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
3
.
1
. Effect of a salt treatment on root RNA populations
To examine the impact of a salt treatment on root RNA populations, 6-week-old tomato plants
were exposed to 170 mM NaCl for 24 h. This concentration of salt has previously been shown to
elicit the accumulation of a number of distinct polypeptides in tomato roots [8]. RNA from salt-
treated and non-treated control roots was used for DD-PCR with four anchor and eight arbitrary
primers in all possible combinations. Parallel com- parisons of treated and control DD-PCR profiles
revealed differences indicative of a salt-induced alteration of the root RNA population. In total,
approximately 2000 products were displayed, 57 of which were induced or up-regulated and 46 de-
pressed or down-regulated. Fig. 1 shows represen- tative gels of DD-PCR products generated from
RNA isolated from salt-treated and non-treated roots.
To determine how rapidly changes in the mRNA population occurred following the onset of
a salt treatment, time course experiments were carried out. RNA was isolated from tomato roots
0, 0.5, 2, 8 and 24 h following the onset of a salt treatment and used for DD-PCR. The results ob-
tained confirmed the majority of the previously observed salt-induced, -enhanced and -repressed
partial cDNAs. In addition, more novel induced or up-regulated cDNA products generated from
RNA isolated between 0.5 and 8 h following the onset of a salt treatment were identified, indicating
Fig. 1. DD-PCR products generated from mRNA isolated from roots exposed to 170 mM NaCl NaCl or MS nutrient solution C for 24 h. DD-PCR was performed with primers [T
11
]GC or [T
11
]CC and TACAACGAGG, respectively A and B and [T
11
]CC and GGAACCAATC C. Duplicate lanes displaying products generated from two independent PCR amplifications are shown. DD-PCR products that exhibited consistent differences between treatments are indicated. Arrows with a closed and open
head indicate cDNAs derived from RNA that was more or less abundant in salt-treated roots, respectively. Arrows marked with an asterisk indicate DD-PCR fragments that were excised from the gel and cloned. The original JWS-17 band is marked.
Fig. 2. DD-PCR products generated from mRNA isolated from non-treated roots C, roots exposed to NaCl for 0.5 h NaCl, 0.5, 2 h NaCl, 2, 8 h NaCl, 8 and 24 h NaCl, 24, and roots exposed to ABA ABA or combined NaClABA NaClABA
treatments for 24 h. DD-PCR was performed with primers [T
11
]CC and TGGATTGGTC A and [T
11
]CC and GGAACCAATC B. Duplicate lanes displaying products generated from two independent PCR amplifications are shown. DD-PCR products that
exhibited consistent differences are indicated. Arrows with a closed and open head indicate cDNAs derived from RNA that was more or less abundant in salt-treated roots, respectively. Arrows marked with an asterisk indicate DD-PCR products that were
excised from the gel and cloned. The original JWS-17 and JWS-19 bands are marked.
that several genes were transiently expressed dur- ing a period of salt-stress see JWS-19 in Fig. 2.
The salt-affected DD-PCR products were catego- rized into salt-induced, up-regulated or down-reg-
ulated groups Table 1. Table 1 also indicates the time-dependent accumulation of various members
of each group in salt-affected roots.
3
.
2
. Effect of an ABA and a combined ABAsalt treatment on root RNA populations
To investigate the role played by ABA in medi- ating salt-induced changes in root RNA popula-
tions, roots were exposed to ABA and combined ABAsalt treatments for 24 h. An ABA treatment
induced and enhanced the accumulation of several cDNAs corresponding to mRNAs whose level was
unaffected by salt Fig. 2, Table 1. In addition to these, ABA enhanced the accumulation of several
cDNA products that were derived from salt-re- sponsive genes Table 1. However in general, an
ABA treatment resulted in a DD-PCR profile with distinct differences compared to that elicited by a
salt treatment Fig. 2. A combined ABAsalt treatment induced similar changes in root RNA
populations to that induced by a salt treatment
alone Fig. 2, Table 1. However, in several in- stances the combined treatment either further en-
hanced or further repressed the accumulation of certain cDNAs over that induced by salt alone see
JWS-17 in Fig. 2, Table 1.
3
.
3
. Cloning and sequencing of salt-responsi6e cDNA fragments
Differential display clearly demonstrated that changes in the mRNA population of roots oc-
curred during a period of salt stress. A total of 46 cDNA products of interest were excised and 40 of
these were successfully re-amplified and cloned. The majority of these represented DD-PCR prod-
ucts with an induced or enhanced accumulation in salt-treated roots. One represented a DD-PCR
product with a repressed accumulation and one other accumulated only in response to ABA. The
insert of each of these clones was sequenced and this revealed that, among the 40 cDNA clones, 20
contained distinct sequences.
Each of the 20 cDNA sequences Table 2 was submitted to the NCBI server for comparison to
sequences residing in the GenBank nucleotide and dbEST databases using BLAST programs [38].
Four DD-PCR products were similar to known sequences deposited in these databases. The nucle-
otide sequence of the partial cDNA JWS-17 was similar to that of laccase genes isolated from vari-
ous plant species. At the amino acid level JWS-17 shared 58 identity with a portion of the N-termi-
nal domain of the laccase from Acer pseudopla- tanus, while the percent identity shared with
laccase
proteins from
Liriodendron tulipifera,
Oryza sati6a, Arabidopsis thaliana and Nicotiana tabacum ranged from 48 to 56. An alignment of
the amino acid sequences is shown in Fig. 3A. The nucleotide sequence of the cDNA insert of JWS-20
was similar to the DNA sequence of a tobacco oxygenase
piox and
Arabidopsis feebly-like
protein. At the amino acid level there was 91 identity between the amino acid sequence derived
from JWS-20 and the C-terminal portion of the tobacco PIOX Fig. 3B. The partial cDNA, JWS-
18, shares 67 and 65 amino acid sequence iden- tity with the central portion of a rice clone and an
Arabidopsis mitotic control protein DIS3 that both share similarity with Schizosaccharomyces pombe
DIS3 Fig. 3C. The JWS-22 insert shares 46, 50 and 41 amino acid sequence identity with the
C-terminus of a yeast DNA repair protein, a hypothetical helicase from Mycoplasma pneumo-
niae and a human helicase-like transcription fac- tor, respectively Fig. 3D. Two other partial
cDNAs were similar to deposited sequences. JWS- 1 shares 89 – 90 nucleotide sequence identity with
a part of five cDNAs derived from Botrytis cinerea grown under conditions of nitrogen deprivation
Table 2. These cDNAs are all similar to an
Table 1 Accumulation of DD-PCR products corresponding to RNA in salt-, ABA- and ABANaCl-treated tomato roots
Type
a
Group ABANaCl
ABA 24 h
8 h 2 h
0.5h Con.
A −
− +
++ ++
− +++
Salt-induced B
− −
− −
++ +
− C
++ +
++ ++
+ −
− D
− +
+ −
− −
− A
+ +
+ ++
++ +
++ Salt up-regulated
+ +
+ ++
++ +
+ B
+ ++
++ C
+ +
+ +
+ +
+ ++
+++ ++
+ D
− −
− +
− +
++ A
Salt down-regulated ++
++ +
+ −
− −
B C
+ +
+ +
− −
− D
+ −
− −
+ +
+ ++
+++ +
+ +++
++ E
+ A
ABA-regulated −
− −
− −
+ −
B +
+ +
+ +
++ +
a
Type ‘A’, ‘B’, ‘C’ etc. correspond to groups of DD-PCR products with different accumulation patterns. ‘+’, ‘++’ etc. denotes the presence and relative intensity of DD-PCR products on differential display gels. ‘−’ denotes the absence of DD-PCR
product.
Fig. 3. A Alignment of the deduced amino acid sequence of JWS-17 with the amino acid sequence of laccases from O. sati6a Os AAC04576; Liriodendron tulipifera Lt AAB17191; A. thaliana At AAC33238 A. pseudoplatanus Ap AAB09228 and
N. tabacum Nt JC5229. Residues that are identical in all the sequences are shaded with black, identical in five out of six sequences are shown as black letters on a dark gray background and in four out of six are shown as black letters on a light gray
background. B Alignment of the deduced amino acid sequence of JWS-20 with the amino acid sequence of PIOX from N. tabacum Nt CAA07589 and A. thaliana feebly-like protein At AAF24612. Residues that are identical in all the three
sequences are shaded in black and those identical in two of the three sequences are shown as black letters on a dark gray background. C Alignment of the deduced amino acid sequence of JWS-18 with the deduced amino acid sequence derived from
an O. sati6a cDNA Os BAA85401 and an A. thaliana mitotic control protein DIS3 At AAD32908. Residues that are identical in all the three sequences are shaded in black and those identical in two of the three sequences are shown as black letters
on a dark gray background. D Alignment of the deduced amino acid sequence of JWS-22 with those of a Saccharomyces cere6isiae DNA repair protein Sc P32849, a Mycoplasma pneumoniae hypothetical helicase Mp P75093, and a Homo sapiens
helicase-like transcription factor Hs S49618. Residues that are identical in all the sequences are shaded in black and those identical in three of the four are shown as black letters on a dark gray background and in two of the four as black letters on a
light gray background.
Table 2 Homology search result for partial cDNAs derived from salt-treated tomato roots
a
Insert size bp CDNA
Homology search result Accession
273 B. cinerea cDNAs: AL114512, AL111963, AL111150, AL113068, AL116383
JWS-1 AW160093
241 AW160094
A. thaliana copia-type polyprotein CAB71063 JWS-3
AW160095 JWS-4
212 No homology
AW160096 JWS-6
261 No homology
196 AW160097
No homology JWS-7
JWS-12 AW160098
217 No homology
239 AW160099
No homology JWS-13
223 JWS-14
No homology AW160100
348 AW160101
No homology JWS-15
356 G. max cDNA AI941153
JWS-16 AW160102
266 AW062236
A. pseudoplatanus diphenol oxidase AAB09228, L. tulipifera laccase AAB17191, JWS-17
O. sati6a laccase AAC04576, L. esculentum ESTs 264578, 256683 322
JWS-18 O. sati6a cDNA AU068209 BAA85401, A. thaliana putative mitotic control
AW160103 protein DIS3 AAD32908
365 JWS-19
L. esculentum ESTs 253019, 244515, 252952, 269112, 245681 AW062237
JWS-20 251
AW062238 N. tabacum PIOX CAA07589; A. thaliana feebly-like protein AAF24612; A.
thaliana cDNAs AI099632, AA042387, Z27292; L. esculentum EST 303018 485
JWS-21 L. esculentum ESTs 244533, 267214, 245863, 245246; A. thaliana hypothetical
AW062239 protein AAD20686; A. thaliana putative protein CAB72159
236 JWS-22
S. cere6isiae DNA repair protein P32849, M. pneumoniae hypothetical helicase AW160104
P75093, H. sapiens helicase-like transcription factor S49618; A. thaliana DNA AB016875
JWS-23 447
AW062240 L. esculentum ESTs 261264, 256193, 283863
298 JWS-24
No homology AW160105
272 AW062241
No homology JWS-26
AW062242 JWS-27
429 L. esculentum ESTs 275366, 242631; L. penellii EST 309721
a
RNA blot hybridization analyses indicated that JWS-17, -19, -20, -21, -26 and -27 correspond to salt-responsive genes. JWS-7 was down-regulated by salt, JWS-13 and -23 correspond to constitutively expressed genes, and JWS-24 was ABA-responsive. No
expression was detected for the remainder.
aconitase gene of Aspergillus terreus. JWS-3 shares 31 amino acid sequence identity with an Ara-
bidopsis hypothetical protein that itself is similar to a putative reverse transcriptase of Arabidopsis
Table 2. Five other cDNAs were similar to vari- ous ESTs of tomato or soybean and hypothetical
proteins of Arabidopsis Table 2. No significant similarity to any sequence residing in the data-
bases was found for the cDNA inserts of the remaining nine clones. It is possible that the short
length of some of the differential display products prevented a higher rate of putative identification.
The sequence for each of the JWS clones has been deposited in the Genbank EST database see
Table 2 for accession numbers.
3
.
4
. RNA blot hybridization analyses The majority of the isolated DD-PCR cDNAs
correspond to mRNAs whose abundance in- creased in salt-treated roots. RNA blot hybridiza-
tion analyses were, therefore, carried out to confirm that the corresponding genes were ex-
pressed in a salt-dependent manner. RNA was isolated from tomato roots treated with 170 mM
NaCl for 0, 0.5, 2, 8, and 24 h and used for RNA blot hybridization analyses. Of the 20 cDNAs, the
expression of the genes corresponding to two was constitutive whereas that of six others was induced
or up-regulated in a salt-dependent manner. Sev- eral others gave no signal on RNA blots or corre-
spond to genes expressed at a very low level in salt-treated roots. It is possible that these do not
represent salt-responsive genes or, that they corre- spond to low abundance mRNAs that are not
readily detected by RNA blot hybridization analyses.
Genes corresponding to the partial cDNA clones JWS-17, -19, -20, -21, -26 and -27 were
clearly expressed at an enhanced level in salt- treated roots Fig. 4. Expression of the gene
corresponding to JWS-17 was detected at 8 h
following a salt treatment when a low level of transcript was present and at 24 h when the level
increased. The estimated size 2.1 kb of the tran- script corresponding to JWS-17 is consistent with
the reported size of laccase mRNA from L. tulip- ifera [41]. A different expression pattern was ob-
served for the gene corresponding to JWS-20 for which mRNA was clearly present at the 0 h time
point. Transcript accumulation was observed at 2, 8 and 24 h after the onset of a salt treatment. The
JWS-20 transcript was approximately 2.3 kb, which corresponds to the reported size of the piox
transcript [42]. A low level of JWS-21 expression was apparent at 0 h and the level increased there-
after for the duration of a 24 h salt treatment. Genes corresponding to JWS-19, -26 and -27
were all transiently expressed in salt-treated roots Fig. 4. Expression of the genes corresponding to
each of these cDNAs was induced rapidly, within 0.5 h of a salt stress and, in the case of JWS-26
and – 27, was apparent at 2 and 8 h after which it declined. Expression of the gene corresponding to
JWS-19 was not apparent beyond the 2 h time point. Two transcripts hybridized to the JWS-19
and -27 probes, both of which accumulated in a transient manner in salt-treated roots.
The expression pattern of the genes correspond- ing to these partial cDNAs was similar to that
observed on the original differential display gel indicating that the overall pattern of displayed
cDNAs on these gels was representative of actual changes in the root RNA population Figs. 2 and
4, compare the expression patterns for JWS-17 and -19.
3
.
5
. Regulation of gene expression by ABA It has been proposed that ABA regulates
changes in gene expression that occur in salt- stressed roots [17]. A comparison of DD-PCR
products generated from salt or ABA-treated roots revealed that distinct RNAs accumulated in
response to each of these treatments Fig. 2, Table 1. This finding suggested that there might be an
ABA-independent component that is involved in the regulation, at least in part, of many of the
salt-induced changes in root RNA population. Therefore, the expression of genes corresponding
to the six salt-responsive cDNAs was examined in the roots of ABA-treated plants as well as those
exposed to a combined ABAsalt treatment. In addition, expression was examined in salt-treated
roots of flacca flc, an ABA-deficient mutant of tomato.
The expression of the genes corresponding to JWS-20 and -21 was responsive to exogenous
ABA Fig. 4. The transcript level induced by ABA was equivalent to that present in salt-treated
roots for JWS-21 but significantly lower for JWS- 20. No expression of genes corresponding to any
of the other partial cDNAs was apparent in ABA- treated roots. The imposition of a salt treatment
together with exogenous ABA elicited an en- hanced transcript level over that present when
either treatment was applied alone for JWS-17,
Fig. 4. Expression of genes corresponding to DD-PCR prod- ucts in salt-treated roots. RNA was isolated from roots
exposed to 170 mM NaCl for 0 h, 0, 0.5 h NaCl, 0.5, 2 h NaCl, 2, and 8 h NaCl, 8, and to MS nutrient solution C,
170 mM NaCl NaCl, ABA ABA and NaCl plus ABA ABANaCl for 24 h. RNA was resolved on denaturing
agarose gels, blotted onto nylon membranes and hybridized with the probes JWS-17, JWS-19, JWS-20, JWS-21, JWS-26
and JWS-27. The blots hybridized with the JWS-17, -21, and -27 probes were exposed to X-ray film for 16 h, those
hybridized with JWS-26 and -19 for three days, and the blot hybridized to JWS-20 was exposed for 5 days. A representa-
tive ethidium-stained agarose gel is shown to indicate approx- imate equal loading of RNA samples.
Fig. 5. Expression of genes corresponding to DD-PCR prod- ucts in salt-treated flacca roots. RNA was isolated from roots
exposed to 170 mM NaCl for 0 h 0, 0.5 h NaCl, 0.5, 2 h NaCl, 2, 8 h NaCl, 8 and 24 h NaCl, 24, and roots
exposed to ABA ABA for 24 h. Blots were hybridized with JWS-17, -19, -20, -21, -26 and -27. Exposure times were the
same as those listed in the legend for Fig. 4. A representative ethidium-stained agarose gel is shown to indicate approximate
equal loading of RNA samples.
sion was apparent in flc roots at the 0 h time-point than in AC roots. In addition, a lower transcript
level corresponding to JWS-21 was apparent at the 0.5 h time point in flc roots relative to AC roots.
In the roots of flc, only the genes corresponding to JWS-20 and JWS-21 were expressed in response to
exogenous ABA.
4. Discussion
