attempted and it succeeded in isolating stress-in- ducible genes from those species. Among the cD-
NAs obtained from the green leaves of N. paniculata, two clones were found with a homol-
ogy to plastidic fructose-1,6-bisphospate aldolase AldP genes.
AldP catalyzes the cleavage of fructose-1,6-bis- phosphate FBP into
D
-glyceraldehyde-3-phos- phate GAP and dihydroxyacetone phosphate
DHAP. Two forms of aldolase are present in higher plants: cytoplasmic and plastidic. The cy-
toplasmic aldolase AldC gene is inducible un- der anaerobic conditions and is considered to
have an important role in producing ATP by stimulating glycolysis under such conditions [5 –
9]. AldP is involved in the photosynthetic carbon reduction cycle and catalyzes the synthesis of
FBP from GAP and DHAP. AldP gene expres- sion is regulated by light [10], but the effect of
other environmental factors on AldP is nu- known. Here, the isolation of two AldP genes
from a salt-tolerant Nicotiana species, N. panicu- lata is reported and their expression regulation
among three Nicotiana species are compared with different salt-tolerance abilities under salt
stress.
2. Materials and methods
2
.
1
. Plant materials Plants were grown from seeds in a greenhouse.
Seedlings were transferred to plastic pots 10 cm in diameter filled with a mixture [1:1 vv] of
vermiculite and Hydroball small particles of brick, Jongkind Ground B.V., Aalsmeer, the
Netherlands 3 weeks after germination. The plants were watered daily with 100 ml of 25
Hoagland’s solution Hoagland’s Solution No. 2, Sigma. They were grown in the greenhouse for
an additional 7 days, and then were transferred to a growth chamber 1 week before stress was
applied. The growth chamber was operated on a 12-h light 23°C12-h dark 20°C cycle. Light
was provided by 400 W metal halide lamps at a constant 500 – 600 mEm
− 2
s
− 1
. Salt stress was initiated 1 h after illumination had started by
watering with 100 ml of 25 Hoagland’s solu- tion containing 250 mM NaCl and 100 ml of the
same solution was watered daily thereafter. Con- trol plants were watered daily with 100 ml of
25 Hoagland’s solution only. Plants for the ex- periment examining organ specificity were grown
in the greenhouse from sowing to anthesis. Plants for the experiment examining the daily
cycle were grown in the growth chamber under the same conditions as above.
3. Differential screening
Total RNA was isolated from green leaves of N. paniculata 24 h after stress was applied, as
described by Ostrem et al. [11]. PolyA
+
RNA was purified using a QuickPrep mRNA Purifica-
tion Kit Amersham Pharmacia Biotech, and a cDNA library was constructed with the polyA
+
RNA using a ZAP-cDNA Synthesis Kit Strata- gene. This library was subjected to a differential
screening. Plaque lifts were done as described in the manufacturer’s manual, except that 3000
plaques were plated per plate. Membranes for hybridization were made in duplicate from each
plate.
32
P-labeled first-strand cDNA probes were prepared from the above polyA
+
RNA from stressed plants and polyA
+
RNA isolated from control plants. The reverse transcription reaction
was done at 37°C for 1 h in 50 ml of reaction mixture containing 50 mM Tris – HCl pH 8.3,
75 mM KCl, 3 mM MgCl
2
, 10 mM dithiothrei- tol, 1 mM dATP, 1 mM dGTP, 1 mM dTTP,
100 ng of polyA
+
RNA, 2.5 mg of random primers 9 mer Takara, Shiga, Japan, 35.5 U
of RNAguard Amersham Pharmacia Biotech, 1.85 MBq of [a-
32
P]dCTP Amersham Pharmacia Biotech, and 10 U of M-MLV reverse transcrip-
tase Toyobo, Osaka, Japan. Unincorporated [a-
32
P]dCTP was removed from the mixture using a Quick Spin Column Boehringer Mannehim af-
ter the reaction. Each set of membranes was hy- bridized with either the control probe or the
stress probe. The membranes were hybridized at 65°C for 20 h in a solution containing 125 mM
Na
2
HPO
4
, 1 mM EDTA, and 7 SDS. After hybridization the membranes were washed twice
at 65°C for 20 min in a solution containing 2 × SSC and 0.1 SDS, and then twice in a solution
containing 0.2 × SSC and 0.1 SDS under the same conditions. Hybridized membranes were
analyzed using FUJX BAS1000 Fuji Photo Film, Tokyo, Japan, and signal strength of cor-
responding plaques was compared.
3
.
1
. DNA and deduced amino acid sequence analyses
Nucleotide sequences of the clones were deter- mined using an automatic DNA sequencer Prism
310, PE Applied Biosystems. DNA and deduced amino acid sequence analyses of obtained genes
and their products, including phylogenetic study, were made using Genetyx-Mac ver. 9.0 Software
Development, Tokyo, Japan. Database searches were made using the GenomeNet WWW Server of
Kyoto University http:www.genome.ad.jp.
3
.
2
. Northern blot analysis Total RNA was isolated from the leaves of
stressed plants and leaves, roots, flowers, stems, or petioles of unstressed plants, as described by Os-
trem et al. [11]. Northern blotting was done with 5 m
g of LiCl-purified total RNA, as described by Vernon and Bohnert [12].
32
P-labeled probes were prepared from the whole cDNA regions using a
Rediprime DNA labeling system Amersham Pharmacia Biotech and were used for hybridiza-
tion. Hybridization was done in the same condi- tion as for differential screening. Hybridized
membranes were analyzed and the signals were quantified using FUJX BAS1000.
3
.
3
. Southern blot hybridization Total DNA was isolated from leaves of each
examined species, as described by Hiei et al. [13]. Southern blot hybridization was done with 20 mg
of total DNA digested with restriction enzymes, using the same method as for Northern blot
analysis.
4. Results
