全 文 ：Received 19 Feb. 2004 Accepted 29 Aug. 2004
* Author for correspondence. Tel: +86 (0)25 84396671; E-mail: < wbshenh@sohu.com >.
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (11): 1307-1315
Nitric Oxide Involved in the Abscisic Acid Induced Proline Accumulation
in Wheat Seedling Leaves Under Salt Stress
RUAN Hai-Hua, SHEN Wen-Biao*, XU Lang-Lai
(College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China)
Abstract: Exogenous nitric oxide (NO) releaser sodium nitroprusside (SNP) with different concentra-
tions from 0.01 to 5.00 mmol/L induced proline accumulation in wheat (Triticum aestivum L. cv. Yangmai
158) seedling leaves under 150 mmol/L salt stress in a dose-dependent manner. It was most effective at
0.1 mmol/L SNP, and the combination treatments with two NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-
tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) and hemoglobin, separately reverted the 0.1 mmol/L SNP
induced proline accumulation. Meanwhile, the proline accumulation induced by NO might be of benefit to
the water retention in wheat seedling leaves when subjected to salinity, and exogenous 0.1 mmol/L SNP
treatment also dramatically activated the synthesis of endogenous abscisic acid (ABA), and the employ-
ment of hemoglobin further indicated that NO might be downstream of the ABA induced proline accumulation
in wheat seedling leaves under 150 mmol/L salt stress, but there did not exist synergism between NO and
ABA signaling toward proline accumulation. Detection of proline synthesis and degradation demonstrated
that exogenous NO induced proline accumulation in a phase dependent manner, mainly by enhancing the
activities of D1-pyrroline-5-carboxylate synthetase (P5CS) within the first 4 d of treatment and inhibiting
activities of proline dehydrogenase (ProDH) 4 d later up to 8 d. And ABA showed a weak effect on P5CS and
ProDH activities in comparison with NO treatment. Additionally, Ca2+ was confirmed as the important
intermediates during the NO signaling pathway in proline accumulation under salinity conditions.
Key words: wheat (Triticum aestivum); nitric oxide (NO); abscisic acid (ABA); proline; salt stress
Salt stress is a key factor that reduces crop production
in agriculture (Zhu, 2002). Plants produce various biochemi-
cal and physiological responses to survive this adverse
environmental condition, such as the accumulation of
proline, glycine betaine and sugar alcohols (Delauney and
Verma, 1993). In fact, free proline accumulates in a wide
variety of higher plants, such as tobacco, soybean, barley,
wheat and rice (Yoshiba et al., 1997). Proline may function
as an osmoticum, a hydroxy-radical scavenger, a compat-
ible solute that protects enzymes and a sink of energy and
reducing power (Verbruggen et al., 1993). Until now, the
metabolism of proline in plants had also been basically re-
viewed (Hare et al., 1999). For example, proline accumula-
tion in response to salt stress in plants is mainly mediated
by two enzymes, D1-pyrroline-5-carboxylate synthetase
(P5CS), a rate-limiting enzyme in proline biosynthesis, and
proline dehydrogenase (ProDH), a mitochondrial enzyme
involved in the first step of the conversion of proline to
glutamic acid (Chen et al., 2001).
Nitric oxide (NO) is a short life bioactive molecule in
plants, convincing data have been obtained by the NO-
induced increase in plant tolerance or resistance against
various abiotic and biotic stresses, such as pathogen
disease (Delledonne et al., 1998), drought (García-Mata and
Lamattina, 2001; Zhang et al., 2003), salinity (Ruan et al.,
2002; 2004; Uchida et al., 2002), UV-B irradiation
(Mackerness et al., 2001) and wounding (Huang et al., 2004).
While abscisic acid (ABA) had been known playing impor-
tant roles in the tolerance of plants to high salinity, there
also exists both ABA-dependent and ABA-independent
signal transduction cascades between the initial signal of
salt stress and the expression of specific tolerance genes
(Shinozaki and Shinozaki, 1997). Meanwhile, it has been
suggested that NO exists net cross talk with ABA, cADPR,
cGMP, ion channels, Ca2+, and others in plants (García-
Mata and Lamattina, 2002; Neill et al., 2002; Correa-
Aragunde et al., 2004). Our previous results (Ruan et al.,
2002) had preliminarily proved that NO promoted the accu-
mulation of proline, which might participate the protective
effect of NO on salt-induced oxidative damage to wheat
seedlings. But how NO regulates the proline accumulation
and its role in ABA pathway underlying proline accumula-
tion under salt stress have not been well documented. In
the present study, we demonstrated the relationship of NO
and ABA in the process of proline accumulation respond-
ing to salt stress, and the effects of NO on the limiting
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041308
enzymes controlling proline metabolism and the intermedi-
ates involving in NO signaling were also investigated.
1 Materials and Methods
1.1 Plant materials and treatments
Sterilized wheat seeds (Triticum aestivum L. cv. Yangmai
158, kindly supplied by Jiangsu Agricultural Institutes,
Jiangsu Province, China) with 0.1% HgCl2, were cleaned in
distilled water and germinated at 25 ℃ in the dark. Selected
the identical buds and grown hydroponically in chamber
(12-h light period, 25 ℃, humidity 60%; 12-h dark period, 18
℃, humidity 60%) with roots submerged in Hoagland solu-
tion under irradiance of 100 µmol.m-2.s-1 provided by fluo-
rescent lamps. The solutions were renewed every day until
the second fully expanded leaves appeared.
SNP ((Na2Fe(CN)5) NO, Merck, Darmstadt, Germany)
was used as NO releaser or donor. 2-(4-carboxyphenyl)-4,
4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO, Sigma,
USA) was used as a specific scavenger of NO. Hemoglobin
(another NO scavenger) and LaCl3 (a blocker of Ca2+
channel) were purchased from Shanghai Boao Ltd., China.
ABA was purchased from Sigma (USA). NaCl was directly
added to Hoagland solutions, and all above chemicals were
added to Hoagland solutions with or without NaCl. Toward
the treatments with sodium nitroprusside (SNP), the solu-
tions were sealed to avoid the NO gas letting out the air.
Moreover, the treatment solutions were changed every day
to maintain the identical concentrations. Finally, the first
leaf of wheat seedlings with two fully expanded leaves was
cut for further investigation after suitable treatment period.
One g of fresh wheat leaves was dried at 70 ℃ for at
least 72 h to determine the dry weight (DW). The following
measurements were expressed as the unit of per g DW in-
stead that of per g FW.
1.2 Determination of proline content
Proline was extracted and its concentration was deter-
mined by the method of Bates et al. (1973). Leaf segments
were homogenized with 3% sulfosalicylic acid (W/V) and
the homogenate was centrifuged at 3 000g for 20 min. The
supernatant was treated with acetic acid and acid ninhydrin,
boiled for 1 h, and then absorbance at 520 nm was
determined. Contents of proline are expressed as µg /g DW.
1.3 Measurement of relative water content (RWC)
RWC was determined by the method of García-Mata
and Lamattina (2001).
1.4 Quantification of ABA
ABA was extracted from leaves with 80% methanol
(V/V) and was quantified by the method of HPLC as de-
scribed by Láng et al. (1994).
1.5 Assay of P5CS activity
Activities of P5CS were assayed according to Zhao and
Liu (2000). One unit (U) of its activity was defined as 0.5
DA 435.g-1 DW .h-1.
1.6 Extraction and active staining of ProDH activity
Extraction and active staining of ProDH activities were
performed as described by Zhao and Liu (2000) and Rayapati
and Stewart (1991) with some modifications. The superna-
tant was the crude enzyme extract. Also the protein in the
supernatant was determined and the equivalent amounts
of protein was loaded during following electrophoresis.
ProDH isozyme was separated by polyacrylamide gel
electrophoresis (PAGE, T=7.5 %). The ProDH activity was
visualized with staining solutions containing 0.1 mol/L
Na2CO3-NaHCO3 (pH 10.3), 8 mmol/L proline and 72
µmol/L 2, 6-dichlorphenolindophenol natriumsalz dihydrat.
The gels were bathed at 30 ℃ for 5 min with staining
solution. Then final concentration of 0.72 mg/mL PMS was
added to it until a clear pink band appeared. Discarded the
solution and cleaned the gel with distilled water three times
to get rid of background. The relative levels of ProDH ac-
tivities were scanned with dual wavelength TLC scanner
(CS-930) at 600 nm, and the data were expressed with its
proportion to that of CK, which was standardized as 100%.
1.7 Protein determination
Protein was determined by the method of Bradford (1976)
with BSA as the standard.
2 Results
2.1 Effect of NO on the content of proline in wheat seed-
ling leaves under salt stress
In previous results (Ruan et al., 2002), we investigated
the protective effects of SNP on wheat seedling leaves
under 150 and 300 mmol/L NaCl salt stress, respectively.
Here, effects of various SNP ranging from 0.01 to 5 mmol/L
on proline accumulation in wheat seedling leaves under
150 mmol/L NaCl salt stress were surveyed. As shown in
Fig.1A, 0.01, 0.10 and 1.00 mmol/L SNP treatment all el-
evated the contents of proline in varying levels through 8 d
of treatments, and 0.10 mmol/L SNP showed the maximum
induction to proline accumulation compared with control
(CK, P < 0.01). Contrary to these, 5 mmol/L SNP treatment
had no obvious effect on the proline content during these
period (Fig.1A). Therefore, it could be summarized that the
increase of proline content by SNP was in a dose depen-
dent manner. However, the proline in wheat seedling roots
was undetectable absolutely (data not shown).
To be sure that the NO releaser SNP induced proline
accumulation in wheat seedling leaves under 150 mmol/L
RUAN Hai-Hua et al.: Nitric Oxide Involved in the Abscisic Acid Induced Proline Accumulation in Wheat Seedling Leaves
Under Salt Stress 1309
NaCl salt stress was specific to NO, the NO scavenger c-
PTIO and hemoglobin (Takahashi and Yamasaki, 2002) were
employed. Figure 1B shows that combination with 0.2 mmol/
L c-PTIO and 0.1 % hemoglobin (W/V) both dramatically
inhibited the 0.1 mmol/L SNP induced proline accumula-
tion by 59.8% and 46.0%, respectively, under salt stress
for 4 d. These further confirmed the specific role of NO on
the proline accumulation in wheat seedling leaves under
salt stress.
Known to all, plant growth responding to salt stress
follows a combined effect of osmotic stress and ionic stress.
With that purpose, we determined the effects of NO donor
SNP on RWC of wheat seedling leaves subjected to 150
mmol/L NaCl salt stress, and found that exogenous 0.1
mmol/L SNP treatment obviously elevated the RWC under
salt stress. For example, the RWC in 0.1 mmol/L SNP treated
leaves after 2 d was approximately 82.5%, which was statis-
tically significant with that of 73.6 % in non-treated leaves
(P < 0.05, Fig.2A). Also, the correlation between the per-
centages of RWC elevation and proline accumulation in-
duced by 0.1 mmol/L SNP under salt stress was analyzed
from the result of Fig.2B. The proline accumulation and
sustentation of water in wheat seedling leaves induced by
NO showed significant positive correlation (Y = 0.410 1 X
+5.363 8, r = 0.914 7, r0.01 = 0.878, Student t-test). These
suggested that the NO induced proline accumulation might
be of benefit to the water retention in seedling leaves when
subjected to salinity, which also partially contributed to
the promotion of salt tolerance exerted by NO (Ruan et al.,
2004).
2.2 Effect of NO in the process of ABA induced proline
accumulation in wheat seedling leaves under salt stress
ABA is an important plant growth regulator in the pro-
cess of plants responding to salinity. We further detected
the effect of exogenous NO on the content of endogenous
ABA in wheat seedling leaves under 150 mmol/L NaCl salt
stress. As shown in Fig.3A, the content of endogenous
ABA was primarily elevated under NaCl salt stress alone
within the first 4 d of treatment, which might be due to an
activation of self-defence in plants responding to severe
environment (Campalans et al., 1999), but it gradually de-
creased after that period time. Compared with 150 mmol/L
NaCl stress alone, the content of ABA was further strik-
ingly enhanced by 0.1 mmol/L SNP treatment under salt
stress through the first 2 d of treatment (P<0.05) and thus
maintained in an approximately equivalent level at the fol-
lowing lag phase. Moreover, it was noticeable that 0.1%
hemoglobin treatment arrested these effects. These further
confirmed that NO could activate the synthesis of ABA,
which plays an important role in plant adaptation to salinity.
It has been reported that ABA could enhance the amount
of proline in plants (Savouré et al., 1997; Yoshiba et al.,
1999). Therefore, effect of NO on the ABA induced proline
accumulation under salt stress was further investigated.
As shown in Fig.3B, the level of proline was significantly
elevated by exogenous supplied ABA in wheat seedling
Fig.1. A. Effects of increasing concentrations (0, 0.01, 0.10, 1 or 5 mmol/L) of SNP on the content of proline in wheat seedling leaves
under 150 mmol/L NaCl salt stress. B. Changes of proline content in wheat seedling leaves after 4 d treated with: Hoagland solution
(control, CK), CK+0.1 mmol/L SNP (S), CK+150 mmol/L NaCl (N), CK+150 mmol/L NaCl+ 0.1 mmol/L SNP (N+S), CK+150
mmol/L NaCl+0.1 mmol/L SNP +0.2 mmol/L c-PTIO (N+S+C), CK+0.2 mmol/L c-PTIO (C), CK+150 mmol/L NaCl+0.1 mmol/L
SNP + 0.1% (W/V) hemoglobin (N+S+H). Bars represent the mean±SE of three independent experiments. One way ANOVA was used
for comparison between the means. Bars with different letters are significantly different at P < 0.05 or P < 0.01.
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041310
leaves under 150 mmol/L NaCl salt stress after 4 d of treat-
ment (P < 0.05). However, NO induced the proline accumu-
lation after 2 d of incubation, and the percentages of induc-
tion were significantly higher than those of ABA treatment.
When combination with 0.1% (W/V) hemoglobin treatment,
the ABA induced proline accumulation was obviously fallen
down after 4 d of treatment. Therefore, it could be conjured
that NO might be essentially required for the ABA-depen-
dent proline accumulation under salt stress. But it was in-
teresting that in the presence of SNP, exogenous ABA el-
evated the same level of proline compared with those of
SNP treatment alone under salinity conditions. The proline
Fig.2. A. Time-dependent changes in relative water content (RWC) of wheat seedling leaves subjected to 150 mmol/L NaCl salt stress.
+ sodium nitroprusside(SNP) (–SNP) represent treatments of Hoagland solution plus 150 mmol/L NaCl with or without 0.1 mmol/L
SNP. Each value is the mean ± SE of three replicates. One way ANOVA was used for comparisons between the means. The data marked
with * and ** are different at a level of significance of P < 0.05 and P < 0.01, respectively. B. Changes of the percentage of RWC elevation
and proline induction by 0.1 mmol/L SNP under 150 mmol/L NaCl salt stress.
Fig.3. A. Exogenous NO induced the synthesis of endogenous ABA in wheat seedling leaves under salt stress. B. Effect of NO in ABA
signaling pathway inducing proline accumulation in wheat seedling leaves under salt stress. Different treatment represents: Hoagland
solution (control, CK), CK+0.1 mmol/L sodium nitroprusside (SNP) (S), CK+150 mmol/L NaCl (N), CK+150 mmol/L NaCl+0.1 mmol/
L SNP (N+S), CK+150 mmol/L NaCl+0.1 mmol/L SNP+ 0.1% hemoglobin (N+S+H), CK+150 mmol/L NaCl+0.1 mmol/L SNP+5 µmol/
L ABA (N+S+A), CK+150 mmol/L NaCl+5 µmol/L ABA (N+A), CK+150 mmol/L NaCl+5 µmol/L ABA+ 0.1% (W/V) hemoglobin
(N+A+H). Bars represent the mean± SE of three independent experiments. One way ANOVA was used for comparisons between the
means. Bars with different letters are significantly different at P < 0.05 or P < 0.01.
RUAN Hai-Hua et al.: Nitric Oxide Involved in the Abscisic Acid Induced Proline Accumulation in Wheat Seedling Leaves
Under Salt Stress 1311
content was increased by 83.8 % in SNP treated wheat leaves
under salt stress, but by ABA only 36.4%, which was much
lower than those of SNP at 8 d. Whereas, when ABA was
combined with exogenous NO treatment, the proline con-
tent was elevated by 80.2%, which was analogous with the
results of NO treatment alone, indicating that NO induced
analogous level of proline under salt stress no matter in the
presence of ABA or not. Also, NO and ABA signaling dis-
played no synergism in proline induction (Fig.3B). Employ-
ment of hemoglobin combined with ABA furthermore illus-
trated the fact that NO might be downstream of the ABA-
dependent proline accumulation under salinity conditions.
2.3 Effects of NO donor and ABA on P5CS and ProDH
activities in wheat seedling leaves under salt stress
P5CS and ProDH appear to catalyze the rate-limiting steps
in proline synthesis and degradation, respectively. Here,
we principally tested the activities of P5CS and ProDH re-
sponding to exogenous NO donor and ABA treatment,
respectively, under salt stress to uncover the reason of the
higher proline accumulation induced by NO donor than
ABA. As shown in Fig.4, SNP and ABA both elevated P5CS
activities in wheat seedling leaves through 4 d of treatment,
and an obvious higher level of induced P5CS activities by
SNP were observed than those of ABA, which might con-
tribute to the higher proline accumulation induced by NO
donor than ABA did in the course of 4 d (Fig.3B). But after
4 d period, both of the regulation of SNP and ABA on P5CS
activities disappeared and showed no statistical changes
compared with those of 150 mmol/L NaCl salt stress alone.
Using the active staining method for ProDH, the single
active band appeared (Fig.5). Stines et al. (1999) also re-
ported that ProDH only existed in mitochondrial and the
MW was about 55 kD. Surprisingly, the ProDH activity in
wheat seedling leaves exhibited the enhancement within 2
d of exposure to 150 mmol/L NaCl salt stress than control,
then reduced gradually by up to 8 d. Meanwhile, employ-
ment of SNP or ABA also induced the ProDH activities
within 4 d of exposure to NaCl salt stress compared with
NaCl treatment alone. After 4 d, ProDH activities were then
depressed by SNP and ABA, respectively, and both of
which might be the major reason to the accumulated pro-
line levels after 4 d treatment by SNP and ABA (Fig.3B). It is
worth mentioning that the level of inhibition to ProDH by
ABA was obviously lower than than of SNP, particularly at
8 d of treatment, which might be responsible for the rapid
accumulation of proline induced by NO donor under salt
stress than those by ABA (Fig.3B). Taken together, it could
be summarized that the enzymes responsible for proline
accumulation under salinity conditions induced by NO
donor and ABA were different in a phase dependent manner.
The weaker roles of ABA to P5CS and ProDH just explained
the reason to the higher induced proline level by NO donor
in wheat seedling leaves when subjected to 150 mmol/L
NaCl salt stress (Fig.3B).
2.4 Role of Ca2+ in NO signaling pathway toward proline
accumulation in wheat seedling leaves under salt stress
As shown in Fig 6, the NO induced proline accumula-
tion was obviously decreased in wheat seedling leaves
under 150 mmol/L NaCl salt stress when subjected to 10
mmol/L LaCl3, a blocker of Ca2+ channel. The content of
proline was decreased by 52.4% by LaCl3 treatment in the
presence of SNP, compared with SNP treatment alone un-
der salt stress at 8 d. Contrary to this, when 50 mmol/L
CaSO4 was added to NaCl solutions, the proline content
was basically elevated to the analogous level in compari-
son with SNP plus NaCl treatment except an obviously lower
level at 8 d. Therefore, it could be concluded that Ca2+
might be tightly associated with the proline accumulation
induced by NO in wheat seedling leaves when subjected to
salt stress, and the signaling cascade of NO might be de-
pendent on the cytosol [Ca2+]c.
3 Discussion
Salinity is one of the major factors limiting agricultural
production around the world and promoting the salt
Fig.4. Effect of NO donor SNP and ABA on the regulation of
P5CS activities in wheat seedling leaves under salt stress. Differ-
ent treatment represents: Hoagland solution (control, CK),
CK+0.1 mmol/L SNP (S), CK+150 mmol/L NaCl (N), CK+150
mmol/L NaCl+0.1 mmol/L SNP (N+S), CK+150 mmol/L NaCl+5
µmol/L ABA (N+A). Bars represent the mean± SE of three in-
dependent experiments. One way ANOVA was used for compari-
sons between the means. Bars with different letters are signifi-
cantly different at P < 0.05 or P < 0.01.
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041312
tolerance of crops has become an efficient pathway for
agriculture. Meanwhile, overproduction of proline has been
confirmed resulting in increased tolerance to osmotic or
salt stress in transgenic plants (Kavi-Kishor et al., 1995;
Zhu, 2002), although there also existed the regards that
proline was an alternative result from adaptive or detrimen-
tal processes responding to osmotic stress (Larher et al.,
2003). In present study, exogenous nitric oxide (NO) re-
leaser SNP with different concentrations from 0.01 to 5
mmol/L induced proline accumulation in wheat seedling
leaves under 150 mmol/L salt stress in a dose-dependent
manner through 8 d of treatment (Fig.1A). Among of these,
the effect of 0.1 mmol/L SNP was the most effective, while
high concentration of SNP (5 mmol/L) had no obvious ac-
tion (Fig.1A). Furthermore, the effect of SNP was specific
for NO because the NO-scavenger c-PTIO and hemoglobin
could reverse the effect of 0.1 mmol/L SNP on induced pro-
line contents (Fig.1B). Meanwhile, the positive correlation
between the enhanced percentage of RWC and proline
induced by NO (Fig.2) ensured the speculation that the
Fig.5. Changes of ProDH activities in wheat seedling leaves responding to NO donor and ABA under 150 mmol/L NaCl salt stress. A,
B, C and D represent the results after 2, 4, 6 and 8 d treatment, respectively, and the corresponding chart at right side of the photographs
were the relative ProDH activity level scanning with dual wavelength TLC scanner (CS-930) at 600 nm. Each lane was loaded equivalent
amount of protein. The different treatments represent: Hoagland solution (control, CK), CK+0.1 mmol/L SNP (S), CK+150 mmol/L
NaCl (N), CK+150 mmol/L NaCl+0.1 mmol/L SNP (N+S), CK+150 mmol/L NaCl+5 µmol/L ABA (N+A).
RUAN Hai-Hua et al.: Nitric Oxide Involved in the Abscisic Acid Induced Proline Accumulation in Wheat Seedling Leaves
Under Salt Stress 1313
induction of proline by NO might be an important mecha-
nism for plants to tolerate severe saline conditions (Ruan
et al., 2002). Also in our experiments, the level of proline in
wheat seedling roots were undetectable (data not shown)
and the proline mainly existed in seedling leaves. It is valu-
able mentioning that we treated wheat seedling roots with
NO releaser, but its eliciting effect on leaves might be due
to the reason that NO was a permeable gas molecule, it
could also transport over short and long distance with as-
sistance of nitrite (Desikan et al. , 2002) and S-
nitrosoglutathione (GSNO) (Reichenbach et al., 2001).
However, these related mechanisms have not been fully
understood yet in plant tissues.
Recently, it has been suggested that there exists net
cross talk between NO and ABA, cGMP, ion channels, Ca2+
and others in plants (García-Mata and Lamattina, 2002; Neill
et al., 2002; Correa-Aragunde et al., 2004). The data in our
experiments also showed that NO could activate the syn-
thesis of endogenous ABA in wheat seedling leaves under
150 mmol/L NaCl salt stress (Fig.3A), which was in agree-
ment with the results of Zhao et al. (2001) that the synthe-
sis of ABA was inhibited by nitric oxide synthase (NOS)
inhibitors in responding to drought stress.
Known to all, ABA has multiple roles in plants when
subjected to salinity, including inducing the accumulation
of proline. Also, proline accumulation appears to be medi-
ated by both ABA-dependent and ABA-independent sig-
naling pathway (Hare et al., 1999). The result of Fig.3B
illustrated that the level of proline was significantly elevated
by exogenous supplied ABA in wheat seedling leaves un-
der 150 mmol/L NaCl salt stress after 4 d of treatment (P <
0.05). Combination with hemoglobin treatment brought up
with the fact that NO was a required factor downstream the
ABA-induced proline accumulation, which was just in co-
incidence with the evidence that NO was a necessarily re-
quired component for the ABA induced stomatal closure
(García-Mata and Lamattina, 2002; Neill et al., 2002).
Therefore, the maintenance of a high level of ABA by NO
might be also an important aspect for the NO promoted salt
tolerance following the reason that ABA has multiple roles
in stress tolerance of plants, such as limiting water loss by
inducing stomatal closure, promoting root morphogenesis
and activating tolerant gene expression (Campalans et al.,
1999). Besides that, NO activated the synthesis of ABA
and might also induce proline accumulation under salt
stress via ABA transduction cascade, which supplied new
evidence for good comprehension of cross talk between
NO and ABA in plant kingdom. Additionally, the phytohor-
mone ABA could enhance the synthesis of NO in reverse
and induce closure of stomates, both NOS and nitrate re-
ductase (NR) have been implicated in these processes
(García-Mata and Lamattina, 2002; 2003; Neill et al., 2002).
Recently, Guo et al. (2003) identified a plant NOS gene
playing a vital role in plant growth, fertility, stomatal move-
ments and ABA signaling. Meanwhile, an inducible plant
NOS gene was also been discovered by viral infection and
encodes a variant of the P protein of glycine decarboxylase
(GDC, Chandok et al., 2003).
ABA and NO both activated the activities of P5CS and
depressed the activities of ProDH in a phase dependent
manner, mainly by enhancing the activities of P5CS within
the first 4 d of treatment and inhibiting activities of ProDH
4 d later up to 8 d. And ABA showed a weaker effect on
P5CS and ProDH in comparison with NO treatment (Figs.4,
5). These could be perfectly explained by above sugges-
tion that NO might be downstream the ABA signaling path-
way toward proline accumulation under salt stress
(Fig. 3B). Uchida et al. (2002) also found that the expres-
sion of P5CS was increased in response to NO in rice seed-
lings leaves under salinity conditions, which could confer
increased tolerance to salt stress. Additionally, our experi-
ments of LaCl3 and CaSO4 (Fig.6) implicated the important
role of Ca2+ in the pathway of NO in proline induction un-
der salt stress. It was noted that plant P5CS and ProDH
Fig.6. Effect of Ca2+ on the proline accumulation induced by
NO in wheat seedling leaves under salt stress. Different treatment
represents: CK+150 mmol/L NaCl (N), CK+150 mmol/L
NaCl+0.1 mmol/L SNP (N+S), CK+150 mmol/L NaCl+0.1
mmol/L SNP+10 mmol/L LaCl3 (N+S+L), CK+150 mmol/L
NaCl+50 mmol/L CaSO4 (N+Ca). Bars represent the mean±SE
of three independent experiments. One way ANOVA was used
for comparisons between the means. Bars with different letters
are significantly different at P < 0.05 or P < 0.01.
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041314
might be co-ordinated by the same signaling cascade, in-
volving both Ca2+ and cADPR (Hare et al., 1999). cADPR
also mediated the ABA signal transduction which elicited
its effect via intracellular Ca2+ release, which was the core
of NO signaling pathway in plants (Durner et al., 1998). In
a word, plants have developed delicate defense system to
survive in nature.
Acknowledgements: We wish to thank FAN Xiao-Rong
in Plant Nutrition Laboratory of Nanjing Agricultural Uni-
versity for her kindly assistance in ABA measurement.
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(Managing editor: HE Ping)