全 文 ：Received 15 Mar. 2004 Accepted 7 Jul. 2004
Supported by the Shaanxi Provincial Natural Science Research Plan (99SM20, 2003C101).
* Author for correspondence. Tel: +86 (0)29 85308451; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (9): 1049-1055
Change of Nitric Oxide and NADPH-diaphorase During the Generation and
the Development of Adventitious Roots in Mung Bean Hypocotyl Cuttings
SHE Xiao-Ping*, HUANG Ai-Xia
(College of Life Sciences, Shannxi Normal University, Xi’an 710062, China)
Abstract: Effects of nitric oxide (NO) donor sodium nitroprusside (SNP), NO specific scavenger c-PTIO
and nitric oxide synthase (NOS) inhibitor L-NAME on the rooting of mung bean (Vigna radiata L.) hypocotyl
cuttings were studied. The spatio-temporal changes of NO and NADPH-diaphorase in the basal part of
cutting were also detected during the adventitious rooting process. The results showed that SNP signifi-
cantly enhanced the adventitious rooting in the range of concentrations tested. NADPH-diaphorase
activity (commonly employed as a marker for NOS) and the fluorescence of NO were respectively observed
in the zone between the vascular bundles of the basal part of cuttings at 24 h and 36 h after cutting. The
root primordium became discernible at 48 h after cutting in the same region, and became more elongate
at 60 h. NADPH-diaphorase activity and NO fluorescence gradually increased during 48-60 h and mainly
distributed in root meristem. L-NAME treatment delayed adventitious root emergency and significantly
reduced the NADPH-diaphorase staining and the fluorescence of NO. The specific NO scavenger, c-PTIO,
also suppressed the fluorescence and inhibited the formation of adventitious roots. These results suggest
that endogenous NO appears to play a key role in the generation and development of adventitious roots,
and the production of NO in this process may be catalyzed by NOS-like enzyme.
Key words: nitric oxide (NO); NADPH-diaphorase; mung bean hypocotyl cutting; adventitious root;
generation; development
Nitric oxide (NO) is an important second messenger in
animal cells (Brunori et al., 1999; Chung et al., 2001) and
accumulating evidence suggested that it was important in
plant cells as well (Beligni and Lamattina, 2001;Wendehenne
et al.,2001). NO was proved to participate in many physi-
ological process in plant, such as growth, development,
abiotic and biotic stresses (Leshem,1996; Beligni and
Lamattina,1999; Ribeiro et al.,1999; Orozco-Cárdenas and
Ryan, 2002; Pagnussat et al.,2002). As a consequence of
the physiological importance of NO, numerous studies have
been focused on the enzyme responsible for its endog-
enous production (Barroso et al.,1999). Nitric oxide syn-
thase (NOS) is responsible for the synthesis of NO, from
the guanidine-nitrogen of L-arginine during the five-elec-
tron oxidation to L-citrulline in animals(Marletta,1993). NOS
activity in plant cells, based on the formation of L-citrulline
from L-arginine, was observed in peroxisomes from leaves
of pea plants (Barroso et al., 1999) and maize cells (Ribeiro
et al.,1999). In lupine roots, a NADPH-diaphorase activity
was also detected, which commonly employed as a marker
for NOS (Cueto et al., 1996). Inhibitor of NOS significantly
decreased NO production in angiosperm and gymnosperm
callus cells and foliar tissues (Pedroso et al., 2000a).These
observations, and the known multifunctional actions of NO
in mammalian cells, indicate that the participation of NOS is
likely to be demonstrated for many cellular processes in
plants (Ribeiro et al., 1999).
More recently, Pagnussat et al. (2002; 2003) demon-
strated that NO mediated the adventitious root generation
induced by IAA, but the spatio-temporal changes of NO
and the sources of NO accompanied adventitious root gen-
eration and development are unknown. In this report, the
histological distribution of NO and NADPH-diaphorase in
different phase of adventitious root organogenesis were
shown. Our results suggest that NO and NOS-like enzyme
as internal factors regulate the generation and develop-
ment of adventitious roots in mung bean hypocotyl
cuttings.
1 Materials and Methods
1.1 Plant materials
Mung bean (Vigna radiata L. cv. Shannong-20) seeds
were soaked for 12 h in distilled water prior to sowing in
sand that was moistened with distilled water. The seed-
lings were maintained at (30 ± 2) ℃ for 48 h in the dark, and
then transferred to a illuminating incubator with (25 ± 2) ℃,
14 h photoperiod at 300 µmol.m-2.s-1 intensity for 3 d. The
hypocotyls of 5-day-old seedlings were excised 6 cm
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041050
below the cotyledonary node, the cotyledons were
removed, and the resulting cuttings consisting of the hy-
pocotyls and the intact epicotyl, with a pair of primary
leaves were used in rooting experiments reported here.
1.2 Cutting treatments
Freshly prepared hypocotyl cuttings were put into 50
mL conical flask containing 30 mL of test solution that cov-
ered upright with the cut end of the hypocotyls 2 cm deep
for 24 h, then renewed with distilled water per day and kept
for 1-7 d under the same conditions as described above
(25 ± 2) ℃, 14 h photoperiod). The test solution contained
different concentration (100-600 µmol/L ) of the NO donor
sodium nitroprusside (SNP, Sigma, USA) , 300 µmol/L NO2-
/NO3- (NOx, as control for NO decomposition)(Beligni et
al.,1999; Pagnussat et al., 2002), 200 µmol/L of the specific
NO scavenger carboxy-PTIO (c-PTIO, Merck, Germany) and
30 mmol/L NOS inhibitor NG-nitro-l-Arg-methyl ester (L-
NAME, Sigma, USA) respectively. Distilled water (H2O)
was as the control. Numbers and length of adventitious
roots more than 1 mm long were calculated. The fresh and
dry weights of adventitious roots were measured.
The endogenous NO and NADPH-diaphorase levels
were analyzed with the cross sections of the cuttings, which
were cultured with H2O, 200 µmol/L c-PTIO or 30 mmol/L L-
NAME, through the basal 2 mm-region of the cuttings ei-
ther immediately or 24, 36, 48 and 60 h after cutting.
1.3 Histochemical localization of NADPH-diaphorase
Cross sections through the basal 2 mm-region of hypo-
cotyl were serially cut at 20 µm using a freezing microtome.
Sections were stained for NADPH-diaphorase activity ac-
cording to the histochemical method from Cueto et al. (1996).
1.4 Imaging of endogenous NO
Two hundred to 250 µm cross sections from the basal 2
mm-region of mung bean cuttings were immersed in 0.1
mol/L Tris-HCl buffer (pH 7.4). After adding the newly de-
veloped NO sensitive fluorophore 4, 5-diaminofluorescein
diacetate (DAF-2DA, Sigma, USA), which allows the de-
tection of NO presence in animal and plant cells (Foissner
et al., 2000), at a final concentration of 10 µmol/L (added
from a 10 mmol/L stock in DMSO), sections were incubated
in the dark for 2 h. The extra probes were removed by
washing the sections at 0.1 mol/L Tris-HCl three times for
15 min and then images were taken using a TCS-SP2 confo-
cal laser scanning microscope (Leica Lasertechnik Gmbh,
Heidelberg), exciting with the 488 line of an argon laser and
dye emission using a 505-530 nm band-pass filter (Foissner
et al., 2000). Plates were produced with Adobe Photoshop
6.0 software and printed on a Kodak dye sublimation printer.
2 Results
2.1 Effects of SNP, c-PTIO and L-NAME on adventitious
root generation
The effect of NO-donor SNP on the generation of ad-
ventitious roots in the cuttings of mung bean seedlings is
dose dependent (Fig.1). In comparison with H2O or 300
µmol/L NO2-/NO3- treatment, 100-600 µmol/L SNP en-
hanced the number of root significantly. The optimal con-
centration of SNP for the initiation of organogenesis was
300 µmol/L. A consequence of NO decomposition is the
generation of NO2- and NO3- (Beligni et al., 1999). In com-
parison with H2O treatment, NO2-/NO3- treatment did not
increase the number of adventitious root. It was shown
that the effect of SNP came from NO and not from NO
decomposition.
In Table 1, four parameters of root growth were consid-
Fig.1. Effect of different sodium nitroprusside (SNP) concen-
trations on rooting of mung bean hypocotyl cuttings. NOx, NO2-
/NO3-.
Table 1 Effects of sodium nitroprusside (SNP, NO donor), c-PTIO (NO scavenger), L-NAME (NOS inhibitor) and H2O on the
initiation and growth of adventitious roots in mung bean hypocotyl cuttings
Treatments Number of roots per Length of root Fresh weight of roots Dry weight of roots
(mmol/L) cutting (mm) per cutting (mg) per cutting (mg)
H2O 6.85±1.23 b 6.09±1.45 b 15.27±1.40 c 0.98±0.14 c
SNP (300) 25.61±8.02 c 6.95±1.36 b 32.40±2.40 d 1.91±0.12 d
c-PTIO (200) 2.20±2.68 a 1.29±0.49 a 6.00±0.37 a 0.38±0.09 a
L-NAME (30 000) 5.20±2.28 b 2.08±0.97 a 9.07±1.17 b 0.69±0.08 b
The values indicated with different letters were significantly different at P = 0.05 by using ANOVA followed by the least significant difference
(LSD) test. Values are means ± SE of 30 cuttings from at least three independent experiments.
SHE Xiao-Ping et al.: Change of Nitric Oxide and NADPH-diaphorase During the Generation and the Development of
Adventitious Roots in Mung Bean Hypocotyl Cuttings 1051
ered (root length, root number, root fresh and dry weight of
per cutting), which showed similar behaviors among the
treatments. SNP treatment produced significant enhance-
ment on parameters of root growth described as above.
The specific NO scavenger, c-PTIO, significantly reduced
the root length and number, root fresh and dry weight of
per cutting. The effects of L-NAME (NOS inhibitor) on the
generation and development of adventitious roots were
similar to that of c-PTIO.
2.2 Dynamic distribution of NADPH-diaphorase during
adventitious root generation and development in mung bean
hypocotyls cuttings
At 0 h after cutting, cross sections through the basal 2
mm-region of hypocotyl showed uniform faint staining and
no pre-formed root initials could be observed (Fig.2). Very
few NADPH-diaphorase positive cell was detected in the
region between the vascular bundles at 24 h after cutting
(Fig.3). The number of cells expressing NADPH-diapho-
Figs.2-7. Light micrographs of NADPH-diaphorase histochemical localization during adventitious rooting process in mung bean
hypocotyl cuttings, visualized as a NBT-formazan precipitation. Cross sections of hypocotyl in the basal part. Scale bar = 50 µm. 2.
Sections of hypocotyls at the time of cutting, showing the ground level of NADPH-diaphorase expression. No pre-formed root initials
were present in the area of root emergence. 3. Sections of hypocotyl at 24 h after cutting, showing increased expression of NADPH-
diaphorase. 4. Showing increased expression of NADPH-diaphorase at 36 h after cutting. 5. Sections of hypocotyl at 48 h after cutting,
showing high expression of NADPH-diaphorase in the adventitious root primordium. 6. Showing high expression of NADPH-diapho-
rase in the adventitious root primordium at 60 h after cutting.7. Sections of hypocotyl treated with 30 mmol/L L-NAME at 48 h after
cutting, showing slightly staining of NADPH-diaphorase. Abbreviations: L-NAME, NG-nitro-1-Arg-methyl ester; V, vascular bundles.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041052
hanced the number, length and weight of adventitious roots
significantly in mung bean hypocotyl cuttings, which im-
ply that endogenous NO maybe play some role in the gen-
eration and development of adventitious roots. Because
the specific NO scavenger c-PTIO and NOS inhibitor L-
NAME caused significant inhibition of adventitious root
formation, endogenous NO appears to play a key role in
the development of adventitious roots (Table 1). The effect
of L-NAME indicates that NOS-like enzyme mediated NO
generation for this process.
NADPH-diaphorase had been used as a reliable marker
enzyme in the identification of NOS-producing cells in ver-
tebrates and insects (Hope et al., 1991; Müller, 1994).
NADPH-diaphorase activity was also detected as a marker
for NOS-like enzyme in plant cells (Cueto et al., 1996). In
view of the facts we examined the histological distribution
of NADPH-diaphorase during adventitious root formation
in mung bean hypocotyls cuttings (Figs.2-7). NADPH-
diaphorase activity was observed in the region where ad-
ventitious roots originated prior to the emergence of root
promordium, and then in the root primordium. With the origi-
nation and development of adventitious roots, the activity
of NADPH-diaphorase and the number of cell expressing
NADPH-diaphorase-positive increased rapidly in the form-
ing root. Most cells in the adventitious root primordium
had a distinctly NADPH-diaphorase positive in the cyto-
plasm and also in the nucleus. And intense staining mainly
distributed in root meristem. Endogenous NO, which de-
tected with NO-sensitive fluorophore DAF-2DA, had simi-
lar distribution with the NADPH-diaphorase activity in this
process (Figs.10-12). L-NAME treatment significantly de-
creased the fluorescence of NO and NADPH-diaphorase
staining, and the formation of root primordia were inhibited.
The results presented above indicate the presence of a
putative NOS-like enzyme and the enzyme might be respon-
sible for the production of NO during adventitious root
generation and development in mung bean hypocotyl
cuttings. Endogenous NO and NOS-like enzyme appears
to be required for adventitious root organogenesis. The
green fluorescence of NO was also detected in the region
where adventitious roots originated and developed (Figs.
rase activity increased obviously in the region between the
vascular bundles at 36 h after cutting (Fig.4). The root pri-
mordium became discernible at 48 h after cutting. All the
cells in the root primordium had a distinctly NADPH-dia-
phorase positive in the cytoplasm and, for most cells, also
in the nucleus (Fig.5). The appearance of the root primor-
dium became more elongate after 60 h, and began differen-
tiation into root cap, cortex and stele, followed by the for-
mation of the root meristem below the root cap (Fig.6).
NADPH-diaphorase positive persisted in the cytoplasm and
the nucleus. Root primordium meristem cells were shown
intense staining (Fig.6). In the cuttings treated by L-NAME,
NADPH-diaphorase positive decreased significantly and
no or very few root primordia were observed (Fig.7).
2.3 Visualization of NO
NO-sensitive fluorophore DAF-2DA allowed real-time
biological imaging of NO in plant cells (Pedroso et al., 2000b).
The present study showed that no fluorescence was ob-
served at 0 h and 24 h after cutting (Figs.8A, 9A).The
green fluorescence of NO, as well as NADPH-diaphorase
staining, was detected in the region between the vascular
bundles at 36 h after cutting (Fig.10A, B). In general, the
cytosol of DAF-2DA-positive cells stained uniformly for
NO. Fluorescence was visibly brighter in the forming root
primordium at 48 h after cutting, especially at small cells
with dense cytoplasm (Fig.11A, B). With the elongation of
root primordium, the number of DAF-2DA-positive cells
increased clearly at 60 h after cutting (Fig.12A, B). And the
root apical meristem cells showed intense fluorescence.
(Figs.11A,12A). When 200 µmol/L c-PTIO or 30 mmol/L L-
NAME was administered to cuttings, the fluorescence was
suppressed completely and no or very few root primordia
were observed at 48 h after cutting (Figs.13A, 14B).
3 Discussion
Though NO mediation of the adventitious rooting pro-
cess induced by auxins has been demonstrated (Pagnussat
et al., 2002; 2003), the role, the histological distribution and
the source of NO during the development of adventitious
roots are still unknown.
In this study, we have shown that NO donor SNP en-
Figs.8-11. Visualization of nitric oxide (NO) in cross sections from the basal part of mung bean hypocotyl cuttings by confocal laser
scanning microscopy using DAF-2DA (see text for details). Scale bar = 80 µm. 8A. Cross sections of hypocotyl at 0 h after cutting,
showing no NO fluorescence. 8B. Bright field image of same section. 9A. Cross sections of hypocotyl at 24 h after cutting, showing no
NO fluorescence. 9B. Bright field image of same section. 10A. Cross sections of hypocotyl at 36 h after cutting, showing NO fluorescence
(green) in the region between the vascular tissues of the hypocotyls. Fluorescence was uniform in cytosol. 10B. Bright field image of the
same section. 11A. Cross sections of hypocotyl at 48 h after cutting, showing bright fluorescence in the forming root primordium. 11B.
Bright field image of same section. V, vascular bundles.
→
SHE Xiao-Ping et al.: Change of Nitric Oxide and NADPH-diaphorase During the Generation and the Development of
Adventitious Roots in Mung Bean Hypocotyl Cuttings 1053
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041054
Figs.12-14. Visualization of nitric oxide (NO) in cross sections from the basal part of mung bean hypocotyl cuttings by confocal laser
scanning microscopy using DAF-2DA. Scale bar = 80 µm.12A. Cross sections of hypocotyl at 60 h after cutting, showing brighter NO
fluorescence in root primordium. Intense fluorescence focused on root apical meristem cells.12B. Bright field image of same section.13A.
Cross sections of hypocotyl treated with 200 µmol/L c-PTIO at 48 h after cutting, showing no fluorescence.13B. Bright field image of
same section. No or very few root primordium were observed.14A. Cross sections of hypocotyl treated with 30 mmol/L L-NAME at 48
h after cutting, showing no fluorescence .14B. Bright field image of the same section. No or very few root primordia were observed. V,
vascular bundles.
10A-12B). With the development of root primordium, the
number of NO-positive cells increased continuously, and
the small cells with dense cytoplasm in root pre-meristem
and meristem showed brighter fluorescence (Figs.11A-
12B). The findings suggested that NO might be relation to
cell division and differentiation in the development of ad-
ventitious roots. C-PTIO suppressed the fluorescence and
inhibited the formation of root primordia (Fig.13A, B). These
results suggested that endogenous NO appears to play a
key role in the adventitious rooting process. Similar results
were reported by Pagnussat et al. (2002). However, they
found that a transient increase in NO level was required in
SHE Xiao-Ping et al.: Change of Nitric Oxide and NADPH-diaphorase During the Generation and the Development of
Adventitious Roots in Mung Bean Hypocotyl Cuttings 1055
adventitious root development induced by IAA, and high
levels of NO was detected at 0 h and then dramatically shut
down at 24 h in water-treated explants of cucumber
(Pagnussat et al., 2002), whereas our data showed that no
fluorescence of NO in histological level was observed in 0
h and 24 h after cutting, and from 36 h to 60 h, the fluores-
cence of NO appeared and increased continuously (Figs.
8-12).
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(Managing editor: HE Ping)