilted on the laboratory bench to
88% of the original fresh weight for drought stress treatments.
Leaves were maintained in a wilted state in plastic bags. Petioles
were placed in 10-' M ABA for ABA treatments. After a 4 h
incubation period, leaflets were removed and were fed 3 gLof
[35S]methionine (45 MCi, 1253 Ci/mmol, Amersham) through
the cut end ofthe petiolule. A 2 h period ofincubation followed.
Leaflets were then frozen in liquid nitrogen.
Protein Extraction and Analysis. Proteins were extracted from
the leaf using a phenol extraction method (18). Briefly, samples
were ground under liquid nitrogen and suspended in extraction
buffer (0.5 M Tris-HCl [pH7.5], 0.1 M KCI, 0.05 M Na2EDTA
[pH 7.4], 2% 2-mercaptoethanol, 0.7 M sucrose). An equal vol-
ume ofwater-saturated phenol was added, and the samples were
mixed at room temperature for 10 min. After centrifugation, the
phenol phase and interface were collected. The phenol phase was
reextracted with extraction buffer. Proteins were precipitated
from the phenol phase with methanol containing 0.77% am-
monium acetate. The pellet was washed with methanol-ammo-
nium acetate and air dried. Incorporation of label into proteins
was determined by TCA precipitation. Protein concentration
was determined using the Peterson assay (15).
Samples containing 350,000 cpm were resuspended in O'Far-
rell (14) lysis buffer and were subjected to two-dimensional
electrophoresis according to O'Farrell (14). Proteins were visu-
alized by autoradiography after the gels were infused with PPO
(0.4% PPO, 55% acetic acid, 15% ethanol, 30% xylene), washed
in water, and dried.
RNA Extraction and in Vitro Translations. Total RNA was
extracted from tomato leaves using a LiCl-phenol extraction
method (16). Briefly, tomato leaves were ground under liquid
nitrogen and were suspended in extraction buffer (50 mM Tris
[pH 9.0], 150 mm LiCl, 5 mm EDTA, 5% SDS). An equal
volume ofphenol:chloroform (1: 1) was added, and after centrif-
ugation, the aqueous phase was recovered. RNA was precipitated
twice in 2 M LiCl and collected by centrifugation. The isolated
RNA was translated in vitro in the presence of [35S]methionine
and [35S]cysteine (10 ,uCi Tran35S-label, ICN, 1105 Ci/mmol)
according to Morch et al. (12). In vitro translation products were
extracted using the phenol extraction method and separated by
two-dimensional gel electrophoresis (14).
1210

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ABA-INDUCED GENE EXPRESSION
70
93kD
69
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93
69
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146
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FIG.
1. In vivo radiolabeled polypeptides from nonstressed leaves
separated by two-dimensional PAGE. Detached leaves from Ailsa Craig
andflacca were kept in water for 4 h. Leaflets were fed [35S]methionine,
incubated for 2 h and polypeptides were separated by two-dimensional
gel electrophoresis. Circled polypeptides were synthesized to a greater
extent in Ailsa Craig. A, Nonstressed Ailsa Craig; B, nonstressedflacca.
ABA Quantitation. ABA was quantified using a competitive
ABA-radioimmunoassay as described by Bray and Beachy (3).
RESULTS
In Vivo Labeled Polypeptides. ABA-deficient mutant and
wild-type leaflets were labeled with [35S]methionine after a 4-h
treatment period, and the polypeptides were separated by two-
dimensional gel electrophoresis. There were no significant differ-
ences in patterns ofpolypeptide accumulation between the non-
stressed flacca and wild type (Fig.
synthesized in the wild type that was not synthesized in the
mutant.
During drought stress, a set of approximately 21 radiolabeled
polypeptides was synthesized in stressed wild type leaves that
was not synthesized or synthesized to a lesser extent in non-
stressed leaves (Figs. IA and 2A). The majority ofthese polypep-
tides ranged from a mol wt of 18 to 24 kD with pIs from 4.25 to
6.7. There were also approximately four polypeptides of higher
mol wt that were synthesized in response to drought stress. A
comparison of polypeptide synthesis during drought stress was
made between the wild type and the ABA-deficient mutant,
flacca. A set of polypeptides was synthesized in the drought-
stressed wild type that was synthesized to a lesser extent in the
drought-stressed flacca (Fig. 2). Several of these polypeptides
were not detected among the polypeptides ofthe stressed mutant.
The set of polypeptides that accumulated only in the wild type
was the set of drought-stress-induced polypeptides. ABA appli-
cation to the wild type and the mutant resulted in synthesis of
this same set of polypeptides (Fig. 3). These results suggest that
1). One polypeptide was
FIG. 2. In vivo radiolabeled polypeptides from drought-stressed leaves
separated by two-dimensional PAGE. Detached leaves from Ailsa Craig
and flacca were wilted to 88% of their original fresh weight and were
then incubated in plastic bags for 4 h. Leaflets were fed [35S]methionine,
incubated for 2 h, and polypeptides were separated by two-dimensional
gel electrophoresis. Circled polypeptides were synthesized to a greater
extent in Ailsa Craig. The polypeptide marked with a triangle was
synthesized to a greater extent inflacca. A, Drought-stressed Ailsa Craig;
B, drought-stressedflacca.
a set of drought-stress polypeptides was synthesized in response
to elevated ABA levels.
Total Protein Synthesis. Total protein synthesis was unaffected
by drought stress and slightly reduced by ABA treatments in the
wild type (Table I). Total protein synthesis was reduced by ABA
and stress treatments in the mutant.
ABA Concentration. ABA accumulation was determined after
drought-stress treatments using a radioimmunoassay (3). ABA
synthesis was induced by drought stress in the wild type (Table
II). There was a 22-fold accumulation of ABA in the drought-
stressed wild type. There was a 3-fold accumulation of ABA in
drought-stressed mutants. The ABA level of the mutant was
approximately 50% ofthe wild type with nonstressed conditions
and 6% of the wild type during drought stress. Feeding ABA to
the leaves resulted in a large increase in ABA concentration of
the leaflet to at least 60,000 ng/g fresh weight.
In Vitro Translation Products. RNA isolated from nonstressed
wild type and mutant leaves was translated in vitro in wheat
germ extract in the presence of[35S]methionineand [35S]cysteine
and separated by two-dimensional gel electrophoresis. One in
vitro translation product was synthesized from RNA isolated
from the nonstressed mutant that was not synthesized from RNA
isolated from the wild type (Fig. 4). The pattern of in vitro
translation products was compared between nonstressed and
stressed wild type. A set of in vitro translation products was
synthesized from RNA isolated from stressed wild type leaves
that was not synthesized or synthesized to a lesser extent from
RNA isolated from the nonstressed leaves (Figs. 4A and 5A).
Comparisons between the genotypes showed that there were also
in vitro translation products that were synthesized from RNA
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Plant Physiol. Vol. 88, 1988
Table II. ABA Concentrations ofDetached Tomato Leafletsfrom Wild
Type (Ailsa Craig) or ABA-Deficient Mutant (flacca)
ABA wasquantified usinganABA-radioimmunoassay. Averageand
SE of four experiments.
93 kD
69
Percent of
Wild-type
Nonstress
Genotype
Treatment
ABA
ng/gfresh wt ± SE
108.4 ± 12.7
2308.5 ± 159.0
57.0 + 8.4
145.8 ± 18.5
Wild type
Nonstress
Water deficit
Nonstress
Water deficit
100
2130
flacca
54
150
4.0
A.
pH
6.5
:i
t69kD
46
30
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A3
FIG. 3. In vivo radiolabeled polypeptides from ABA-treated leaves
separated by two-dimensional PAGE. Detached leaves were treated with
10-' M ABA for 4 h. Leaflets were fed [35S]methionine for 2 h and
polypeptides were separated by two-dimensional gel electrophoresis.
Circled polypeptides were not synthesized in nonstressed leaves and
correspond to the drought-induced polypeptides. A, ABA-treated Ailsa
Craig; B, ABA-treatedflacca.
Table I. Effect ofDrought Stress andABA Treatments on Protein
Synthesis ofDetached Tomato Leaves ofthe Wild Type (Ailsa Craig)
and theABA-Deficient Mutant (flacca)
[35S]Methionine was fed to the leaflets of both genotypes after 4 h of
the different treatments. Incorporation into protein was determined by
TCA precipitation after a 2-h incubation period. Average and SE of six
replications.
Percent of
Nonstressed
Control
% ± SE
100
93 ± 3
97 ± 14
94 ± 8
85 ± 10
68 ± 11
Genotype
Treatment
Wild type
Nonstress
ABA
Water deficit
Nonstress
ABA
Water deficit
flacca
isolated from drought-stressed wild type that were synthesized to
a much lesser extent from the RNA isolated from the stressed
mutant (Fig. 5). The in vitro translation products that were
synthesized only in the wild type were the same products that
were newly synthesized during periods of stress. The treatment
of leaves with ABA for 4 h prior to RNA isolation resulted in
the accumulation ofthis same set of in vitro translation products
in the mutant and wild type that was produced in the wild type
during drought stress (Fig. 6).
14
B.
r
69
46
Vq
30
14
FIG. 4. In vitro translation products of mRNA from nonstressed
leaves separated by two-dimensional PAGE. Detached leaves were in-
cubated for 4 h in water, RNA was isolated from the leaves and was in
vitro translated in the presence of [35S]methionine and [35S]cysteine. In
vitro translation products were separated by two-dimensional gel electro-
phoresis. The circled in vitro translation product was synthesized to a
greater extent inflacca. A, Nonstressed Ailsa Craig; B, nonstressedflacca.
DISCUSSION
Plants respond to alterations in the environment through
changes in metabolic pathways and alterations in other physio-
logical and developmental responses. One means of response is
an alteration of synthesis of specific polypeptides. New polypep-
tides are synthesized in response to drought, salt, and temperature
stress (9, 11, 17, 18). This report also identifies a set ofpolypep-
tides that are synthesized in response to drought stress in tomato.
It is unknown ifthese polypeptides are involved in adaptation to
the stress, and the mechanism of induction is not understood.
During drought stress, the ABA concentration of the plant
increases significantly. Neill and Horgan (13) reportedthatflacca
does not produce additional ABA when it is stressed. However,
there was a small increase in ABA concentration offlacca during
DH
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Page 4

ABA-INDUCED GENE EXPRESSION
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FIG. 5. In vitro translation products ofmRNA from drought-stressed
leaves separated by two-dimensional PAGE. Detached leaves were
drought stressed for 4 h, RNA was isolated from the leaves and in vitro
translated in the presence of [35S]methionine and [35S]cysteine. In vitro
translation products were separated by two-dimensional gel electropho-
resis. Circled in vitro translation products were synthesized to a greater
extent in Ailsa Craig. A, Drought-stressed Ailsa Craig; B, drought-stressed
flacca.
drought stress in these experiments. The difference in these
results may be due to the environmental conditions in which the
plants are grown or the experimental protocols.
An increase in ABA during drought stress is thought to cause
stomatal closure, but it is unknown if there are any additional
responses to the increased ABA concentration. The use of an
isogenic mutant has made it possible to show that the changes
in polypeptide synthesis during stress are probably a result of
increased ABA levels. The ABA-deficient mutant flacca pro-
duced a decreased amount of ABA when it was stressed and
synthesized a specific set of drought-induced polypeptides to a
much lesser extent. Becauseflacca did produce someABA during
stress, the limited production of stress proteins was expected. It
is suggested that elevated ABA levels during drought stress result
in induction of a specific set ofABA-induced polypeptides. The
nature and role of the polypeptides during stress is currently
unknown.
It is generally thought that total protein synthesis is decreased
by stress (8). However, total protein synthesis was only slightly
reduced by drought stress in these experiments and the proteins
that were synthesized during periods of nonstress were not sig-
nificantly reduced.
The accumulation of proteins in response to ABA may be
regulated from transcription to accumulation ofthe mature gene
product. In vivo labeling studies have shown that there is a
difference in accumulation of gene products during stress in the
mutant compared with the wild type. However, these studies do
FIG. 6. In vitro translation products of mRNA from ABA-treated
leaves separated bytwo-dimensional PAGE. Detached leaves were treated
with IO-' M ABA for 4 h, RNA was isolated from the leaves and in vitro
translated in the presence of [35S]methionine and [35S]cysteine. In vitro
translation products were separated by two-dimensional gel electropho-
resis. Circled in vitro translation products correspond to the drought-
induced in vitro translation products. A, ABA-treated Ailsa Craig; B,
ABA-treatedflacca.
not address the level ofthe regulation. In vitro translation prod-
ucts were used to show that there were differences in accumula-
tion ofmRNAs during drought stress among the mutant and the
wild type. A set of mRNAs was increased during drought stress
in response to an increase in the ABA concentration. This
accumulation of specific mRNAs may be due to increased tran-
scription or altered post-transcriptional processing that promote
specific mRNA accumulation'. However, this does not eliminate
the possibility that ABA regulates gene expression at the trans-
lational level; it merely suggests that some regulation occurs at
the level ofmRNA accumulation. In order to continue to study
the mechanism ofgene regulation during drought stress by ABA,
genes that are induced by ABA are currently being isolated.
Acknowledgments-The author would like to thank Stephani Allison for her
excellent technical assistance and Patti Fagan for typing this manuscript.
LITERATURE CITED
1. BEWLEY JD, KM LARSEN, JT PAPP 1983 Water-stress-induced changes in the
pattern ofprotein synthesis in maize seeding mesocotyls: a comparison with
the effects ofheat shock. J Exp Bot 34: 1126-1133
2. BRADFORD KJ 1983 Water relations and growth of the flacca tomato mutant
in relation to abscisic acid. Plant Physiol 72: 251-255
3. BRAY EA, RN BEACHY 1985 Regulation by ABA of,%-conglycinin expression
in cultured developing soybean cotyledons. Plant Physiol 79: 746-750
4. CLOUTIER Y 1983 Changes in the electrophoretic pattern ofthe soluble proteins
of winter wheat and rye following cold acclimation and desiccation stress.
Plant Physiol 71: 400-403
5. DASGUPTA J, JD BEWLEY 1984 Variations in protein synthesis in different
regions of greening leaves of barley seedlings and effects of imposed water
stress. J Exp Bot 35: 1450-1459
4.0
A.
6.5
4.0
A.
I
pH
6.5
B.
69
46
30
69
46
30
14
14
I
1213
'WI

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Plant Physiol. Vol. 88, 1988
6. DHINDSA RS, RE CLELAND 1975 Water stress and protein synthesis. I. Differ-
ential inhibition of protein synthesis. Plant Physiol 55: 778-781
7. GULICK P, J DVORAK 1987 Gene induction and repression by salt treatment
in roots of the salt-sensitive Chinese Spring wheat and the salinity-tolerant
Chinese Spring x Elytrigia elongata amphiploid. Proc Natl Acad Sci USA
84: 99-103
8. HANSON AD, WD HITZ 1982 Metabolic responses of mesophytes to plant
water deficits. Annu Rev Plant Physiol 33: 163-203
9. HEIKKILA JJ, JET PAPP, GA SCHULTZ, JD BEWLEY 1984 Induction of heat
shock protein messenger RNA in maize mesocotyls by water stress, abscisic
acid and wounding. Plant Physiol 76: 270-274
10. JACOBSEN JV, AD HANSON, P CHANDLER 1986 Water stress enhances expres-
sion of an a-amylase gene in barley leaves. Plant Physiol 80: 350-359
11. KEY JL, CY LIN, YM CHEN 1981 Heat shock proteins of higher plants. Proc
Natl Acad Sci USA 78: 2356-2530
12. MORCH MD, G DRUGEON, W ZAGORSKI, AL HAENNI 1986 The synthesis of
high-molecular-weight proteins in the wheat germ translation system. Meth-
ods Enzymol 118: 154-164
13. NEILL SJ, R HORGAN 1985 Abscisic acid production and water relations in
wilty tomato mutants subjected to water deficiency. J Exp Bot 36: 1222-
1231
14. O'FARRELL PH 1977 High resolution two-dimensional electrophoresis of pro-
teins. J Biol Chem 250: 4007-4021
15. PETERSON GL 1977 A simplification of the protein assay method of Lowry et
al., which is more generally applicable. Anal Biochem 83: 346-356
16. PREscorr A, C MARTIN 1987 A rapid method for the quantitative assessment
of levels of specific mRNAs in plants. Plant Molecular Biology Reporter 4:
219-224
17. RAMAGOPAL S 1987 Differential mRNA transcription during salinity stress in
barley. Proc Natl Acad Sci USA 84: 94-98
18. RAMAGOPAL S 1987 Salinity stress induced tissue specific proteins in barley
seedlings. Plant Physiol 84: 324-331
19. SINGH NK,AK HANDA, PM HASEGAWA, RA BRESSAN 1985 Proteinsassociated
with adaptation of cultured tobacco cells to NaCl. Plant Physiol 79: 126-
137
20. SINGH NK, PC LAROSA, AK HANDA, PM HASEGAWA, R BRESSAN 1987
Hormonal regulation of protein synthesis associated with salt tolerance in
plant cells. Proc Natl Acad Sci USA 84: 739-743
21. SINGH NK, CA BRACKER, PM HASEGAWA, AK HANDA, S BUCKEL, MA
HERMODSON, F REGNIER, R BRESSAN 1987 Characterization of osmotin: a
thaumatin-like protein associated with osmotic adaptation in plant cells.
Plant Physiol 85: 529-536
22. TAL M 1966 Abnormal stomatal behavior in wilty mutants of tomato. Plant
Physiol 41: 1387-1391
23. TAL M, Y NEVO 1973 Abnormal stomatal behavior and root resistances and
hormonal imbalance in three wilty mutants of tomato. Biochem Gent 8:
29 1-300
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BRAY