Nitric Oxide Mediates Gravitropic Bending in
Soybean Roots1[w]
Xiangyang Hu2, Steven J. Neill, Zhangcheng Tang, and Weiming Cai*
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200032, China (X.H., Z.T., W.C.);
and Centre for Research in Plant Science, University of the West of England, Bristol BS16 1QY,
United Kingdom (S.J.N.)
Plant roots are gravitropic, detecting and responding to changes in orientation via differential growth that results in bending
and reestablishment of downward growth. Recent data support the basics of the Cholodny-Went hypothesis, indicating that
differential growth is due to redistribution of auxin to the lower sides of gravistimulated roots, but little is known regarding the
molecular details of such effects. Here, we investigate auxin and gravity signal transduction by demonstrating that the
endogenous signaling molecules nitric oxide (NO) and cGMP mediate responses to gravistimulation in primary roots of
soybean (Glycine max). Horizontal orientation of soybean roots caused the accumulation of both NO and cGMP in the primary
root tip. Fluorescence confocal microcopy revealed that the accumulation of NO was asymmetric, with NO concentrating in the
lower side of the root. Removal of NO with an NO scavenger or inhibition of NO synthesis via NO synthase inhibitors or an
inhibitor of nitrate reductase reduced both NO accumulation and gravitropic bending, indicating that NO synthesis was
required for the gravitropic responses and that both NO synthase and nitrate reductase may contribute to the synthesis of the
NO required. Auxin induced NO accumulation in root protoplasts and asymmetric NO accumulation in root tips.
Gravistimulation, NO, and auxin also induced the accumulation of cGMP, a response inhibited by removal of NO or by
inhibitors of guanylyl cyclase, compounds that also reduced gravitropic bending. Asymmetric NO accumulation and
gravitropic bending were both inhibited by an auxin transport inhibitor, and the inhibition of bending was overcome by
treatment with NO or 8-bromo-cGMP, a cell-permeable analog of cGMP. These data indicate that auxin-induced NO and cGMP
mediate gravitropic curvature in soybean roots.
Plant growth and development are profoundly
influenced by gravity. When roots are gravistimulated
by horizontal orientation, they respond by bending
via differential growth so as to recover their normal
orientation with respect to gravity, the gravitropic setpoint angle (Perbal and Driss-Ecole, 2003). Gravity
perception and responses probably involve mechanosensing in the root cap and changes in calcium and pH
(and other second messengers) that result in relocation
of auxin efflux carriers and subsequent lateral (downward) transport of auxin, inducing differential growth
and downward bending (Blancaflor and Masson, 2003;
Ottenschläger et al., 2003; Perbal and Driss-Ecole,
2003).
1
This work was supported by the Chinese Academy of Sciences
(grant no. KSCX2–SW–322), the Shanghai Institute of Plant Physiology and Ecology, and the National Natural Sciences Foundation of
China (grant no. 39770199). Collaboration between the United
Kingdom and China was supported by The Royal Society and The
Biotechnology and Biological Sciences Research Council.
2
Present address: Department of Botany and Plant Sciences,
University of California, Riverside, CA 92521.
* Corresponding author; e-mail [email protected]; fax 86–21–
54924015.
[w]
The online version of this article contains Web-only data.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.104.054494.
Although recent data provide support for the
Cholodny-Went hypothesis, one of the oldest and
best known in plant science, explaining root gravitropism by downward redistribution of auxin, the precise
details of auxin signaling remain somewhat unclear. In
this study, we report that gravistimulation induced the
asymmetric accumulation of nitric oxide (NO) on the
lower side of the apical region of soybean (Glycine max)
seedling roots. NO is a diffusible multifunctional
molecule involved in numerous processes in bacteria,
fungi, animals, and plants. In plants, NO is an endogenous signaling molecule implicated in a growing
number of developmental and physiological processes, including plant defense, root initiation, stomatal closure in response to abscisic acid, salt tolerance,
seed germination, nutrition, and flowering (Lamattina
et al., 2003; Neill et al., 2003; He et al., 2004; Wendehenne
et al., 2004). Potential sources of NO in plants include
NO synthase (NOS; Guo et al., 2003) and nitrate
reductase (NR), in addition to other potential enzymatic and nonenzymatic sources (Neill et al., 2003;
Bethke et al., 2004). In mammalian cells, it is known
that NO activates the enzyme guanylyl cyclase (GC),
resulting in the accumulation of cGMP. NO was
reported to induce a transient increase in cGMP in
tobacco (Nicotiana tabacum) cells, and specific GC
inhibitors were able to suppress NO-induced gene
expression (Durner et al., 1998). Moreover, stomatal
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Hu et al.
closure and programmed cell death that are both
inducible by NO can also both be inhibited by GC
inhibitors, such inhibition being reversible by addition
of cell-permeable cGMP analogs (Neill et al., 2003).
NO is synthesized in roots (Pagnussat et al., 2002;
Guo et al., 2003), and both NO and cGMP mediate
auxin-induced root organogenesis (Pagnussat et al.,
2003), but the roles of NO and cGMP in gravity
responses are not yet known. Here, gravistimulation
of soybean roots induced asymmetric NO accumulation, and direct NO application to the lower side
of horizontal roots enhanced gravitropic curvature,
whereas application to the upper side suppressed it.
Auxin induced NO accumulation in root protoplasts,
and asymmetric auxin application to root tips resulted
in asymmetric NO accumulation. Moreover, treatments that removed NO or inhibited its synthesis
also inhibited gravitropic bending. In addition, both
gravistimulation and auxin induced the accumulation
of cGMP. Together, these data indicate that NO and
cGMP mediate auxin-induced gravitropic bending in
soybean roots.
RESULTS
Gravistimulation and Auxin Induce NO
Accumulation in Soybean Root Tips
Gravistimulation induced rapid and substantial NO
accumulation in the apical regions (Zone 1) of soybean
primary roots. NO also accumulated in more basal
root tissue (Zone 2) but to a lesser extent (Fig. 1a).
To determine a potential role for NO in gravitropic
bending, we used the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide
(cPTIO), the NOS inhibitors Nv-nitro-L-Arg (L-NNA)
and S,S#-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU), and the putative NR inhibitor
NaN3. To assess any requirement for lateral auxin transport, we used N-1-naphthylphthalamic acid (NPA).
Dose-response data for these compounds are shown in
Supplemental Figure 1. Figure 1b shows that pretreatment with all these compounds (cPTIO at 50 mM,
L-NAA and NaN3 at 20 mM, PBITU and NPA at 30 mM)
significantly reduced both gravitropic bending and
NO accumulation (P , 0.05; one-way ANOVA followed by a Tukey’s test). Treatment with both a NOS
and NR inhibitor did reduce both NO content and
gravicurvature more than when each compound was
applied singly, although inhibition was still not total
(Fig. 1b). We also tested the effects of these compounds
alone at the above concentrations on soybean root
growth: there were no negative effects (P , 0.05).
Confocal microscopy using the NO indicator dye
diaminofluorescein diacetate (DAF-2DA) demonstrated that NO accumulation was asymmetric, with
NO being detected predominantly in the lower cells
(Fig. 1c). Moreover, NO accumulation appeared to
begin in the most apical region of the root (Fig. 1).
664
Gravity-induced increases in fluorescence were not
observed when 4-aminofluorescein diacetate (4-AF
DA, the negative control for DAF-2DA) was used
(data not shown). Pretreatment with NaN3, L-NNA,
cPTIO, or NPA all reduced NO fluorescence (Fig. 1c).
At low concentrations (1 and 5 mM), NPA had a greater
effect on NO content than it did on gravibending
(Supplemental Fig. 1). At these concentrations of NPA,
NO distribution in the roots was still asymmetric (data
not shown), whereas NO asymmetry was disrupted at
higher concentrations, offering a potential explanation
for this discrepancy. We also determined the effects of
gravistimulation on asymmetric NO accumulation
using the hemoglobin assay. Although there were
some differences in the absolute amount of NO (for
example, Fig. 1a, 1.7 6 0.18 nmol/g; and Fig. 1d, 3.1 6
0.23 nmol/g [averaged between upper and lower
halves], both after 12 h of gravity treatment; note
that these data were obtained at different times using
different batches of plant material and assay components), the trends in [NO] were reproducible and
confirmed that the NO content of the lower half of
horizontal roots increased considerably following
gravistimulation. Taken together, these data indicate
that NO is required for the gravitropic response and
suggest that auxin redistribution activates NO synthesis, potentially via both NOS- and NR-like enzymes.
We next examined the effect of auxin on NO
accumulation in both intact roots and root protoplasts.
The dose-response data for auxin-induced NO accumulation are presented in Supplemental Figure 2.
Auxin at 5 mM significantly induced NO accumulation
in the root tip. Furthermore, when the auxin was
applied unilaterally to roots via agar blocks, NO
accumulation was restricted to the side of the root in
contact with the auxin-containing agar (Fig. 2, a and b).
Auxin also induced rapid NO generation in root
protoplasts (Fig. 2c). Replacing DAF-2DA with 4-AF
DA did not give rise to fluorescence in the auxintreated protoplasts (data not shown). NaN3 and
L-NAA treatment suppressed auxin-induced NO generation but not completely, whereas treatment with
cPTIO strongly suppressed it (Fig. 2d).
NO Modulates Root Extension and Gravitropic Bending
The data in Figure 1d show that the average NO
concentration in the upper side of roots was about
0.25 nmol/g, whereas that in the lower side was
about 8 nmol/g. To determine the effects of exogenous
NO on gravitropic responses, we first assessed its effects on root extension. Guo et al. (2003) reported that
NOS-deficient Arabidopsis (Arabidopsis thaliana) mutant plants do not synthesize NO in the roots and
have a short root phenotype that can be reversed
by treatment with the NO donor sodium nitroprusside (SNP). In our experiments with soybean roots,
incubation in solutions of SNP at very low concentrations ([NO] ranging from 0.1–0.75 nmol/g in the
Plant Physiol. Vol. 137, 2005
Nitric Oxide and Gravitropism
Figure 1. Gravistimulation induces the rapid accumulation of NO required for gravitropism in soybean roots. a, Kinetics of NO
accumulation in Zone 1 (d) or Zone 2 (s) from horizontal roots. ;, Vertical roots. b, Correlation between NO accumulation in
Zone 1 (black bars) and gravitropic curvature (white bars). Roots were pretreated as indicated for 12 h and then placed
horizontally for 12 h (50 mM cPTIO; 20 mM L-NAA and NaN3; 30 mM PBITU and NPA; 100 mM ODQ and LY83583). On x axis: C,
Control; G, gravistimulated. Values are the mean 6 SE for five independent experiments. Data analyzed by one-way ANOVA
followed by Tukey’s test. Different symbols indicate significant differences between treatments (P , 0.05). c, Gravistimulation
induces asymmetric NO accumulation. Soybean roots were loaded with DAF-2DA and gravistimulated by orientating
horizontally. Fluorescence intensity of dissected root tips was observed at the indicated times by confocal fluorescence
microscopy. Effects of pretreatment with 20 mM NaN3, 20 mM L-NNA, 50 mM cPTIO, and 10 mM NPA are shown. The pictures were
taken after 6 h treatment. Scale bar 5 1 mm. Experiments were repeated at least five times with similar results. d, Asymmetric
accumulation of NO in root Zone 1. Soybean roots were gravistimulated for different times, bisected horizontally with a razor
blade, and NO content assayed. ;, Vertical roots; s, lower half of horizontal root; d, upper half of horizontal root.
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Hu et al.
graviresponding roots and, strikingly, reduced when
the NO was applied to the upper side (Fig. 3b). We also
determined the effects of asymmetric NO application
on the inhibition of gravitropism brought about by
NPA: supplying NO from 0.5 mM SNP or from 5 mM
SNP in agar to the upper or lower side, respectively, of
NPA-treated roots overcame the effects of NPA (Fig.
3c). Applying PBTIU, cPTIO, or NaN3 via agar blocks
to the lower side of horizontal roots suppressed root
bending, suggesting that reducing NO accumulation
in the lower side of horizontal roots reduces root
bending. Application of these inhibitors to the upper
side of horizontal roots had no significant effects,
either positive or negative (data not shown).
NO Effects Are Mediated by cGMP
Figure 2. Auxin induces NO accumulation. a and b, Soybean roots
were loaded with 10 mM DAF-2DA for 30 min and then control buffer
(A) or 5 mM IAA (B) applied via agar block to the left-hand side of the
vertically orientated root. NO was imaged after 30 min. Scale bar 5
1 mm. Experiments were repeated five times with similar results. c,
Protoplasts were loaded with DAF-2DA and then incubated in 5 mM IAA
for 30 min prior to flow cytometry. Experiments were repeated three
times with similar results. d, Protoplasts were loaded with DAF-2DA
and then incubated with 5 mM IAA in combination with 20 mM L-NAA,
20 mM NaN3, or 50 mM cPTIO. Experiments were repeated three times
with similar results.
root) actually promoted growth (Fig. 3a). However, the
root elongation response to NO was positive or negative depending on NO concentration: at concentrations greater than 1 nmol/g, growth was increasingly
inhibited (Fig. 3a, P , 0.01). He et al. (2004) have
recently reported a similar biphasic response of Arabidopsis roots to NO. Treatment with the SNP analog
sodium ferrocyanide that does not release NO had no
effect (data not shown). These results suggest that the
low concentration of NO in the upper side could
promote root elongation, whereas the high concentration of NO in the lower side would suppress
elongation, thus effecting gravitropic bending. To
demonstrate that NO alone can modulate gravitropic
responses, we applied NO via agar blocks containing
SNP at a high concentration (5 mM). Bending was
enhanced when NO was applied to the lower side of
666
cGMP is an intracellular signaling molecule often
synthesized in response to increasing NO concentrations (Neill et al., 2003). Consequently, we assessed the
potential involvement of cGMP in gravitropism. Gravistimulation induced a rapid and substantial increase in
the endogenous root cGMP content, particularly in
apical tissue (Fig. 4a). This accumulation was asymmetric, similar to that seen for NO (Fig. 4b). Again, and
in a similar manner, there were some differences in the
absolute amounts of cGMP, e.g. average [cGMP] total
root in Figure 4b is approximately 80 pmol/g and
approximately 45 pmol/g in Figure 4a. Potential reasons for these differences relate to the use of different
plants and materials, as mentioned above. Treatments
that inhibited NO accumulation and gravitropic bending (Fig. 1b) also inhibited the increase in endogenous
cGMP (Fig. 4c). Treatment with both inhibitors of NOS
and NR had a slightly greater effect than treatment with
either alone (Fig. 4c). In addition, 1H-[1,2,4]-oxadiazole[4,3-a]quinoxalin-1-one (ODQ) and 6-anilino-5,8quinolinedione (LY83583), inhibitors of the cGMP synthesizing enzyme GC (Neill et al., 2003), prevented
increases in cGMP (Fig. 4c) and inhibited bending (Fig.
1b), although they had little effect on NO accumulation
(Fig. 1b). Incubation of roots in either SNP or auxin
induced a rapid and substantial increase in endogenous cGMP content (Fig. 5, a and b; see Supplemental
Fig. 3 for auxin dose response). Application of cGMP
(as the cell-permeable analog 8-bromo-cGMP) to roots
pretreated with NPA had the same effect as did
exogenous NO and overcame the inhibition of gravitropic bending by NPA (Fig. 3c).
DISCUSSION
Our data demonstrate that gravistimulation of soybean primary roots induces asymmetric NO generation, NO accumulating in the lower half of horizontally
orientated roots. That such NO accumulation is required for a full gravitropic response is indicated by the
reduction in gravitropic bending effected by treatments
that reduce NO production. Several potential sources of
Plant Physiol. Vol. 137, 2005
Nitric Oxide and Gravitropism
Figure 3. Effect of NO on root extension and of asymmetric NO and cGMP application on root curvature of graviresponding
soybean roots. a, Effects of incubation in SNP on root extension. Roots were incubated in SNP at various concentrations and then
root [NO] (d) and extension (s) measured after 48 h. b, Effects of NO at high concentration on root bending. Agar blocks
containing 5 mM SNP (resulting in approximately 5 nmol/g NO in roots after 48 h) were placed on the lower (d) or upper side (s)
of horizontally orientated soybean roots and gravitropic curvature measured at the indicated times. ;, Control root showing
normal gravitropism. c, Asymmetric applications of NO and inhibitors. Roots were pretreated with NPA and then gravistimulated
(E). Agar blocks containing 5 mM (B) or 0.5 mM (C) SNP were placed on the lower (B) or upper (C) side, or blocks containing 50 pM
8-Br-cGMP (D) were placed on the lower side of NPA-pretreated horizontal roots. For inhibitor experiments, agar blocks
containing 20 mM PBTIU (F), 20 mM L-NAA (G), 20 mM NaN3 (H), and 50 mM cPTIO (I), were placed on the lower side of
gravistimulated roots. A, Control with no pretreatment. Black bars, 3-h treatment; gray bars, 6-h treatment; and white bars, 12-h
treatment. Root curvature was measured at the indicated times. Values are the means 6 SE for five independent experiments.
NO exist in plants, including NR and a NOS, AtNOS1
(Guo et al., 2003; Neill et al., 2003). Inhibition of NO
accumulation by NOS inhibitors suggests that one
source of NO is a NOS-like enzyme, but whether or
not the enzyme activity in soybean roots is similar to
AtNOS1 is not yet known, although AtNOS1 is expressed in Arabidopsis roots (Guo et al., 2003). It is
likely that NR also contributes to NO production in
roots, as treatment with the putative NR inhibitor NaN3
reduced both the increases in [NO] and bending. NO
generation via NR is known to occur in roots (Dordas
et al., 2003), and it may be that NR is responsible for
basal NO production that in some way is required for
NO generation in addition to elevated NOS activity. It is
interesting that NO was visible in the root tip of control
roots and still present after L-NAA treatment but not
after treatment with NaN3 (Fig. 1b). Nonenzymatic NO
generation via reduction of apoplastic nitrite has been
reported recently, with functions for apoplastic NO in
seed germination and root development being suggested (Bethke et al., 2004). Some apoplastic NO production is a possibility in our study because the
inhibitors of NR or NOS did not completely prevent
root bending and NO generation. The pH of the root
cap in Arabidopsis can decrease from 5.5 to 4.5 within
2 min of gravitropic stimulation (Fasano et al., 2001),
potentially providing the necessary acidification for
apoplastic NO formation.
The involvement of NO in gravitropism is also
indicated by the modulation by exogenous NO of
gravitropic bending. At concentrations greater than
500 nM, exogenous NO (approximately 5 nmol/g
internal [NO]) inhibits soybean primary root extension, and application to the lower or upper sides of
Figure 4. Gravistimulation induces the rapid accumulation of cGMP in soybean roots. a, Kinetics of cGMP accumulation in
Zone 1 (d) or Zone 2 (s) from horizontal roots following gravistimulation. ;, Vertical roots. Values are the means 6 SE for five
independent experiments. b, Asymmetric distribution of cGMP in root Zone 1. Soybean roots were gravistimulated for different
times, bisected horizontally with a razor blade, and cGMP content assayed. ;, Vertical roots; s, lower half of horizontal root; d,
upper half of horizontal root. c, Roots were pretreated as indicated for 12 h and then placed horizontally for 12 h. On x axis: CK,
Control; G, gravistimulated. Data analyzed by one-way ANOVA followed by Tukey’s test. Different symbols indicate significant
differences between treatments (P , 0.05).
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Figure 5. NO and auxin stimulate cGMP accumulation in soybean
roots. a, Roots were treated with SNP (d, control; s, 50 nM; =, 100 nM;
;, 500 nM). b, Roots were treated with 5 mM auxin (d, IAA; s, control)
and cGMP content assayed at different times. Values are the means 6 SE
for five independent experiments.
graviresponding roots enhances or reduces bending,
respectively. Arabidopsis Atnos1 mutants defective in
NO synthesis have shorter roots, a defect restored by
exogenous NO (Guo et al., 2003). Our data are consistent with this and with those of He at al. (2004),
suggesting that NO can exert positive or negative
effects on root growth depending on its concentration
and interactions with other signaling molecules, similar to auxin itself (Fu and Harberd, 2003).
Our data also show clearly that auxin stimulates NO
generation in soybean roots. Although the effects of
auxin on NO generation were not reported for Arabidopsis roots (Guo et al., 2003), auxin has previously
been shown to stimulate NO production in cucumber
(Cucumis sativus) hypocotyls, and this NO was required for adventitious rooting (Pagnussat et al., 2003).
Auxin did not stimulate NO production in tobacco
suspension cultures (Tun et al., 2001), suggesting that
the effects on NO production may be cell specific or
signal specific. Graviperception occurs in the columella cells of the root cap, whereas differential growth
occurs in the extension zone (Blancaflor and Masson,
2003). Recent data show that the auxin efflux regulator
protein PIN3 relocalizes laterally following gravisti668
mulation (Friml et al., 2002) and that auxin is transported differentially from columella to lateral root cap
cells (Ottenschläger et al., 2003). Inhibition of NO
accumulation by NPA, an auxin efflux inhibitor
(Ottenschläger et al., 2003), indicates that lateral auxin
transport is required for asymmetric NO generation.
Moreover, the location of NO accumulation is consistent with the site of maximum endogenous auxin
accumulation. Subsequent basipetal transport of auxin
to cells of the extension zone results in a much lower
auxin content there than in the lateral root cap
(Ottenschläger et al., 2003); thus, lower amounts of
NO also would be expected. Stimulation of NO production by auxin potentially involves some cooperation between NOS and NR, as NPA and both NOS and
NR inhibitors all blocked NO accumulation. Moreover,
the inhibitory effects of NPA on gravitropic bending
and NO accumulation were somewhat greater than
those of the NO synthesis inhibitors, suggesting that
auxin-induced acidification may also contribute to NO
production during graviresponses.
cGMP is a second messenger generated in response
to NO (Neill et al., 2003), and its presence in plants has
been established unequivocally (Newton et al., 1999).
Here, gravistimulation induced asymmetric cGMP
similar to the increases in [NO]. In addition, treatment
with either NO or auxin also stimulated cGMP accumulation in soybean roots. Inhibition of increased
cGMP content by the GC inhibitors ODQ and
LY83583 suggests that auxin and NO activate a GClike enzyme. Such an enzyme has been identified
recently in Arabidopsis, but its activation characteristics have not yet been defined fully (Ludidi and
Gehring, 2002). The induction of cGMP synthesis in
stomatal guard cells by auxin has already been indicated via pharmacological studies (Cousson and
Vavasseur, 1998). Inhibition of NO synthesis by NPA,
coupled with the lack of effect of ODQ and LY83583 on
NO accumulation and the restoration of gravitropic
bending by 8-Br-cGMP to NPA-treated roots, indicate
that auxin induces NO production that in turn leads to
cGMP synthesis.
While the data reported herein do show that NO
and cGMP are required for full gravitropic responses,
the lack of complete inhibition by inhibitors of NO and
cGMP synthesis indicate that additional mechanisms
may mediate auxin effects in root cells. In fact, NO is
often generated at the same time as reactive oxygen
species (ROS) such as hydrogen peroxide, for example,
during pathogen responses or abscisic acid-induced
stomatal closure (Neill et al., 2003). Root gravitropism
appears to be another example of a physiological
process in which both NO and ROS play key roles,
as ROS were reported recently to be required for
auxin-mediated gravitropic responses in maize (Zea
mays; Joo et al., 2001). Preliminary data indicate that
ROS scavengers do indeed reduce gravitropic bending
in our system (data not shown).
In summary, our data implicate NO as another signal
mediating root responses to gravity and auxin, with
Plant Physiol. Vol. 137, 2005
Nitric Oxide and Gravitropism
several potential sources of NO. The recent cloning of
NO biosynthetic enzymes from Arabidopsis will no
doubt facilitate further experiments to discern the roles
of NO signaling in roots.
MATERIALS AND METHODS
Plant Materials
Soybean seeds (Glycine max cv Williams 82) were soaked in distilled water
for 6 h, placed between wet paper towels held between plastic sheets mounted
vertically in trays, and germinated for 2 d (at this point there was one primary
and some secondary roots).
Root Treatments
To induce gravitropism, the trays were turned so that the roots were
orientated horizontally. For subsequent analyses, the root was excised at the
indicated times and then divided into two parts, Zone 1 and Zone 2, as
described by Joo et al. (2001), that were immediately frozen in liquid nitrogen.
Zone 1 represented the apical 4 mm of the root, including the root cap,
meristem, and sometimes part of the elongation zone. Zone 2 (4–8 mm)
represented the rest of the elongation zone.
To pretreat with various chemicals, attached roots (vertically orientated)
were dipped in solutions of the appropriate chemicals (L-NNA, PBITU, cPTIO,
NPA, ODQ, LY83583) for 12 h. For direct application to roots, agar blocks (0.8%
[w/v], 5 mm 3 5 mm 3 20 mm) were used. Filter-sterilized solutions were
added to cooled, molten agar in MES/KCl buffer (10 mM MES, pH 6.15, 10 mM
KCl, 50 mM CaCl2) to give the required final concentrations. All manipulations
were done in a dark room under dim green light at 24°C 6 1°C. Chemicals
were dissolved in distilled water (SNP, indole-3-acetic acid [IAA]) or 1%
dimethyl sulfoxide (L-NAA, PBITU, cPTIO, NPA, ODQ, LY83583, 8-Br-cGMP).
Control treatments involved addition of equivalent amounts of 1% dimethyl
sulfoxide or distilled water.
Determination of Curvature and Elongation of
Soybean Roots
After various treatments and at various times, images of growing roots
were recorded with a cooled CCD camera (CCD512BKS; Princeton Instruments, Trenton, NJ) and analyzed using Adobe Photoshop 5.0 (Adobe
Systems, Mountain View, CA). Curvature and elongation were measured
using a protractor and ruler. All manipulations were done in the darkroom
under dim green light at 24°C 6 1°C.
Preparation of Soybean Root Protoplasts and
Flow Cytometry
Protoplasts were prepared from 2-d-old soybean roots (1 g) essentially as
described by Joo et al. (2001). Protoplasts were separated from the partially
digested tissues by passage through a nylon mesh (75 mm) and pelleted at 60g
for 5 min. The protoplasts were washed three times with MES/KCl buffer
supplemented with 0.45 M mannitol and 1 mM CaCl2 and stored in the dark.
Protoplasts were incubated in 10 mM DAF-2DA in MES/KCl buffer supplemented with 0.45 M mannitol and 1 mM CaCl2 for 10 min and then washed in
buffer (3 3 5 min) prior to incubation in buffer plus or minus 5 mM IAA.
Fluorescence intensity was measured by flow cytometry using a FACScan
(Becton-Dickinson, Franklin Lakes, NJ) with excitation and emission settings
of 488 and 530 nm, respectively. Counting of cells stopped at 30,000. Gating
(gate set at 75 mm) was performed prior to data collection. All experiments
were repeated three times with similar results.
ACKNOWLEDGMENTS
We thank R. Desikan and J. Hancock (University of the West of England,
Bristol) for their helpful comments on the manuscript.
Received October 8, 2004; returned for revision December 16, 2004; accepted
December 20, 2004.
NO Assay
LITERATURE CITED
Primary root tissue (approximately 100 mg; Zone 1 or Zone 2) was
homogenized in 1 mL of extraction buffer (10 mM Tris-Cl, pH 8.0, 1 mM
phenylmethylsulfonyl fluoride, 10 mM MgSO4, 5 mM KCl, 5 mM NaCl
containing 10 mM oxyhemoglobin and 10 units/mL of catalase). The extracts
were then centrifuged (15,000g, 10 min) at 4°C and the supernatants used for
NO determination using the hemoglobin assay (Delledonne et al., 1998). To
assess the asymmetric distribution of NO (and cGMP) in the root tip, the roots
were bisected into upper and lower halves with a razor blade prior to freezing
and analysis.
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