全 文 ：Received 3 Mar. 2004 Accepted 12 Jul. 2004
Supported by the State Key Basic Research and Development Plan of China (G2000048704).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (9): 1009-1014
Effects of Exogenous Nitric Oxide on Photochemical Activity of
Photosystem Ⅱ in Potato Leaf Tissue Under Non-stress Condition
YANG Jia-Ding*, ZHAO Ha-Lin, ZHANG Tong-Hui, YUN Jian-Fei
(Cold and Arid Regions Environmental and Engineering Research Institute, The Chinese Academy
of Sciences, Lanzhou 730000, China)
Abstract: Under non-stress condition, effects of exogenous nitric oxide (NO) on chlorophyll fluores-
cence parameters in detached leaves and leaf discs of potato (Solanum tuberosum L.) were surveyed.
Results showed that the maximal quantum efficiency (Fv/Fm) and the effective quantum efficiency (F PSⅡ)
of photosystem Ⅱ (PSⅡ) were reduced by exogenous NO under illumination (150 mmol.m-2.s-1, 25 ℃).
This influence was related not only to the concentration of sodium nitroprusside (SNP, a NO donor)
solution, but also to the active duration of NO on leaf tissue. Results with leaf discs showed that the
effects of SNP on F PSⅡ could be prevented by bovine hemoglobin (a powerful NO scavenger), while a
mixture of NO2- and NO3- (the decomposition product of NO or its donor SNP) had much less influence on
F PSⅡ than SNP, indicating that effects of exogenous SNP on PSⅡ photochemical activity was mainly due
to NO generation. Under light (150 mmol.m-2.s-1, 25 ℃) for 4 h or longer period, the non-photochemical
quenching (NPQ) in SNP-soaked leaves was statistically similar to that in H2O-soaked control, but F PSⅡ
and the proportion of open reaction centers (measured as qP) were lower than control, respectively. After
25 min dark-adaptation, the maximal fluorescence (Fm) in SNP treatment (8 and 12 h illumination duration)
was significantly lower than the control, while the initial fluorescence (Fo) in SNP and H2O-treated leaves
had no significant difference. Therefore this indicated that under the present experimental condition, the
NO-affected site might not be the PSⅡ reaction centers. On the donor side of PSⅡ, NO putatively
influenced the light-harvesting capacity of leaves under light; on the acceptor side, NO-affected sites were
some components of electron transport chain after QA, i.e. NO enhanced the reductive degree of reaction
centers through blocking the electron transport after QA, thus reducing the photochemical activity of PSⅡ.
Key words: nitric oxide; photosystem Ⅱ (PSⅡ); quantum efficiency; potato leaf tissue; non-stress
condition
.Rapid Communication.
As a free-radical molecule, nitric oxide (NO) is either
protective or toxic depending on its applied concentration
and different tissues it acts (Beligni and Lamattina, 1999).
Mounting evidence proved NO to be involved in many
plant physiological and metabolic processes. It could act
as a signal molecule in plant defense interactions with mi-
croorganisms (Dangl, 1998; Durner et al., 1998), or as a
compound with hormonal properties (Leshem and
Haramaty, 1996), affecting growth and development, such
as photomorphogenesis (seed germination, de-etiolation,
inhibition of hypocotyl elongation) (Beligni and Lamattina,
2000), organ maturation and senescence (e.g. root, leaf and
fruit) (Leshem, 1996; Leshem et al., 1998; Tu et al., 2003), or
as both antioxidant and anti-stress agent in tolerance re-
sponses to abiotic stresses (Beligni and Lamattina, 1999;
García Mata and Lamattina, 2001; Uchida et al., 2002). So, it
is becoming increasingly apparent that NO is a functional
metabolite in plants (Durner and Klessig, 1999). As for plant
respiration, NO was shown to affect mitochondrial func-
tionality and reduce total cell respiration due to strong in-
hibition of the cytochrome pathway (Zottini et al., 2002). It
was also shown that NO was a reversible inhibitor of pho-
tosynthetic ATP synthesis in chloroplasts (Takahashi and
Yamasaki, 2002). The electron transport activity in photo-
system Ⅱ (PSⅡ) determined from chlorophyll a fluores-
cence was inhibited by NO and ATP synthesis activity
under illumination was severely inhibited by NO and com-
pletely recovered by removing NO (Takahashi and
Yamasaki, 2002).
Interestingly, in some previous reports (Beligni and
Lamattina, 1999; García Mata and Lamattina, 2001), the de-
tached leaves or leaf discs were submerged or floated in a
NO donor — sodium nitroprusside (SNP) solution under
light for different time (from several hours to several days),
then the variations of some physiological parameters in
plant tissues were assayed. However, was the leaf
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041010
photochemical capacity influenced or not during such treat-
ment process? Hereafter we report that under non-stress
conditions, exogenous NO treatment can influence the pho-
tochemical activity of detached potato leaf tissue under
light for several hours, and the NO-affected sites were also
discussed.
1 Materials and Methods
1.1 Plant materials
Potato (Solanum tuberosum L.) tubers were grown in a
sterile mixture of soil:vermiculite (3:1, V/V) and the result-
ing plants were maintained at 25 ℃ with a 14-h photoperiod
for 30 d. Fully expanded healthy leaves with similar posi-
tion on seedlings were selected for further experiments.
1.2 Treatments and measurements
Sodium nitroprusside (SNP, Merck; Darmstadt, Germany)
was used as NO donor. Bovine hemoglobin (Hb) was from
Jingke-Hongda Biotechnology Company (Beijing, China).
NaNO2 and NaNO3 were of analytical purity and from Beijing
Chemical Reagent Company (Beijing, China).
Potato leaves were excised and the petioles were sub-
merged in tubes containing either distilled H2O, SNP solu-
tion or other chemicals with certain concentrations. In each
treatment, 4 sets of leaves (3-4 leaves per set) were laid
under light (150 mmol·m-2·s-1, 25 ℃) successively for 0.2,
4, 8, 12 h respectively. At the end of illumination, the effec-
tive quantum efficiency of PSⅡ (F PSⅡ) of treated leaves
was determined using a portable fluorometer (Handy Plant
Efficiency Analyzer, Hansatech Instruments Ltd., UK) un-
der light (150 mmol·m-2·s-1, 25 ℃). Then the leaves were
transferred to dark room (25 ℃) for 25 min. The maximal
quantum efficiency of PSⅡ (Fv/Fm) was determined in the
dark. Additionally two sets of leaves submerged respec-
tively in distilled H2O and SNP (150 mmol/L) were put all
along in dark serving as dark-incubation control. The val-
ues of Fv/Fm were measured at 0.2, 4, 8, 12 h in the dark
condition respectively. For each leaf, two measurements
were taken on its middle parts across the midrib. In all chlo-
rophyll fluorescence determination, one single excitation
of actinic light (0.8 s) with saturating photosynthetic pho-
ton flux density (PPFD) (3 000 mmol·m-2·s-1) was used. For
Fv/Fm measurement, the pre-illumination light was 0.6
mmol·m-2·s-1 (0.8 s). For F PSⅡ under light, the pre-illumi-
nation light was 150 mmol·m-2·s-1 (0.8 s).
The initial fluorescence (Fo) and maximal fluorescence
(Fm) in dark, and stable fluorescence (Fs) and maximal fluo-
rescence (Fm) under light, as well as F PSⅡ (calculated as
(Fm-Fs)/Fm) and Fv/Fm (calculated as (Fm-Fo)/Fm), were
automatically recorded by the fluorometer. Quenching due
to non-photochemical dissipation of absorbed light energy
(NPQ) was calculated as (Fm –Fm)/ Fm (Bilger and
Björkman, 1990). As an indication of the proportion of open
PSⅡ reaction centers, photochemical quenching (qP) was
calculated as F PSⅡ /(Fv/Fm) according to Maxwell and
Johnson (2000).
To test whether NO itself or the decomposition product
of NO or its donor SNP (a mixture of NO2- and NO3-) was
responsible for the variations of fluorescence parameters,
potato leaves were excised, midribs removed, and cut into
discs with a diameter of 1.5 cm. The leaf discs were floated
respectively in Petri dishes containing distilled water, SNP
(150 mmol/L), SNP (150 mmol/L) plus bovine hemoglobin (4
g/L) (SNP/Hb), and a mixture of 150 mmol/L NaNO2 and 150
mmol/L NaNO3 (NO2-/NO3-) under light (PPFD 150
mmol·m-2·s-1) as above. Then F PSⅡ of leaf discs were
assayed at 0.2, 4, 8, 12 h after illumination.
1.3 Statistical analysis
Each experiment was repeated at least three times. Val-
ues are expressed as means ± SD. The statistical signifi-
cance of differences between H2O treatment and SNP treat-
ment was based on the t-test of mean values. The analysis
was carried out using the analytical tool of the EXCEL pro-
gram package. Prior to analysis, data to be compared were
tested on homogeneity of variance (F-test).
2 Results
2.1 Effect of SNP solution with different concentrations
on the maximal quantum efficiency (Fv/Fm)
As shown in Fig.1, after 8 h illumination and 25 min
dark-adaptation, Fv/Fm in leaves was declined with the in-
crease of SNP concentration. Fv/Fm in treatment with 150
Fig.1. The maximal quantum efficiency of PSⅡ (Fv/Fm) in
potato leaves submerged in sodium nitroprusside (SNP) solu-
tions with different concentrations and illuminated for 8 h (PPFD
150 mmol·m-2·s-1, 25 ℃), then adapted in dark for 25 min. Pre-
sented asterisks indicated the levels of significance: *P < 0.05 and
**P < 0.01.
YANG Jia-Ding et al.: Effects of Exogenous Nitric Oxide on Photochemical Activity of Photosystem Ⅱ in Potato Leaf Tissue
Under Non-stress Condition 1011
mmol/L or higher concentration of SNP had a significant
difference compared to that of H2O control (P<0.05 or 0.01).
F PSⅡ measured under light exhibited a similar trend. Re-
ferred to the reported experimental method (García Mata
and Lamattina, 2001; Zhang et al., 2003), the SNP concen-
tration was selected as 150 mmol/L in the hereafter
experiments. Considering that 0.5 mmol/L NO could be re-
leased from 150 mmol/L SNP solution (García Mata and
Lamattina, 2001), the lowest concentration of NO to induce
significant decline in Fv/Fm was 0.5 mmol/L NO under the
present experimental condition.
2.2 Fluorescence parameters due to 150 mmol/L SNP
treatment
2.2.1 Fv/Fm in leaves undergone dark incubation or light
illumination As shown in Table 1, when leaves were dark-
incubated for 0.2, 4, 8, 12 h, the values of Fv/Fm in SNP-
treated leaves had no significant difference, although it
was slightly smaller, compared to the H2O-treated control.
For leaves which were dark-adapted after illumination,
Fv/Fm between SNP-treated and control leaves exhibited a
significant difference (P < 0.01) after 8 and 12 h of
illumination, i.e. SNP-treated leaves had a lower Fv/Fm. The
original fluorescence (Fo) of SNP-treated leaves and con-
trol had no significant difference (Table 1), whereas the
maximal fluorescence (Fm) after 8 and 12 h illumination was
significantly smaller in SNP-treated leaves (P < 0.05).
2.2.2 F PSⅡ under illumination When measured under
light condition (150 mmol·m-2·s-1), F PSⅡ was significantly
lower in SNP-treated leaves than that of control after 4, 8,
12 h of illumination (P < 0.01) (Table 1). Associating with
the equivalent Fv/Fm after dark-adaptation, it was interest-
ingly shown that significant lower F PSⅡ in SNP-treated
leaves after 4 h illumination was relative to a Fv/Fm with no
statistical difference from that of control. This meant that
the significant lower F PSⅡ in NO-treated leaves after 4 h
illumination was putatively due to some reversible inacti-
vation of photosynthetic apparatus. This inactivation could
be released during dark-adaptation. While for leaves illumi-
nated for 8 and 12 h, the decrease of F PSⅡ in SNP treat-
ment should be due to some irreversible influence to pho-
tosynthetic apparatus by NO, which could not be released
during dark-adaptation.
2.2.3 NPQ and qP in leaves under illumination NPQ
had no statistical difference between SNP-treated leaves
and control (Table 1). However, as an indication of the pro-
portion of open PSⅡ reaction centers, qP was significantly
decreased, i.e. the proportion of closed reaction centers
was larger in SNP-treated leaves (P < 0.01) after 4 h or longer
illumination.
2.3 Variations of F PSⅡ in leaf discs with different treat-
ments
Taking the distilled water treatment as the control, val-
ues of F PSⅡ in potato leaf discs in four different treat-
ments at 0.2 h had no statistical difference, whereas after 4
h or longer illumination, F PSⅡ values in SNP treatments
decreased about 64%-67% and only about 4%-9% in SNP/
Hb or NO2-/NO3- treatments (Table 2).
3 Discussion
In mammals, NO is produced from arginine by the
Table 1 Effects of 150 mmol/L SNP solution on fluorescence parameters under different treatment conditions
Treatment Fluorescence Treatment duration (h)
condition parameters 0.2 4 8 12
Dark-incubated Fv/Fm H 0.843±0.004 0.845±0.006 0.846±0.006 0.843±0.006
S 0.842±0.004 0.844±0.005 0.842±0.005 0.842±0.005
Illuminated F PSⅡ H 0.625±0.009 0.639±0.022 0.583±0.090 0.564±0.066
S 0.600±0.042 0.528±0.076** 0.440±0.092** 0.449±0.041**
NPQ H 0.582±0.107 0.543±0.091 0.556±0.133 0.484±0.069
S 0.596±0.065 0.480±0.180 0.631±0.204 0.495±0.038
qP H 0.740±0.012 0.757±0.027 0.688±0.106 0.666±0.078
S 0.710±0.051 0.625±0.090** 0.514±0.107** 0.536±0.049**
Dark-adapted Fv/Fm H 0.845±0.004 0.844±0.004 0.847±0.002 0.847±0.002
after illumination S 0.845±0.004 0.845±0.002 0.838±0.006** 0.838±0.006**
F o H 633±16 637±18 624±8 627±6
S 635±17 632±9 631±19 637±14
Fm H 4 088±1 4 086±1 4 087±1 4 087±1
S 4 087±1 4 086±1 3 899±196* 3 929±118*
H stands for control treated with distilled H2O. S stands for the leaves treated with 150 mmol/L SNP. Presented asterisks are used to identify
the levels of significance. *P < 0.05 and **P < 0.01 between H and S at certain treatment duration. Fv/Fm, the maximal quantum efficiency;
F PSⅡ, the effective quantum efficiency; NPQ, non-photochemical quenching; qP, photochemical quenching; Fo, the initial fluorescence; Fm,
the maximal fluorescence.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041012
enzyme NO synthase. In plants, NO can be generated from
nitrate using nitrate reductase (Yamasaki, 2000) and nonen-
zymatic reduction of apoplastic nitrite (Bethke et al., 2004).
The pathogen-induced, NO-synthesizing enzyme in plants
was demonstrated as a variant form of the P protein of
glycine decarboxylase and only a few of the critical motifs
associated with NO production in animals could be recog-
nized (Chandok et al., 2003). Also, Guo et al. (2003) identi-
fied a plant nitric oxide synthase gene (AtNOS1) in
Arabidopsis and the enzyme protein expressed by this gene
has no sequence similarities to those of their mammalian
counterparts. All these reports suggest multiple pathways
for NO generation in plants and different biochemical mecha-
nisms from those in animals.
Photosynthesis is one of the most important metabolic
processes for energy transformation in plants. Using the
thylakoid membrane of spinach leaves as the experimental
material, Takahashi and Yamasaki (2002) reported that NO-
pretreatment for only 3 min could reduce the electron trans-
port activity (determined from chlorophyll fluorescence)
under light, but has no effects on Fv/Fm. However, in the
present work, chlorophyll fluorescence measurements on
detached whole potato leaves revealed that exogenous NO
could reduce not only F PSⅡ, but also Fv/Fm (Fig.1). This
effect was dependent on the concentration of NO donor
SNP solution and NO active duration on leaf tissues. For
example, submerging in 150 mmol/L SNP for 8 or 12 h under
illumination could significantly reduce the leaf Fv/Fm (Table
1), but no statistical difference between SNP treatment and
control was caused by 0.2 or 4 h illumination. The factors
underlying the difference between the present result and
that of Takahashi and Yamasaki (2002) were maybe due to
different materials (thylakoid membranes vs whole leaves),
active duration of exogenous NO (3 min vs 8 h or longer).
The result using leaf discs (Table 2) showed that bovine
hemoglobin, a powerful NO scavenger (Takahashi and
Yamasaki, 2002), could significantly reverse the effects of
SNP, suggesting that variations of F PSⅡ in SNP treatment
was mainly due to NO production. Although the mixture of
NO2- and NO3- induced a slight decline in F PSⅡ values,
this trend was not as apparent as in SNP treatment (Table
2), indicating that F PSⅡ variations could not be attributed
to the decomposition of NO or its donor SNP which gener-
ates NO2- and NO3- (Beligni and Lamattina, 1999). So, it
was suggested that NO itself influenced the photochemi-
cal activity in potato leaf tissues.
As for a PSⅡ unit, the excited reaction center is called
open when QA is in its oxidized form. If QA is already reduced,
then the excited reaction center is called closed (Strasser et
al., 2000). A lower proportion of PSⅡ reaction centers that
are open (indicated by qP) in SNP treatments (4, 8 and 12 h)
under illumination (Table 1) meant that QA in oxidative form
decreased, and recycle rate of oxidative and reductive QA
was slowed down due to NO treatment, indicating an inhib-
ited electron transport behind QA (Strasser et al., 2000). So
it was suggested that the main sites of NO to affect elec-
tron transport were on the acceptor side of PSⅡ, i.e. NO
treatment increased the reductive proportion of QA by
blocking the electron transport after QA, thus partially clos-
ing the reaction centers and reducing the photochemical
activity of PSⅡ.
Decrease in Fo was suggested to be associated with an
increased capacity for energy dissipation within light-har-
vesting complexes (Demmig-Adams, 1990), and increase in
Fo to be related to partly reversible inactivation or irrevers-
ible damage in the reaction centers of PSⅡ (Yamane et al.,
1997). In the present study, although NO soaking under
illumination for 4 h or longer would significantly induce a
lower F PSⅡ, Fo in SNP treatment and control after dark-
adaptation were statistically similar to different treatment
duration, meaning that PSⅡ reaction centers might not be
irreversibly influenced by NO treatment. So the decrease of
Fv/Fm in SNP treatment with 8 and 12 h illumination and
dark adaptation, being significantly lower than equivalent
control, was mainly due to the maximal fluorescence (Fm)
decrease (Table 1). Considering that Fm reflects the elec-
tron transport from QA to the electron transfer chain (Ortiz
and Cardemil, 2001), the present result strongly suggested
Table 2 Values of F PSⅡ in leaf discs submitted to different treatments with various illumination durations
Treatments
Duration under illumination
0.2 h 4 h 8 h 12 h
Distilled water 0.715 ± 0.018 a 0.714 ± 0.023 a 0.672 ± 0.039 a 0.668 ± 0.058 a
SNP 0.713 ± 0.022 a 0.260 ± 0.035 b 0.246 ± 0.025 b 0.223 ± 0.024 b
SNP/Hb 0.710 ± 0.027 a 0.649 ± 0.071 c 0.643 ± 0.039 c 0.643 ± 0.058 a
NO2-/NO3- 0.713 ± 0.031 a 0.693 ± 0.036 a 0.643 ± 0.052 c 0.630 ± 0.050 c
Leaf discs were treated as described in “Materials and Methods”. In each column, values followed by the same letter were not significantly
different at P < 0.05 (n = 10). The concentration of SNP solution was 150 mmol/L, SNP/Hb stood for SNP (150 mmol/L) plus bovine
hemoglobin (4 g/L), NO2-/NO3- stood for a mixture of 150 mmol/L NaNO2 and 150 mmol/L NaNO3. SNP, sodium nitroprusside.
YANG Jia-Ding et al.: Effects of Exogenous Nitric Oxide on Photochemical Activity of Photosystem Ⅱ in Potato Leaf Tissue
Under Non-stress Condition 1013
that the electron transport through QA was irreversibly in-
hibited by NO after 8 h or longer illumination and this inhi-
bition could not be released by subsequent dark adaptation.
This should be related to the differences in the redox states
and heterogeneity of the filling up of the PSⅡ acceptor
side (Strasser et al, 1995).
In addition, compared with control, SNP treatment re-
duced F PSⅡ significantly, while NPQ had no statistical
difference (Table 1). These two aspects indicated that the
absorbed energy by PSⅡ light-harvesting chlorophyll
(i.e. the energy used for photochemical reaction plus that
used for non-photochemical quenching) was lower in SNP-
treated leaves, putatively indicating that NO could reduce
the light-absorbing capacity of PSⅡ under the light.
Although it was concluded that the NO-affected sites
were downstream to QA in present experiment, the details
of the process and possible mechanisms are still not known.
According to the reported characteristics of NO, it was
suggested that following four sites were to be affected
possibly by NO. (1) NO could release the bound bicarbon-
ate (HCO3-) in PSⅡ(e.g. non-heme iron complex, QB bind-
ing site, water-oxidizing complex), which stimulates elec-
tron transport through PSⅡ (Bilger and Björkman, 1990).
So NO would retard electron transport at QB, the second-
ary electron acceptor of PSⅡ. (2) NO has an intense affin-
ity to iron. Some heme protein and non-heme iron protein
are usually the NO target (Tu et al., 2002). So cytb559, cytb6/
f and other iron-containing components in electron trans-
port chain would be attacked by NO, thus influencing elec-
tron transport through PSⅡ. (3) Strasser et al. (2000) once
mentioned that the redox state of plastoquinone (PQ or
PQH2) can influence the fluorescence intensity mainly at
Fm. Associating the Fm variations (Table 1), it was indicated
that maybe plastoquinone was an affected active site by
NO. (4) Because NO is a reversible inhibitor of photosyn-
thetic ATP synthesis in chloroplasts (Takahashi and
Yamasaki, 2002), reduced rate of photosynthetic phospho-
rylation and enzymatic process of carbon reduction
(photosynthetic dark reaction) would also indirectly influ-
ence the quantum efficiency of PSⅡ.
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