number of cDNA clones corresponding to salt-re- sponsive genes have been identified and isolated
from cDNA libraries constructed using RNA from salt-treated plant tissues [9 – 13]. Those that have
been isolated from salt-treated roots include ger- min [14], the early salt-stress responsive cDNAs
from Lophopyrum elongatum [9] and several cD- NAs associated with salt-tolerance in rice [15,16].
The expression of many of these genes in roots is responsive to abscisic acid ABA and it has subse-
quently been proposed that ABA is the primary regulator of salt-induced changes in gene expres-
sion [17]. ABA is known to regulate the expression of many genes in response to other environmental
stresses, particularly water-deficit-stress [18]. How- ever, in salt-challenged plants, a role for ABA has
largely been based on enhanced gene expression in response to the application of exogenous ABA to
unstressed plant tissues. Several studies have ad- dressed the role of endogenous ABA in regulating
salt-responsive genes, however they, have exam- ined expression in either leaves or whole seedlings
[19 – 22]. Very few studies have examined directly the role of endogenous ABA in eliciting salt-in-
duced changes in gene expression in roots.
The recently developed differential display or DD-PCR technique [23] provides a sensitive and
flexible approach to the identification of differen- tially expressed genes. This method has been used
successfully to identify several cDNAs correspond- ing to genes regulated by gibberellic acid [24,25],
ozone [26], salt [27], heat [28], senescence [29], sucrose [30] and those differentially expressed dur-
ing development [31 – 34]. Differential display was used in this study to analyze changes in mRNA
populations that occur in salt- and ABA-treated tomato roots and to identify novel salt-responsive
genes. DD-PCR and RNA blot hybridization data are presented that indicate that a substantial pro-
portion of the gene expression that occurs in salt- treated
roots appears
to be
regulated independently of ABA.
2. Methods
2
.
1
. Materials Seeds of tomato Lycopersicon esculentum Mill.
cv. Ailsa Craig and the near-isogenic ABA-defi- cient mutant flacca flc were germinated in moist-
ened vermiculite contained within a plastic grid 1.5 × 1.5 cm that was lined with a plastic mesh
and housed in a plastic tray. Upon germination, the roots grew down through the mesh to contact
the Murashige and Skoog MS nutrient solution 23-strength, [35] in the tray below. The MS
nutrient solution was changed twice weekly and aerated. Plants were maintained in a growth cham-
ber Conviron Basic Model I25L Incubator in the light at 25°C, 70 relative humidity for 16 h; in
the dark at 21°C and 70 relative humidity for 8 h.
2
.
2
. Experimental treatments Six-week-old plants were used for all the experi-
ments. A salt treatment was imposed by the addi- tion of NaCl to the MS nutrient solution to reach
a final concentration of 170 mM. Plants were exposed to NaCl for varying periods of time rang-
ing from 0 to 24 h. Exogenous ABA and com- bined NaClABA treatments were imposed by
exposing plants to 100 mM ABA mixed isomers, + −
cistrans ABA; Sigma and 100 mM ABA together with 170 mM NaCl, respectively. Plants
were exposed to these treatments for 24 h during which time aeration of the media was maintained.
Control plants were transferred to and maintained in MS nutrient solution for the duration of the
experimental period. Following each treatment the roots were harvested and frozen in liquid N
2
be- fore storing at − 80°C until needed. All treat-
ments were performed at least twice.
2
.
3
. RNA extraction Frozen roots were ground to a fine powder in
liquid nitrogen. Total RNA for DD-PCR was extracted using the Plant RNeasy System Qiagen,
Mississauga, Ontario, Canada following the man- ufacturer’s instructions. Total RNA for Northern
blot hybridization analyses was extracted using the LiCl-phenol method described by Prescott and
Martin [36].
2
.
4
. Differential display Differential display was carried out according to
Liang and Pardee [23] and Bauer et al. [37]. A DD-PCR primer set was obtained from the Bio-
technology Laboratory University of British Co-
lumbia, Vancouver, BC, Canada. Each reverse transcription RT reaction contained 5 mM KCl,
10 mM Tris – HCl pH 8.3, 4 mM MgCl2, 20 mM dNTPs, 1.5 mM anchor primer T
11
GG, T
11
GC, T
11
CG, or T
11
CC, 0.2 mg RNA, 20 units RNase inhibitor Perkin Elmer, Foster City, CA, USA
and 50 U MuLV reverse transcriptase Perkin Elmer, Foster City, CA, USA. The RT reaction
was performed at 37°C for 60 min followed by incubation at 95°C for 5 min. The subsequent
PCR contained 2 mM MgCl
2
, 0.25 mM arbitrary primer
TACAACGAGG, TGGATTGGTC,
CTTTCTACCC, TTTTGGCTCC,
GGAAC- CAATC, AAACTCCGTC, TCGATACAGG, or
TGGTAAAGGG, 1.5 mM anchor primer, 20 mM dNTPs, 2 ml RT products, 0.5 U DNA polymerase
supplied with its own buffer Ultratherm, BioCan Scientific, Mississauga, Ontario, Canada, and
0.074 mBq
33
P-dATP Amersham, B’aie d’Urfe, Quebec, Canada. Each PCR cycle consisted of
94°C for 30 s, 40°C for 2 min and 72°C for 30 s, the reaction was subjected to 40 cycles with a 5
min extension at 72°C following the final cycle. One-fifth of the PCR products was loaded on a
6 acrylamide8.3M urea gel. The gel was run for 3.5 h at 55 W constant power after which it was
transferred to Whatman 3MM paper, dried under vacuum at 80°C for 2 h, and exposed to an X-ray
film Kodak X-Omat blue XB-1 for 24 – 48 h. Bands of interest were excised from the gel and the
resulting gel slices were boiled in 100 ml dH
2
O for 15 min. After centrifugation, the supernatant was
directly used for reamplification, which was per- formed using the same primer set and PCR condi-
tions as described above except the dNTP concentration was 200 mM and no isotope was
added. RT reactions were performed at least twice for each RNA sample, and the subsequent PCR
step was duplicated at least twice for each primer combination.
2
.
5
. Cloning, sequencing and analyses Reamplified cDNA products were cloned into
plasmid vectors using the TA Cloning System from Invitrogen San Diego, CA, USA. The
cloned partial cDNA sequences were sequenced using an ABI 377 automatic sequencer. The nucle-
otide sequences obtained were submitted to the NCBI server for BLASTN and BLASTX searches
against nucleotide and protein sequences deposited in various databases [38].
2
.
6
. RNA blot hybridization analyses Total RNA 20 mg was size separated on a
formaldehyde denaturing 1.2 agarose gel accord- ing to Sambrook et al. [39]. RNA was capillary
transferred to a positively charged nylon mem- brane Boehringer Mannheim, Laval, Quebec,
Canada using 20 × SSC as the transfer medium. RNA was fixed to the membrane by UV-crosslink-
ing for two min UV Stratalinker 2400 followed by baking at 80°C for 30 min. Membranes were
prehybridized in 100 mM tetrasodium pyrophos- phate, 50 mM sodium phosphate, 7 SDS, 1 mM
EDTA at 65°C for 2 h FSB [40]. Partial cDNA inserts were labeled with 1.85 MBq a-
32
P-dCTP Amersham, B’aie d’Urfe, Quebec, Canada using
the Random Prime-it kit Stratagene, La Jolla, CA, USA. Hybridization continued in the same
buffer containing 10
7
– 10
8
cpm
32
P-labelled probe for 16 h at 65°C. Membranes were washed two
times in FSB1 SDS at 65°C for 45 min each time and then once in the same buffer at 68°C for
45 min. The washed membranes were exposed to autoradiography film Kodak X-Omat blue XB-1
with a single intensifying screen at − 80°C. All RNA blots were performed twice.
3. Results
