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
4
.
1
. Isolation and sequence analyses of AldP genes In differential screening of a leaf cDNA library
from N. paniculata, which had been stressed for 24 h by adding 250 mM NaCl to the nutrient solu-
tion, 2 × 10
5
plaques were screened and 61 inde- pendent clones were isolated. By exploratory
partial sequence analyses, two clones were found to have a high homology to aldolases from higher
plants. A comparison of the complete nucleotide sequences of these clones showed that the clones
are products of different genes, NpAldP
1
and NpAldP
2
. The nucleotide sequences for these clones were deposited in the EMBLGenBank
DDBJ under accession numbers AB027001 and AB027002. These clones had 1384 and 1431 bp,
respectively excluding poly adenylation sites, and had a reading frame of 395 and 398 amino acids,
respectively. They are 75 homologous at the nucleotide level and 91 identical at the amino
acid level. An amino acid sequence comparison with known aldolases from higher plants showed a
putative transit peptide region at the amino termi- nal end of these products, which led to the conclu-
sion that these products were AldP Fig. 1a. A phylogenetic study based on the amino acid se-
quences also supported this conclusion Fig. 1b. Their similarity with other AldP and AldC was
70 – 85 and 54 – 60, respectively.
4
.
2
. Gene complexity Hybridization of NpAldP
1
or NpAldP
2
to total DNA of N. paniculata, N. excelsior, and N.
arentsii was done under high stringency condi- tions. NpAldP
1
cDNA has no EcoRI site and three HindIII sites, and NpAldP
2
cDNA has one EcoRI site and one HindIII sites. Therefore, the
band patterns in N. paniculata indicate this species has one or two copies of the AldP1 and AldP2
genes Fig. 2, but further information is needed about restriction sites of their genomic sequences
to give more precise conclusions. There were a few bands in each lane in N. arentsii, and this pattern
indicates that this species has one or two copies of AldP1 and AldP2 genes Fig. 2. The band pat-
terns in N. excelsior were distinct from those of N. paniculata or N. arentsii; there were several bands
in each lane Fig. 2. This result indicates that the gene structure of N. excelsior is distinct from the
other two species and has several copies of AldP1 and AldP2 genes.
4
.
3
. Organ specificity of NpAldP
1
and NpAldP
2
expression The organ specificity of NpAldP
1
and NpAldP
2
gene expression was investigated in green leaves, roots, flowers, stems, and petioles of N. paniculata.
These genes showed a similar organ specificity Fig. 3. Accumulations of the transcripts of both
Fig. 1.
Fig. 2. Southern blot analysis of AldP1 and AldP2 genes. Twenty mg of genomic DNA from N. paniculata Np, N.
excelsior Ne, or N. arentsii Na was digested with the restriction enzymes E; EcoRI, H; HindIII and subjected to
agarose gel electrophoresis. DNA was blotted onto nylon membranes and was hybridized with
32
P-labeled probes.
genes were high in green leaves, low in flowers, stems, and petioles, and undetectable in roots. As
expected, high mRNA accumulations were ob- served in organs with a high chloroplast content.
4
.
4
. mRNA Accumulation of AldP genes under salt stress
Time courses of the mRNA levels of AldP genes under salt stress were analyzed by Northern blot
analysis in N. paniculata, N. excelsior, and N. arentsii Fig. 4a. The signals were quantified, and
the stresscontrol ratios Fig. 4b and relative strengths Fig. 4c at each time point were plotted.
In Fig. 4c, signal strength of the control at 0 h in each blot was taken as unity. N. paniculata and N.
arentsii showed similar expressions of each aldo- lase gene: the amount of AldP1 mRNA gradually
decreased, but the accumulation of AldP2 slightly increased under salt stress Fig. 4b. In contrast,
salt stress distinctively affected accumulations of mRNA of AldP genes in N. excelsior: the mRNA
level of NeAldP
1
decreased to 50 of the control within 3 h and gradually recovered by the fifth
day; the mRNA level of NeAldP
2
decreased to less than 50 of the control within 3 h, but it recov-
ered within 24 h and exceeded the control level thereafter, reaching 250 of the control Fig. 4b.
Fig. 4c, which shows the relative strength of each signal, also indicates that the mRNA amount of
NeAldP
2
was distinctly increased by salt stress. These results showed that the AldP2 gene was
up-regulated under salt stress in these species with different profiles, while the AldP1 gene was nega-
tively affected by salt stress.
4
.
5
. Daily changes of NpAldP
1
and NpAldP
2
mRNA accumulation Fig. 5 shows the daily cycles of NpAldP
1
and NpAldP
2
mRNA accumulation in green leaves of unstressed N. paniculata. The mRNA amounts
Fig. 3. Northern blot analysis of NpAldP
1
and NpAldP
2
for organ specificity. Five mg of LiCl-purified RNA prepared
from each organ was resolved on formaldehyde agarose gel and blotted onto nylon membranes. Northern hybridization
was done with
32
P-labeled probes. L, leaves; R, roots; F, flowers; S, stems; P, petioles. The bottom panel shows an
EtBr-stained gel.
Fig. 1. a Alignment of deduced amino acid sequences of NpAldP
1
and NpAldP
2
products compared with the deduced amino acid sequences of plastidic RICCHLALD, SOALDCHL and cytoplasmic RICAC1, SOALDCYT aldolases from higher plants.
Asterisks and dots indicate identical and homologous residues, respectively. b Phylogenetic tree of plant aldolases. Plastidic and cytoplasmic groups are enclosed in boxes. STPLASALD; Solanum tuberosum Y10380, PSALDIA; Pisum sati6um M97476,
PSALDIB; Pisum sati6um M97477, RICCHLALD; Oryza sati6a D13513, SOALDCHL; Spinacia oleracea X66814, CRALD- CHL; Chlamydomonas reinhardtii X69969, MZEALD; Zea mays M16220, OSFBPA; Oryza sati6a X53130, RICAC1; Oryza
sati6a D50301, MCAF3124; Mesembryanthemum crystallinum AF003124, SOALDCYT; Spinacia oleracea X65742, PSR- NAF16B; Pisum sati6um X89828.
Fig. 4.
Fig. 5. Northern blot analysis of NpAldP
1
and NpAldP
2
for daily cycles. Total RNA was isolated from green leaves of N.
paniculata at the indicated time for 2 consecutive days. Five m
g of LiCl-purified RNA was resolved on formaldehyde agarose gel and was blotted onto nylon membranes. Northern
hybridization was done with
32
P-labeled probes. L1, 1 h after illumination start; L7, 7 h after illumination start; D1, 1 h
after illumination termination.
for Southern analysis require 90 homology or higher to hybridize. The band patterns of AldP1
and AldP2 were different from each other in the other two species Fig. 4, indicating that plastidic
aldolase genes can be grouped in two sub-families in these species, too. Although more work is re-
quired to determine the exact number of copies of each gene in each species, N. excelsior seems to
have more copies than the other two species. N. paniculata and N. arentsii have 24 chromosomes,
whereas N. excelsior has 38 [20]. Therefore the genome structure of N. excelsior is predicted to be
different from the other two. This difference in N. excelsior may reflect the distinct gene dosage.
Effects of environmental stress on AldP gene expression have not yet been reported. It was
found that AldP2 was up-regulated by salt stress Fig. 4, which is the first reported instance of
environmental stress inducing AldP gene expres- sion. Expression of AldP1 genes was suppressed
by salt stress. Although some characteristics, such as amino acid sequences of their products, organ
specificity, and daily cycles, are very similar, AldP1 and AldP2 might have different physiologi-
cal roles. AldC gene expression is induced under anaerobic conditions to facilitate glycolysis and
produce ATP in higher plants [5 – 9]. Because AldC and AldP are involved in different metabolic
pathways and catalyze the reaction in opposite directions from each other, the physiological
meaning of AldP induction must be different from that of AldC.
One interpretation is that the AldP regulation itself is not involved in the stress tolerance mecha-
nisms represented by osmoprotectant production, but that the regulation is derivative. This would be
supported by the observation that AldP2 behavior under salt stress seems to correlate with the char-
acteristics of each species. The ability to accumu- late dry matter is similar in N. paniculata and N.
arentsii, approximately 60 of the control plants 9 days after stress initiation, although the former is
stress tolerant and the latter is stress sensitive. were high under illumination 1 and 7 h-illumina-
tion for lanes L1 and L7, respectively and de- creased in the dark 1 h-dark for lane D1. This
result suggests that the decrease in signal 8 h after the stress initiation in the control plants Fig. 4c
is due to the daily cycles.
5. Discussion
