hydrogen peroxide–mediated activation of map kinase 6 ...hydrogen peroxide–mediated activation...
TRANSCRIPT
Hydrogen Peroxide–Mediated Activation of MAP Kinase 6Modulates Nitric Oxide Biosynthesis and Signal Transductionin Arabidopsis W
Pengcheng Wang,a Yanyan Du,a Yuan Li,b Dongtao Ren,b and Chun-Peng Songa,1
a Laboratory of Plant Stress Biology, Department of Biology, Henan University, Kaifeng 475001, Chinab State Key Laboratory of Plant Physiology and Biochemistry, China Agricultural University, Beijing 100193, China
Nitric oxide (NO) is a bioactive molecule that functions in numerous physiological and developmental processes in plants,
including lateral root development. In this study, we used biochemical and genetic approaches to analyze the function of
Arabidopsis thaliana mitogen-activated protein kinase 6 (MPK6) in the regulation of NO synthesis in response to hydrogen
peroxide (H2O2) during lateral root development. In both mpk6 mutants studied, H2O2-induced NO synthesis and nitrate
reductase (NR) activity were decreased dramatically. Furthermore, one NR isoform, NIA2, was required for the MPK6-
mediated production of NO induced by H2O2. Notably, NIA2 interacted physically with MPK6 in vitro and in vivo and also
served as a substrate of MPK6. Phosphorylation of NIA2 by MPK6 led to an increase in NR activity, and Ser-627 was
identified as the putative phosphorylation site on NIA2. Phenotypical analysis revealed that mpk6-2 and mpk6-3 seedlings
produce more and longer lateral roots than wild-type plants did after application of the NO donor sodium nitroprusside or
H2O2. These data support strongly a function of MPK6 in modulating NO production and signal transduction in response to
H2O2 during Arabidopsis root development.
INTRODUCTION
Nitric oxide (NO) has been characterized recently as an important
signal molecule that mediates many developmental and physi-
ological processes in plants, which include seed germination,
lateral root initiation, flowering, stomatal closure, and responses
to abiotic stresses (He et al., 2005; Simpson, 2005; Libourel et al.,
2006; Lombardo et al., 2006; Neill et al., 2008). In plant cells, NO
is produced mainly via two distinct enzymatic pathways: the
Arg-dependent nitric oxide synthase (NOS) pathway and the
nitrite-dependent nitrate reductase (NR) pathway. In addition,
nonenzymatic processes contribute to the synthesis of NO in
plants (Neill et al., 2003, 2008; Wilson et al., 2007). Although pre-
vious findings have indicated the existence of NOS in plants, no
gene or protein with sequence homology to known mammalian-
type NOS has been found (Guo et al., 2003; Crawford, 2006). In
Arabidopsis thaliana, NOS1/NOA1, which was identified origi-
nally as a potential NOS (Guo et al., 2003), was found to be
unable to bind and oxidize Arg to NO andwas later shown to be a
circularly permuted GTPase (Moreau et al., 2008).
NR is the only enzyme that has been shown to catalyze the
production of NO in plant defense responses. Although the
primary function of NR in plants is to convert nitrate to nitrite, it
can also convert nitrite to NO in vitro and in vivo (Dean and
Harper, 1988; Desikan et al., 2002). Bursts of NO that are induced
by auxins, elicitors, abscisic acid (ABA), or hydrogen perox-
ide (H2O2) are dependent on NR activity (Bright et al., 2006;
Yamamoto-Katou et al., 2006; Kolbert et al., 2008). InArabidopsis,
NR is encoded by two genes, NIA1 and NIA2 (Campbell, 1999).
Deficiency of NIA1 and NIA2 results in a significant reduction in
NO synthesis (Bright et al., 2006; Modolo et al., 2006). Further
investigations have revealed that NIA1 and NIA2 contribute
differently to the synthesis of NO in different tissues. During
stomatal closure induced by ABA, NIA1 plays the major role in
NO production (Bright et al., 2006), although NIA1 is known to be
less abundant and less active than NIA2 in seedlings (Wilkinson
and Crawford, 1991).
Mitogen-activated protein kinase (MAPK) cascades are con-
served pathways that transduce environmental stimuli into
intracellular responses in many organisms, including humans,
Drosophila melanogaster, yeast, and plants. Each MAPK cas-
cade is composed of three kinases. MAPKs are activated
through phosphorylation by upstreamMAPK kinases (MAPKKs),
which are in turn activated by MAPKK kinases (MAPKKKs)
(Ichimura et al., 2000). In Arabidopsis, there are 20 MAPKs, 10
MAPKKs, and ;60 MAPKKKs (Ichimura et al., 2000). It is well
documented that MAPK cascades play key roles in innate
immunity and environmental stress responses and are also in-
volved in the regulation of plant growth and development (Tena
et al., 2001; Zhang and Klessig, 2001; Jonak et al., 2002; Mishra
et al., 2006; Colcombet and Hirt, 2008). Mitogen-activated
protein kinase 6 (MPK6) is a well-characterized MAPK in
Arabidopsis. It can be activated by various abiotic and biotic
stresses, which include low temperature, low humidity, hyper-
osmolarity, touch, wounding, microbial elicitors, and oxidative
1 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Chun-Peng Song([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.072959
The Plant Cell, Vol. 22: 2981–2998, September 2010, www.plantcell.org ã 2010 American Society of Plant Biologists
stress (Ichimura et al., 2000; Nuhse et al., 2000; Desikan et al.,
2001; Yuasa et al., 2001). MPK6 is also involved in the signaling
and responses of the plant hormones ethylene and jasmonic
acid (Liu and Zhang, 2004; Seo et al., 2007; Takahashi et al.,
2007; Joo et al., 2008; Yoo et al., 2008). Emerging evidence has
also indicated a novel function of MPK6 in plant growth and
development. For example, MPK6 and MPK3 are required for
specification of cell fate during stomatal development, the
formation of anther lobes and differentiation of anther cells, and
development of ovules (Wang et al., 2007; Hord et al., 2008;
Wang et al., 2008). Several substrates of MPK6 have been
identified by biochemical and genetic analyses in Arabidopsis;
these include the rate-limiting enzyme of ethylene biosynthesis,
1-aminocyclopropane-1-carboxylic acid synthase (ACS) (Liu
and Zhang, 2004), the transcriptional regulators ethylene-
insensitive 3 (Yoo et al., 2008) and ethylene response factor
104 (Bethke et al., 2009), and PHOS32 (a 32-kD phosphorylated
protein) (Merkouropoulos et al., 2008). Furthermore, 39 pro-
teins were isolated as potential substrates of MPK6 from
protein microarrays that included 1690 Arabidopsis proteins,
which indicates that MPK6 acts as a universal regulator in plant
stress responses, as well as during growth and development
(Feilner et al., 2005).
Previous findings have indicated that NO can activate MAPK
cascades; for example, it activates salicylic acid–induced pro-
tein kinase (SIPK) in tobacco (Nicotiana tabacum) and a 46-kD
MAPK in maize (Zea mays) and Arabidopsis (Clarke et al., 2000;
Kumar and Klessig, 2000; Zhang et al., 2007a). Surprisingly,
recent evidence also indicates that MAPKs play a critical role in
regulating the production of NO (Asai et al., 2008). In tobacco,
the elicitor INF-1, produced by Phytophthora infestans, can
induce the activation of MAP or ERK kinases (MEK) MEK1 and
MEK2 and also enhances the production of NO (Yamamoto
et al., 2004). Constitutive activation of MEK2 enhances NO
synthesis dramatically. Furthermore, silencing of SIPK and the
SIPK homolog, NTF4, a MAPK that is activated by MEK2 in
tobacco, markedly reduces the production of NO. Synthesis of
NO that is induced by INF1 and Solanum tuberosum (St)
MEK2DD is decreased significantly in plants in which NOA1 is
silenced, which suggests that NOA1 is involved in the process
(Asai et al., 2008). However, tungstate, an inhibitor of NR, can
also suppress the production of NO that is induced by INF1 and
St MEK2DD, which suggests that NR also participates in the NO
burst. Although the regulation of NO synthesis might involve the
posttranscriptional modification of NR, details of the mecha-
nism remain unclear.
In the study reported here, we investigated the roles of the
MAPK cascade in the production of NO induced by H2O2 in
Arabidopsis. We found that MPK6 was involved in regulating
H2O2-induced synthesis of NO. We identified a direct interaction
between MPK6 and NIA2 in vitro and in vivo and demonstrated
that MPK6 could phosphorylate NIA2, with Ser-627 being the
putative phosphorylation site of NIA2. Phosphorylation of NIA2
by MPK6 not only increased the activity of NIA2 and the pro-
duction of NO dramatically, but also led to morphological
changes in the root system of Arabidopsis. Our findings suggest
a novel model for the involvement of MPK6 in NO synthesis and
signal transduction during root development.
RESULTS
mpk6Mutants Are Defective in H2O2-Induced
NO Generation
A recent study has indicated that MAPK signaling regulates the
production of NO and NADPH oxidase–dependent oxidative
bursts in tobacco (Asai et al., 2008). In Arabidopsis, MPK6 is the
functional ortholog of the tobacco protein SIPK (Tena et al., 2001;
Ichimura et al., 2002), and MPK6 can be activated by oxidative
stress (Desikan et al., 2001; Yuasa et al., 2001). To investigate the
role of MPK6 in NO synthesis, we measured the H2O2-induced
production of NO in the roots of MPK6-deficient mutants using
the NO-specific fluorescent indicator diaminofluorescein diace-
tate (DAF-2 DA). DAF-2 DA is membrane permeable and is
deacetylated by intracellular esterases to nonfluorescent 4,5-
diaminofluorescein (DAF-2). However, in the presence of NO and
O2, DAF-2 is converted to the fluorescent triazole derivative DAF-
2 T. This increases the quantum yield of fluorescence >180-fold,
which allows the measurement of NO levels in the cell cytoplasm
(Kojima et al., 1998). The roots of seedlings were loaded with
DAF-2 DA, and changes in fluorescence induced by NO were
examined by confocal laser scanning microscopy. Exogenous
application of H2O2 enhanced the relative fluorescent intensity of
DAF-2 in root cells of both mpk6-3 and wild-type seedlings; the
relative fluorescent intensity corresponds to the intensity for
each pixel averaged over awhole root (Figure 1A).When catalase
(CAT), a widely used H2O2 scavenger, was applied alone or
together with 10 mM H2O2, H2O2-induced fluorescence was
almost completely abolished (Figure 1B). Furthermore, appli-
cation of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-
oxyl-3-oxide (c-PTIO), which is a specific scavenger of NO,
eliminated the DAF-2 fluorescence induced by H2O2 in the wild
type and mpk6-3 (Figure 1B). These results suggest that DAF-2
fluorescence intensity reflects the concentration of NO in the
roots.
As shown in Figure 1, NO was synthesized more rapidly after
H2O2 treatment in wild-type than inmpk6-3 roots (Figures 1A and
1C, significantly different according to the Steel-Dwass test, P#
0.05). The effect of H2O2 on promotion of NO production in a
time-dependent manner (Figure 1C) was significant (P# 0.05) at
a concentration of H2O2 > 10 mM in roots of the wild type. The
pixel intensity of root cells frommpk6-3was 40% lower than that
from thewild type after 20min of treatment with H2O2 (Figures 1A
and 1C, significantly different according to the Steel-Dwass test,
P# 0.05). In the presence of 0.5 to 2mMH2O2, NO generation in
the wild type and mpk6-3 mutants was further enhanced (see
Supplemental Figure 1A online).
In mammalian systems, PD 098059 has been used widely as
an inhibitor of specificMAPKKs (and thus activation ofMAPKs) to
establish the physiological role of MAPKs in various types of cell
(Alessi et al., 1995). When roots were preincubated with 50 mM
PD 098059 for 30 min, the increase in NO production induced by
H2O2 was suppressed by 46% in the wild type (P # 0.05),
whereas the fluorescence intensity in mpk6-3 was almost unaf-
fected by treatment with PD 098059 (P # 0.05) (Figure 1D). NO
production of the roots in the wild type and mpk6-3 treated with
PD 098059 alone was almost equal to that of untreated controls.
2982 The Plant Cell
The data suggest that the H2O2-mediated NO generation is
dependent on the activation of MPK6.
Encouraged by the above results, we assayed the MPK6
kinase activity induced by H2O2 by measuring the levels of
phosphorylation in wild-type and mpk6-3 mutant plants that
were exposed to different concentrations of exogenous H2O2.
Exogenous application of H2O2 promoted the kinase activity of
MPK6 in a dose-dependent manner (see Supplemental Figure
1B online). The obvious promotion of MPK kinase activity was
observed at 30 min after treatment with 10 mM H2O2 (see
Supplemental Figure 1B online). Under the same conditions,
the NO production was significantly increased (Figure 1C).
Application of 2 mM H2O2 resulted in the strongest activation
of MPK6 (Figure 1E; see Supplemental Figure 1B online).
Consistent with the above results, application of H2O2 to
wild-type plants stimulated MPK6 activity significantly in a
time-dependentmanner (Figure 1E). The activation ofMPK6 by
H2O2 was rapid, with the maximum level of phosphorylation
occurring 30 min after the addition of H2O2 (Figure 1E). Acti-
vation of MPK6 was still detectable at 60 min. In contrast with
MPK6 activity, MPK3 activity was weak and relatively unstim-
ulated by H2O2 under the same experimental conditions (Fig-
ure 1E).
Rapid activation of MPK6 by H2O2 was abolished completely
in the mpk6-3 mutant (Figure 1F; see Supplemental Figure 1B
online). These data show that regulation of H2O2-induced NO
production is disrupted inmpk6-3, indicating that active MPK6 is
required for H2O2-induced NO synthesis in Arabidopsis.
Figure 1. mpk6 Mutants Produce Less Nitric Oxide than Wild-Type Plants in Response to H2O2.
(A) Images of H2O2-induced NO production in wild-type andmpk6-3 plants. Roots fromwild-type andmpk6-3mutants were loaded with DAF-2 DA, and
NO synthesis was monitored after the addition of 10 mM H2O2 or a water control. The micrographs show pairs of representative bright-field and
fluorescence images of the roots of wild-type and mpk6-3 plants in six independent experiments. Bar = 100 mm.
(B) Effects of CAT and c-PTIO on DAF-2 fluorescence in wild-type and mpk6-3 plants in the presence and absence of H2O2. Control represents wild-
type and mutants treated with water. Average pixel intensities of DAF-2 DA fluorescence were calculated after addition of CAT or c-PTIO for 30 min.
Values are means (6SD) from whole root regions (from maturation zone to meristematic zone) from six independent experiments.
(C) Time course of NO production expressed as the pixel intensities of DAF-2 DA fluorescence in the roots of wild-type and mpk6-3 plants before and
after addition of H2O2. Wild-type andmpk6-3 plants were treated with 10 mMH2O2 for the times indicated. Signal intensities were quantified over whole
roots from micrographs taken under confocal laser scanning microscopy. Values are means (6SD) from six independent experiments.
(D) Effects of PD 098059 on NO production in wild-type andmpk6-3 plants in the presence and absence of H2O2. Average pixel intensities of DAF-2 DA
fluorescence were calculated after addition of PD 098059, H2O2, or water control for 30 min. Values are means (6SD) from whole root regions from six
independent experiments.
(E) H2O2-induced activation of MPK6 and MPK3 in vivo. Proteins were extracted from wild-type seedlings treated with 2 mM H2O2 for the time
indicated. In-gel kinase assays were performed as described in Methods, and MBP was used as the substrate.
(F) Mutation of mpk6 abolishes H2O2-activated MPK6. The wild-type and mpk6-3 plants were treated with 2 mM H2O2 for 20 min, and the activity of
MPK6 was detected by in-gel kinase assay using MBP as the substrate.
MPK6 and Nitric Oxide Signaling 2983
NIA2 Is Required for the MPK6-Mediated Increase in NO
Production Induced by H2O2
As mentioned above, NOS and NR are the two potential enzy-
matic sources of NO in planta (Desikan et al., 2002; Guo et al.,
2003). However, the identity of Arabidopsis NOA1 as an authen-
tic NOS has been questioned recently (Moreau et al., 2008). To
determinewhether the decrease in H2O2-inducedNOproduction
in mpk6-3 was due to a change in NR activity, NO production
was measured after treatment with tungstate (an inhibitor of NR).
Tungstate treatment resulted in a 61% reduction in H2O2-
induced NO production in wild-type plants (Figure 2A, black
column 1 versus black column 2, significantly different according
to the Steel-Dwass test, P# 0.05). The amount of NO produced
in response to H2O2 in the kinase-deficient mutant mpk6-3 was
similar to that produced in wild-type plants pretreated with
tungstate (black column 2 versus black column 3). These data
suggest that MPK6 is involved in NO synthesis induced by H2O2
through the action of NR.
Given that NR is encoded by two genes, NIA1 and NIA2, in
Arabidopsis (Campbell, 1999), deficiency of both NIA1 and NIA2
results in significant reduction in NO generation (Bright et al.,
2006; Modolo et al., 2006). To test which gene functions in
MPK6-mediated NO synthesis, the T-DNA insertion mutants
nia1-3, nia1-4, and nia2-1, nia2-2 and the double mutantmpk6-3
nia2-1, mpk6-3 nia2-2 were used for measurements of NR
activity and NO production. Since similar results were obtained
with these double mutants, we presented the data only from the
analysis of nia1-2, nia2-1, and nia2-1mpk6-3. Real-time RT-PCR
analysis indicated that the genes NIA1, NIA2, and MPK6 were
knocked out in these single and double mutants (see Supple-
mental Figure 2 and Supplemental Methods online). In the nia1
mutants, H2O2-induced production of NO was almost unaf-
fected, perhaps even higher than that in the wild type (Figure 2A,
black column 5 versus black column 1), whereas NO production
was inhibited dramatically in the nia2mutants (37.5%, P# 0.05;
black column 6 versus black column 1). In the nia1-2 nia2-5
doublemutant, the level of NO production was even lower than in
the single mutant and was almost equal to that in the wild-type
plants treated with tungstate (Figure 2A, the last black column
versus black column 2). These results suggest that NIA2 plays an
important role in nitrite-dependent NO synthesis in response to
exogenous H2O2 in roots.
Next, we measured the NR activity in wild-type, mpk6-3, and
NIA-deficient plants. As mentioned above, tungstate treatment
has been used to produce nonfunctional NR. In our working
conditions, NR activity in wild-type and mpk6-3 mutants was
almost inhibited for 30 min after 200 mM tungstate treatment
(see Supplemental Figure 4 online; Figure 2B, columns 2 and 4).
Furthermore, the activity of NR was rapidly enhanced in wild-
type, nia1-3, and nia2-1 plants when exogenous H2O2 was
applied (significantly different according to the Steel-Dwass test,
P# 0.05) (Figure 2B, the black column versus the white column).
An increase in total NR activity of 177, 238, and 161% was
observed inwild-type, nia1-3, and nia2-1 seedlings, respectively,
in response to H2O2 treatment. By contrast, there was only a
slight increase in total NR activity inmpk6-3 andmpk6-3 nia2-1 in
response to treatment with H2O2. Regardless of the total NR
activity or level of NO production, these increases were signif-
icantly lower than those observed in wild-type and nia1-3 plants.
As mentioned above, in the mpk6-3 nia2-1 double mutant, the
level of NO synthesis after exposure to H2O2 was very similar to
that measured in the mpk6-3 and nia2-1 single mutants, which
indicated that it is mainly the NIA2 isoform that is responsible for
the production of NO in response to H2O2 in roots. The nia1-2
nia2-5 double mutant, which lacked both NIA genes, showed
diminished NR activity (Figure 2B, last column), but NO produc-
tion was still detectable, which indicated that NO might also be
generated nonenzymatically or by an as yet unknown source in
root cells (Crawford, 2006).
To confirm that NR activity and NO synthesis induced by H2O2
correlate with the activation of MPK6, we also determined the
level of H2O2-induced MPK6 activity in wild-type and mpk6 and
nia mutant plants. As shown in Figure 2C, H2O2 increased the
level of MPK6 phosphorylation in wild-type plants, nia mutants,
and wild-type plants treated with tungstate. The activation of
MPK6 in response to H2O2 was abolished completely in the
mpk6-3 and mpk6-3 nia2-1 mutants. Furthermore, after treat-
ment with the NO donor sodium nitroprusside (SNP) for the times
indicated (5 to 60 min), slight activation of MPK6 was observed
by in-gel kinase assay (Figure 2D), indicating that there might be
a feedback loop regulating MPK6 activity. The H2O2-mediated
MPK6 activation correlated well with NO synthesis and NR ac-
tivity, but the expression of NIA2 was not affected (see Supple-
mental Figure 3 online), which suggests that MPK6 is involved
in the NR-mediated production of NO via posttranscriptional
modulation.
MPK6 Interacts with NIA2
After we established that MPK6 was involved in the regulation of
H2O2-induced NO synthesis catalyzed by NIA2, we investigated
whether MPK6 interacted with NIA in vitro or in vivo. Cotrans-
formation of yeast Y190 with these constructs (pAS-MPK6 or
pAS-MPK3 with pACT-NIA1 or -NIA2) yielded His/Trp/Leu auxo-
trophs that were also positive for b-galactosidase expression
(Figure 3A), which demonstrated that MPK6 and MPK3 inter-
acted physically with NIA1 and NIA2. In addition, cotransforma-
tion of a construct that expressed the control bait protein MPK4
with pACT-NIA1 or NIA2 did not result in detectable b-galacto-
sidase activity. These results show thatMPK6 andMPK3 interact
with both NIA1 and NIA2 in the yeast two-hybrid system (Figures
3A and 3B).
To confirm the interaction between MPK6 and NIA1/NIA2, we
also performed a glutathione S-transferase (GST) pull-down
assay.MPK6 protein labeledwith biotinylated Lyswas incubated
together with GST-NIA1 or GST-NIA2 fusion protein, using GST
alone as the negative control. As shown in Figure 3C, NIA1 and
NIA2 could bind to MPK6 and MPK3, but GST could not. The
binding of NIA1 toMPK6 andNIA2 toMPK3wasweaker than that
of NIA2 and NIA1, respectively (Figure 3C).
To determine whether MPK6 and NIA2 interacted in vivo, we
monitored the association of transiently expressed MPK6 and
NIA2 in protoplasts of Arabidopsis leaves using bimolecular
fluorescence complementation (BiFC) (Walter et al., 2004). To
carry out BiFC, the coding region of yellow fluorescent protein
2984 The Plant Cell
(YFP) was split between two vectors: the N-terminal 155 amino
acids were encoded by pSPYNE, and the C-terminal 84 amino
acids were encoded by pSPYCE. Fusion of these two segments
to two separate proteins that interact resulted in the reconstitu-
tion of YFP fluorescence when the proteins were coexpressed.
MPK6 and NIA1 or NIA2 were cloned into pSPYNE and pSPYCE
to give constructs that encoded the proteins MPK6-YNE and
NIA1-YCE or NIA2-YCE, respectively. MPK6-YNE and NIA2-
YCE were coexpressed in protoplasts of Arabidopsis leaves. As
expected, we observed reconstituted fluorescence in the cyto-
plasm (Figure 3D, a). However, coexpression of MPK6-YNE and
NIA1-YCE did not reconstitute fluorescence (Figure 3D, b). In
addition, no YFP signal was detected with the combinations of
MPK6-YNE and pSPYCE or NIA1-YCE/NIA2-YCE and pSPYNE
(Figure 3D, c to e). Consistent with the localization of the inter-
action signal, confocal microscopy of protoplasts transformed
with MPK6, NIA2, or NIA1 fused with green fluorescent protein
(GFP) showed that the MPK6, NIA1, and NIA2 fusion proteins
were also localized in the cytoplasm. These results confirmed
that MPK6 and NIA2 can interact in planta.
Interaction of MPK6 and NIA2 Requires Hinge 2 and
FAD Domains
To identify the domains of NIA2 that interactedwithMPK6, seven
fragments that covered various regions of NIA2 were tested for
their ability to interact with MPK6 (Figure 4A). The C-terminal
region (amino acids 621 to 917), which contained hinge 2, FAD,
and NADH binding domains, could interact with MPK6, but the
N-terminal region (amino acids 1 to 334), which contained an
Mo-molybdopterin domain, and the middle region (amino acids
335 to 620), which contained dimer, hinge 1, and cytochrome b
domains, could not (Figure 4B). Furthermore, deletion of the
NADH binding domain (amino acids 781 to 917) did not affect the
interaction between C-terminal regions with MPK6 protein,
which suggested that the hinge 2 and FAD domains (amino
acids 621 to 780) were required for recognition of and interaction
with MPK6.
It is well known that two regions within the substrate are
involved in recognition and binding by MAPK: docking se-
quences and the phosphorylation site (Sharrocks et al., 2000).
Generally, sequence analysis indicated that the docking se-
quence contains a region rich in basic amino acids, an LxL motif,
and sometimes a triplet of hydrophobic residues (Jacobs et al.,
1999; Sharrocks et al., 2000). The hinge 2 and FAD binding
domain of NIA2 contain a typical RKFRFALPVEDMVLGL se-
quence (Figure 4C), which is similar to the MAPK docking
sequence in mammals and plants (Kiegerl et al., 2000; Sharrocks
et al., 2000). Four potential sites of phosphorylation by MAPK
Figure 2. Involvement of NIA2 in MPK6-Modulated NO Biosynthesis in
Response to H2O2.
(A) NO production in the wild type, mpk6-3, and nia single or mpk6-3
nia2-1 and nia1-2 nia2-5 double mutants. Average pixel intensities of
DAF-2 DA fluorescence were measured at 30 min after exposure to
exogenous 10 mM H2O2 or a water control. For tungstate treatment,
seedlings were loaded with 200 mM tungstate for 30 min before addition
of H2O2. Values are means (6SD) from whole root regions from six
independent experiments.
(B) H2O2-induced increase in NR activity in the wild type, mpk6-3, and
nia single or mpk6-3 nia2-1 and nia1-2 nia2-5 double mutants.
Arabidopsis plants were treated with 10 mM H2O2 or a water control
for 30 min. Each value represents the mean (6SD) of three independent
experiments.
(C) Effects of H2O2 on MPK6 phosphorylation in wild-type, mpk6-3, nia,
and mpk6-3 nia2-1 mutants. The wild-type and mutant plants were
treated with 2 mM H2O2 or a water control for 20 min. For tungstate
treatment, seedlings were loaded with 200 mM tungstate for 30 min
before addition of H2O2. The activity of MPK6 was detected by in-gel
kinase assay using MBP as the substrate.
(D) Effects of the NO donor SNP on MPK6 phosphorylation. Proteins
were extracted from wild-type seedlings treated with 20 mM SNP at the
indicated times. In-gel kinase assays were performed with MBP as the
substrate.
MPK6 and Nitric Oxide Signaling 2985
(Ser-627, Thr-649, Thr-715, and Thr-799), where Ser or Thr
residues are followed by Pro, are located in this region, which
indicates that NIA2 could be a substrate of MPK6 in Arabidopsis.
However, the triplet of conserved hydrophobic residues (MVL) of
NIA2 corresponded to QQL in NIA1 at the C-terminal end of
docking motif (Figure 4C), which is not a hydrophobic triplet. In
addition, NIA1 lacks the obvious LxL motif. Perhaps these
differences may determine that NIA2, not NIA1, interacts with
MPK6 in vivo.
PhosphorylationofNIA2byMPK6EnhancesNRActivity and
NO Production
Previous studies have indicated that the activity of NR can be
regulated by phosphorylation of a Ser residue in the hinge
1 region (Su et al., 1996; Kanamaru et al., 1999). On the basis
of previous results and those presented above, we hypothesized
that NIA2might be a substrate ofMPK6 and a target of theMAPK
cascade. To test this hypothesis, we performed an in vitro
phosphorylation assay. The recombinant His-tagged C-terminal
fragments NIA2c (amino acids 335 to 620) and NIA2d (amino
acids 621 to 917), as well as NIA1c (amino acids 338 to 623) and
NIA1d (amino acids 624 to 917), were used as substrates. As
shown in Figure 5A, recombinant MPK6 that was activated by
MKK5DD, which is a constitutively active MAPKK, could phos-
phorylate NIA2d but not NIA2c. However, NIA1d and NIA1c
could not be phosphorylated by activated MPK6 (Figure 5C). In
addition, MPK3 could phosphorylate neither NIA2c nor NIA2d
(Figure 5B). These data demonstrate that MPK6 is the kinase
responsible for the phosphorylation of NIA2.
To characterize further the MAPK pathway involved in the
regulation of NR activity and NO synthesis, we measured NR
activity in the conditional gain-of-function (dexamethasone
[DEX]-inducible) MKK5DD transgenic plants and in mpk6-3/
MKK5DD transgenic plants (Ren et al., 2008). Following DEX
treatment, the constitutively active MKK5DD induced the activa-
tion of MPK6 in a time-dependent manner (Figure 5D, top panel),
which resulted in a corresponding rapid increase in NR activity
when compared with the same line treated with water as a
control (Figure 5E). However, DEX could not stimulate MPK6
activation (Figure 5D, bottom panel) or NR activity (Figure 5E) in
MKK5DD/mpk6-3 plants. In addition, NR activity was increased
significantly (612%) in 35S-NIA2 transgenic plants, which over-
expressedNIA2 (Figure 5F, column 3 versus column 1, P# 0.05).
Figure 3. MPK6 Interacts with NIA2 in Vitro and in Vivo.
(A) MPK6 and MPK3 interacted with NIA1 and NIA2 in the yeast two-
hybrid system. Yeast strains that contained pAS-MPK6 or pAS-MPK3 as
bait and pACT-NIA1/2 as prey were grown for 48 h on synthetic defined
(SD) medium that lacked Trp and Leu (left panel) and were assayed for
LacZ expression by a filter-lift assay for b-galactosidase activity (b-gal;
right panel). pAS-MPK4 and the empty bait vector were used as negative
controls. A blue color indicates interaction. b-gal, b-galactosidase ac-
tivity.
(B)Quantitative analysis of b-galactosidase activity of the yeast strains in
liquid culture showing the interaction between MPK3, MPK6, and MPK4
and NIA1 or NIA2. Values are means of data from three independent
experiments. Error bars indicate SD.
(C) MPK3 and MPK6 proteins labeled with biotinylated-Lys were pulled
down by GST-NIA1 and GST-NIA2 but not by GST.
(D) NIA2, but not NIA1, interacted with MPK6 in vivo as determined by
BiFC. (a) The YFP signal in the cytoplasm indicates a positive interaction
between MPK6 and NIA2; (b) no YFP signal was detected in protoplasts
cotransformed with MPK6-YNE and NIA1-YCE; (c) and (d) no YFP signal
was detected in protoplasts cotransformed with SPYNE and NIA2-YCE
or NIA1-YCE; (e) no YFP signal was detected in protoplasts cotrans-
formed with MPK6-YNE and SPYCE; (f) to (h) localization of GFP signals
from NIA1, NIA2, and MPK6 fused with GFP. Left panels, fluorescence
images under confocal microscopy; right panels, bright-field images of
the cells. Bars = 5 mm.
2986 The Plant Cell
Similar results were obtained with a second line that overex-
pressedNIA2 (Figure 5F, column 2). By contrast, evenwhenNIA2
was overexpressed in anMPK6-deficient background, as shown
by immunoblotting (Figure 5F, bottom panel), the NR activity only
elevated slightly when comparedwithwild-type ormpk6mutants
(Figure 5F, top panel). Similar results were obtained for the NO
production in these transgenic plants (Figure 5F, middle panel).
Overexpression of NIA2-enhanced NO production and muta-
tions inMPK6 prevented the increase of NO production, even in
the background of NIA2 overexpression. These data suggest
that the MKK5/MPK6 pathway mediates the activation of NR
through phosphorylation of NIA2.
Phenotypic Characterization of thempk6Mutants
Responsive to NO and H2O2
The results of several previous studies have suggested that NO
affects lateral root development in a dose-dependent manner
(Correa-Aragunde et al., 2004). In wild-type Arabidopsis plants,
treatment with SNP enhanced the initiation and growth of lateral
roots in a concentration-dependent manner (Figures 6A and 6B).
At concentrations#20 mMSNP,mpk6-2 andmpk6-3were more
sensitive to SNP than thewild-type plants regarding promotion of
lateral root growth. On average, the total length of lateral roots in
the mutant plants was;2.6-fold longer than that in the wild type
(Figure 6B, significantly different according to the Steel-Dwass
test, P# 0.05). By contrast, in treatments with$20 mMSNP, the
growth-promoting effects of SNP in lateral roots were gradually
decreased. Compared with untreated seedling roots, treatment
with 50mMSNP significantly inhibited growth of lateral roots (P#
0.05) (Figure 6B).
Next, we examined carefully the phenotypes of the mpk6
mutant after treatment with exogenous H2O2. Under control
conditions, mpk6 and wild-type plants showed similar rates of
germination and growth. However, after 2 weeks of H2O2 treat-
ment, mpk6-2 and mpk6-3 seedlings had produced more and
longer lateral roots than wild-type seedlings (Figures 6A and 6C,
significantly different according to the Steel-Dwass test, P #
0.05). For example, with 2 mM H2O2, the mean length of lateral
roots in the wild type was 8.21 6 2.14 cm, whereas in mpk6-2
andmpk6-3, the mean lengths were 15.46 2.35 and 16.16 2.64
cm, respectively. The effects of H2O2 on lateral root elongation
were dose dependent, andH2O2 concentrations of 4mMormore
inhibited lateral root initiation entirely in both wild-type andmpk6
mutant plants. These results reveal that lateral root development
in mpk6-2 and mpk6-3 seedlings is more sensitive to H2O2 than
that in wild-type seedlings.
To test whether the root phenotype of the mpk6 mutants
resulted from a deficiency of NR activity, tungstate was used to
inhibit the total NR activity in both wild-type and mpk6 plants.
Application of tungstate totally abolished the difference in the
lateral root phenotype between wild-type and mpk6 plants in
response to H2O2 (see Supplemental Figure 5 online). Further-
more, application of the NO scavenger c-PTIO eliminated the
inhibition by H2O2 partially and also abolished the lateral root
phenotype of mpk6-2 and mpk6-3 (Figures 6A and 6E), which
suggests that the lateral root phenotypes of mpk6 mutants are
critically dependent on the concentration of NO.
Ser-627 of NIA2 Is a Putative Phosphorylation Site and Its
Modifications Affect NR Activity and Lateral
Root Development
To identify which amino acid residue is required for MPK6
phosphorylation, recombinant NIA2d proteins were generated
by mutating four putative Ser (S) or Thr (T) residues to Ala (A)
by site-directed mutagenesis and were used as substrates for
MPK6 phosphorylation assay. As shown in Figure 7A, MPK6
could not phosphorylate the S627A mutant, whereas T649A,
T715A, and T799A mutants could still be phosphorylated. This
result indicated that the Ser-627 residue in hinge 2 domain of
Figure 4. MPK6 Interacts with the Hinge 2 and FAD Domains of NIA2.
(A) Schematic representation of seven fragments that covered various
regions of the NIA2 protein and were used for yeast two-hybrid assays.
FL, full-length NIA2 protein (amino acids 1 to 917). (a) N-terminal region
and Mo-molybdopterin domain (amino acids 1 to 334); (b) all regions
except those contained in fragment a (amino acids 335 to 917); (c) dimer,
hinge 1, and cytochrome b domains (amino acids 335 to 620); (d) hinge
2, FAD, and NADH binding domains (amino acids 621 to 917); (e) hinge 2
and FAD domains (amino acids 621 to 780); (f) NADH binding domain
(amino acids 781 to 917).
(B)Quantitative analysis of b-galactosidase activity of the yeast strains in
liquid culture showing the interaction between MPK6 and the seven
fragments of NIA2. Values are means (6SD) of data from three indepen-
dent experiments. The results show that fragment e is required for
interaction between MPK6 and NIA2.
(C) Sequence of the putative docking domain in NIA1 and NIA2. The first
amino acid in each sequence used for alignment is indicated, and
conserved residues are highlighted (red). A triplet of conserved hydro-
phobic residues (MVL) in NIA2 corresponded to QQL in NIA1 (blue). f
represents a hydrophobic amino acid.
MPK6 and Nitric Oxide Signaling 2987
NIA2 is the unique MPK6 phosphorylation site. Interestingly, NIA1
lacks several adjacent Ser residues in this region, which may be
required for MPK6 recognization and phosphorylation (Figure 7B).
To confirm Ser-627 as the specific phosphorylation site in
NIA2, Arabidopsis transgenic plants were generated by intro-
ducing recombinant FLAG-tagged full length NIA2WT and NIA2A,
which has Ser-627 replaced by Ala. NIA2WT and NIA2A proteins
were immunoprecipitated from these transgenic seedlings and
served as MPK6 substrates in vitro phosphorylation assays.
Although both mutants produced a complete NR protein,
Figure 5. Phosphorylation of NIA1 and NIA2 by MPK3 and MPK6.
(A) and (B) Activated MPK6 phosphorylated NIA2 in the C-terminal region, but activated MPK3 could not phosphorylate NIA2. Fragments c and d of
NIA2 are described in Figure 4 and correspond to amino acids 335 to 620 and 621 to 917, respectively. Recombinant His-tagged MPK6 was activated
and then used to phosphorylate His-tagged NIA2c and NIA2d in the presence of [g-32P]ATP. After electrophoresis, the phosphorylated proteins were
visualized by autoradiography. Reactions with various components omitted (�) were used as controls. Immunoblotting with anti-Flag and anti-His
antibodies was performed to show the loading of Flag-tagged MKK5DD and His-tagged MPK6, NIA2c, and NIA2d, respectively.
(C) Activated MPK6 could not phosphorylate NIA1 in the C-terminal region. The assay was performed as described in (A).
(D) Treatment with DEX enhanced the phosphorylation of MPK6 in MKK5DD mutants. Protein extracts from MKK5DD and MKK5DD/mpk6-3 seedlings
were treated with 2 mM DEX for the indicated times, and 10 mg protein extract was used for SDS-PAGE. Activated MPK6 and MPK3 were detected by
immunoblotting using the phospho-p44/42 MAPK antibody.
(E) Phosphorylation of NIA2 by MPK6 increases NR activity. MKK5DD andMKK5DD/mpk6-3 seedlings were treated with DEX (2 mM). The NR activity in
each mutant was determined at the indicated time point. NR activity was measured as described in Figure 2B. Each value represents the mean (6SD) of
three independent experiments.
(F)MPK6 is required for full activation of NR and NO generation. The NR activity (top panel) and NO production (middle panel) in 35S-NIA2 andmpk6-3/
35S-NIA2 transgenic plants were measured as described in Figure 2. Each value represents the mean (6SD) of three independent experiments. The NR
activity and NO production in the wild type were set as 100%. Protein extract (10 mg) from 35S-NIA2 and mpk6-3/35S-NIA2 transgenic plants was
separated by electrophoresis, and immunoblotting with anti-Flag antibody was performed to determine the amount of Flag-tagged NIA2 proteins
(bottom panel).
2988 The Plant Cell
activated MPK6 could phosphorylate only NIA2WT, not NIA2A
mutant protein (Figure 7C, top panel). For the nontransgenic
plants, no FLAG-tagged protein was immunoprecipitated and no
phosphorylated band was observed. Alternatively, the same
results were obtained when MYC-tagged NIA2WT and NIA2A
expressed in Pichia pastoris (see Supplemental Figure 6 online).
To determine whether phosphorylation of Ser-627 affects NR
activity, Ser-627 was also replaced by Asp (D), creating NIA2D,
which mimics NIA2 phosphorylated at Ser-627. Consistent with
above results, the NR activity in the yeast cells containing NIA2D
was increased by 3.5-fold compared with that of NIA2A (Figure
7D). Similarly, the phosphomimic NIA2D showed substantially
greater NR activity in transgenic plants (Figure 7E, top panel). A
5.15-fold increase in NO production was observed in NIA2WT
plants in response toH2O2 treatment. By contrast, therewas only
a slight increase in NOcontent inNIA2A plants (Figure 7E, bottom
panel). This increase was significantly lower than that observed
in NIA2D. By contrast, the much higher NR activity and NO
production in NIA2D transgenic plants were observed even
without H2O2 treatment.
To investigate whether modification of Ser-627 in NIA2 also
has a function in planta, 5-d-old seedlings of overexpression
lines harboring replacements of Ser-627 were transferred to
Murashige and Skoog (MS) medium without or with 15 mM SNP.
It was previously reported that lateral root growth in Arabidopsis
was induced by SNP treatment (Correa-Aragunde et al., 2004).
Under our experimental conditions, lateral roots of wild-type
seedlings treated with 15 mM SNP for 7 d were markedly longer
than those without SNP; their length increased from 2.296 0.42
to 4.18 6 1.01 cm (Figures 7F and 7G). Similarly, lateral root
length in all overexpression lines of NIA2 (NIA2A, NIA2WT, and
NIA2D) was increased to;37.5 to 50.5%comparedwith thewild
type. The increase in lateral root length in NIA2 overexpression
lines is associated with the increased NO production and higher
NR activity (Figures 5F and 7E). Surprisingly, following SNP
treatment, both the number and length of lateral roots in NIA2D,
the Ser-627 phosphomimic, were decreased compared with
those of NIA2A plants (3.63 6 0.68 cm in NIA2A versus 1.87 60.31 cm in NIA2D) (Figures 7F and 7G, significantly different
according to the Steel-Dwass test, P # 0.05). This stands in
contrast with wild-type plants in which significantly longer lateral
roots were observed. This inhibited lateral root growth suggests
an increased NO concentration in NIA2D seedlings, presumably
due to their increased sensitivity to exogenous NO derived from
SNP. These results suggest that Ser-627 of NIA2 is essential site
for MPK6 phosphorylation.
DISCUSSION
In Arabidopsis, MPK6 is involved in a number of signaling
pathways; it activates downstream partner proteins in response
to various stresses and during various developmental processes
(Ichimura et al., 2000; Nuhse et al., 2000; Yuasa et al., 2001). The
biochemical and genetic data obtained in this study reveal a key
function of MPK6 in NO biosynthesis and signal transduction
during root development. The involvement of NO in lateral root
development, described herein, is likely to be a result of the
modulation of NIA2 activity by MPK6.
MPK6Activity Appears to Be Redox Regulated in Root Cells
It has been reported that reactive oxygen species (ROS) act
upstream of the MAPK cascade in plant development and stress
responses (Lu et al., 2002; Yoshioka et al., 2003; Desikan et al.,
2004; Zhang et al., 2006). We found that H2O2 significantly
stimulated MPK6 activity in an in-gel kinase assay (Figures 1E
Figure 6. Changes in Morphology of the Root System in Wild-Type and
mpk6-3 Plants in Response to NO and H2O2.
(A) The number and length of lateral roots was greater in mpk6 mutant
plants than in wild-type plants after treatment with SNP or H2O2. Five-
day-old seedlings of the wild type,mpk6-2, andmpk6-3were transferred
to vertical MS plates containing (top to bottom): no additions (control);
15 mM SNP, 2.5 mM H2O2, or 2.5 mM H2O2 plus 0.5 mM c-PTIO. The
photographs were taken at 10 d (top two panels) or 15 d (bottom two
panels) after transfer. Bar = 0.5 cm.
(B) Average length of the lateral roots of all plants on MS medium
supplemented with SNP at the indicated concentrations. Data represent
means (6SD) of three independent experiments (;20 plants per point).
(C) Average length of the lateral roots of each type of plant on MS
medium supplemented with H2O2 at the indicated concentrations. Data
represent means (6SD) of three independent experiments (at least 30
plants).
(D) c-PTIO treatment abolished the H2O2-induced differences in lateral
roots between the wild type andmpk6mutant. Five-day-old seedlings of
the wild type, mpk6-2, and mpk6-3 were transferred to MS plates
(control) or vertical MS plates that contained 2.5 mM H2O2 and 0.5 mM
c-PTIO. Data represent means (6SD) of three independent experiments
(at least 30 plants).
MPK6 and Nitric Oxide Signaling 2989
and 1F). More and longer lateral roots were observed in mpk6
plants than in wild-type plants after treatment with H2O2 (Figure
6). These data indicate that theMPK6 cascade might be part of a
redox-sensitive signaling pathway.
In HeLa cells treated with lysophosphatidic acid, a potential
signaling molecule that stimulates cell proliferation, H2O2 medi-
ates the activation of the epidermal growth factor receptor and
MAPK (Cunnick et al., 1998). The kinase-activating role of H2O2 in
these cells was demonstrated by blocking the production of
H2O2with diphenylene iodonium, an inhibitor of NADPH oxidase,
by preventing the accumulation of H2O2 withN-acetylcysteine or
CAT, or by introducing exogenous H2O2 (Cunnick et al., 1998;
Greene et al., 2000; Rhee et al., 2000). In Arabidopsis, MPK3 and
MPK6 have been shown to be activated by H2O2 via the activa-
tion of NPK1-related protein kinase 1, anMAPKKK (Kovtun et al.,
2000). The observations that H2O2 strongly induces Arabidopsis
NDPkinase 2 (NDPK2) and that NDPK2 interacts specifically with
MPK3 as well asMPK6 (Moon et al., 2003) suggest that oxidative
stress activates NDPK2, possibly by activating the MAPK cas-
cade. Alternatively, changes in redox state can be detected by
Figure 7. Ser-627 of NIA2 Was a Putative Site for MPK6 Phosphorylation and Essential for the Efficient Activation of NR and Development of Lateral
Roots.
(A) Ser-627 in the hinge 2 domain of NIA2 is the MPK6 phosphorylation site. Recombinant His-tagged MPK6 was activated and then used to
phosphorylate His-tagged NIA2d protein with either the S627A, T649A, T715A, or the T799A mutation in the presence of [g-32P]ATP. After electro-
phoresis, the phosphorylated proteins were visualized by autoradiography. Immunoblotting with anti-Flag and anti-His antibodies was performed
to show the loading of Flag-tagged MKK5DD and His-tagged MPK6 and NIA2d, respectively.
(B) Sequence alignment shows three Ser residues adjacent to the phosphorylation site are missing in NIA1 protein. The first amino acid in each
sequence used for alignment is indicated, and the phosphorylation site is underlined.
(C) Phosphorylation of native NIA2 protein by MPK6. Flag-tagged full-length NIA2WT and NIA2S627A protein were immunoprecipitated from DEX-treated
NIA2WT and NIA2A transgenic plants and were used for the phosphorylation assay as described in (A). Only wild-type NIA2 protein could be
phosphorylated by MPK6. Immunoblotting with anti-Flag antibodies was performed to show the loading of NIA2 protein.
(D) NR activity was stimulated by the phosphorylation of NIA2-Ser627. Five micrograms of purified NIA2A and NIA2D protein from P. pastoris was used
for NR activity assay as described in Figure 2B. Each value represents the mean (6SD) of three independent experiments.
(E) Phosphorylation and substitution of Ser-627 of NIA2 affected NR activity and NO production. The NR activity (top panel) and NO production (bottom
panel) in NIA2A, NIA2WT, and NIA2D transgenic plants treated with 10 mMH2O2 or a water control were measured as described in Figure 2B. Each value
represents the mean (6SD) of three independent experiments.
(F) Photography of transgenic plants containing the modifications of Ser-627 of NIA2 on an MS agar plate (control, top) and an MS agar plate
supplemented with 15 mM SNP (bottom). Five-day-old seedlings of the wild-type, NIA2A, NIA2WT, and NIA2D transgenic plants were transferred to the
plate. The photographs were taken 10 d after the transfer. Bar = 0.5 cm.
(G) Average of the total length of lateral roots of wild-type and transgenic plants on MS medium (control) or MS supplemented with 15 mM SNP. Data
represent means (6SD) of three independent experiments (;20 plants per point).
2990 The Plant Cell
protein Tyr phosphatase 1, which can inactivate MPK6 (Gupta
and Luan, 2003). Here, it is important to consider an important
link between the phosphorylation- and redox-based signaling
pathways. Many kinases have a conserved Cys pair in their
active domain, and oxidation of this pair potentiates their activity
(Sitia and Molteni, 2004). Thus, it is easy to envision how redox
changes could affect many aspects of cell physiology. We
expected that the MPK6 cascademight also be redox regulated.
Indeed, we found that MPK6 was activated by H2O2 and that
more NO accumulated in response to H2O2 (Figure 1). A feed-
back loop situation appears to exist wherebyNOcan activate the
MAPKs, as has been reported previously (Clarke et al., 2000;
Kumar and Klessig, 2000; Zhang et al., 2007a). In fact, NO
transiently activatedMPK6 in our working conditions (Figure 2D).
The findings support the idea that changes in the redox state
might be the molecular switch for activation of MPK6. A possible
explanation for this is that a feedback loop regulates the redox
state of cells to activate MAPK cascade.
NIA2 Is Responsible for the Production of NO in Root Cells
in Response to H2O2
The genetic data have demonstrated that NR, which catalyzes
the conversion of nitrite to NO in a reaction that depends on
NADP(H), was the major source of NO in plants (Yamasaki and
Sakihama, 2000; Rockel et al., 2002; Bright et al., 2006). In plant
cells, the amount of NR is regulated at the transcriptional and
posttranscriptional level; for example, the expression of Arabi-
dopsis NIA1 and NIA2 is induced by light, nitrate, and sucrose
(Cheng et al., 1992; Campbell, 1999). The activity of NR protein
is also regulated at a posttranscriptional level; for example, Ser-
534 in hinge 1 region of Arabidopsis NIA2 can be phosphory-
lated by a calcium-dependent protein kinase (Su et al., 1996).
Deficiency of NIA1 had almost no affect on NR activity and NO
synthesis in the roots (Figure 2B), whereas, inmpk6-3 and nia2-1
mutants, total NR activity and NO synthesis increased only
slightly in response to H2O2 treatment. In the mpk6-3 nia2-1
doublemutant, the level of NO synthesis was very similar to that
in the mpk6-3 and nia2-1 single mutants following treatment
with H2O2. These data imply strongly that NIA2 is responsible
for the production of NO in root cells in response to exogenous
H2O2.
Although our data showed that H2O2-induced NO production
in roots resulted mainly from MPK6 activating NR by phosphor-
ylation of Ser-627 in the hinge 2 region of NIA2, this does not rule
out the alternative pathway to stimulate NO generation. The fact
that NO production was not completely abolished bymutation of
mpk6 or by addition of the MAPKK inhibitor to wild-type plants
(Figures 1A and 1D) indeed implies that there is an alternative
route to activate NO production. It is well documented that the
regulatory Ser-534 phosphorylation site, occurring in a region of
hinge 1 in the NR molecule (Campbell, 2001), is important for
binding of a 14-3-3 protein, leading to a catalytically inactive
complex (NR+pSer534+14-3-3) that modulates NR activity in
response to light and other environmental signals (Su et al., 1996;
Moorhead et al., 1996). A recent report indicated that oxidation of
surface-exposed Met-538 in NR can inhibit the phosphorylation
of nearby sites (Ser-534) (Hardin et al., 2009), suggesting that
Met-538 oxidation may play a role as a redox switch in regulation
of NR phosphorylation.
Guard cells of the nia1 nia2 double mutant do not generate NO
in response to H2O2, and stomatal apertures in nia1 nia2 leaves
treated with H2O2 do not differ from those in untreated controls
(Bright et al., 2006), suggesting that NO production via NR is
required for H2O2-mediated stomatal closure. However, pre-
vious data have indicated that the nia1 dissociation insertion
mutant (nia1::Ds nia1::DS) (Parinov et al., 1999; Wang et al.,
2004) appears to be deficient in ABA and H2O2-induced stomatal
closure and NO generation, whereas the nia2 deletion mutant
responds normally to ABA and H2O2 (Bright et al., 2006). The
simplest explanation is that NIA1 might mediate ABA- or H2O2-
activated NO synthesis in guard cells.
Given that NIA1 and NIA2 are both expressed in guard cells, it
is likely that protein–protein interactions and differential activities
of these proteins are important for their different roles. Indeed,
we found a strong interaction between MPK6 and NIA2, but not
MPK6 and NIA1, in vivo (Figure 3D). In Arabidopsis, NIA2 repre-
sents ;90% of the total NR activity (Wilkinson and Crawford,
1991), and NIA2 is expressed mainly in roots rather than leaves
(Cheng et al., 1991). Thus, although NIA1 is required for effective
ABA signal transduction in guard cells during stomatal closure
(Bright et al., 2006), our data suggest that NIA2 is the main target
of MPK6 with respect to NO biosynthesis in root cells. The fact
that significant NR activity was observed in nia2mutants (Figure
2B) might be because whole seedlings were used in the mea-
surement. NIA1 and NIA2 have similar amino acid sequences,
but there are distinct regions that differ between the two proteins
and that influence the protein partners with which they interact
with and their activation characteristics. In addition, NIA1 and
NIA2 are expressed in different tissues. These differences might
explain why NIA1 and NIA2 have distinct signal transduction
pathways and roles in N assimilation.
NIA2 Is the Target for theMKK5-MPK6Module in Root Cells
Complete MAPK cascades that function in innate immunity, eth-
ylene signaling, and stomatal development in plants have been
elucidated (Asai et al., 2002; Ouaked et al., 2003; Bergmann
et al., 2004; Wang et al., 2007). All theseMAPK cascades involve
MPK6 and/or MPK3, which indicates that the MPK3/MPK6
module has important multifunctional abilities. It has been well
documented that ROS act upstream ofMAPK cascades and that
H2O2 induces the synthesis of NO (Lu et al., 2002; Yoshioka et al.,
2003; Desikan et al., 2004; Bright et al., 2006; Zhang et al., 2006).
We extend these observations to establish that H2O2 and NO
signaling are linked through the activation of MPK6.
We found that MPK6 interacted strongly with NIA2, and NR
activity was increased by H2O2 treatment in amanner dependent
onMPK6 (Figure 2C).We demonstrated that NIA2, not NIA1, was
the substrate of MPK6 (Figure 5) and that the S627A mutant of
NIA2 failed to be phosphorylated by MPK6 (Figure 7C; see
Supplemental Figure 6 online). Intriguingly, phosphorylation of
NIA2 also dramatically increased NR activity and NO production
in root cells (Figure 7E). These results clearly demonstrate that
NIA2 is the substrate of MPK6, and phosphorylation of NIA2 is
required for H2O2-induced NO production. As mentioned above,
MPK6 and Nitric Oxide Signaling 2991
NR activity is known to be regulated by reversible phosphory-
lation (Su et al., 1996; Campbell, 1999). This reversible mecha-
nism allows NR activity to bemodulated more rapidly than can be
achieved by protein degradation or synthesis (Campbell, 2001;
Kaiser and Huber, 2001). Thus, regulation of NR by the MPK6-
mediated cascade may play a key role in the posttranslational
regulation of nitrate reduction and NO production, but more de-
tailed studies are needed to establish the mechanisms involved.
Our analysis of the role of MPK6 activation in response to H2O2
helps us to position H2O2 and NO in the signaling scheme for
specific responses. Although it is well established that H2O2
induces the synthesis and accumulation of NO, it also has been
suggested that NO modulates H2O2 levels in the guard cells of
Vicia faba (She et al., 2004; He et al., 2005). However, NO does
not appear to induce the production of H2O2 inArabidopsis guard
cells (Bright et al., 2006) or in maize mesophyll cells (Zhang et al.,
2007a). In addition, we did not observe this NO-induced H2O2
production in V. faba guard cells (Lu et al., 2005). Thus, the
function and relationship between NO and H2O2 remain topics of
debate. Here, we found that H2O2 induced the rapid activation of
MPK6 (Figures 1C and 1D), which resulted in increase in NR
activity and NO production.
NO has long been defined as a positive regulator in lateral root
development when applied exogenously (Pagnussat et al., 2002;
Correa-Aragunde et al., 2004). Surprisingly, unlike the NO promo-
tion effects seen in wild-type plants, NIA2D transgenic plants
showed the growth retardation phenotypes of lateral root in
response to exogenously applied NO (Figures 7F and 7G). One
possibility is that there is a bell-shaped curve of dose response for
NO, similar to that for cytokinin in root growth (Werner et al., 2001).
Indeed, bell-shaped NO response curve for lateral root growth in
mpk6 mutants was observed (Figure 6B). Because the level of
endogenous NO might be nearly optimal for growth in NIA2
overexpression lines, supplying the NIA2D plants with exogenous
NO (15mMSNP)may cause overly high concentrations of NO that
may be inhibitory. Similarly, without SNP treatment, lateral root
growth in all overexpression lines of NIA2 (NIA2A, NIA2WT, and
NIA2D) was strongly stimulated compared with the wild type
(Figures 7F and 7G). These data are consistent with the previous
observations (Pagnussat et al., 2002; Correa-Aragunde et al.,
2004). By contrast, we did not find the differences in lateral root
growth between wild-type and transgenic plants after application
of H2O2 (see Supplemental Figure 7 online), probably due to the
consistent activation of NR in the overexpression lines. Together,
these data strongly support this notion that lateral root growthmay
require a minimum concentration of NO to promote growth but
that root growth is inhibited in NIA2D by SNP concentrations that
promote elongation in the wild type.
Similarly, the observation that mpk6 mutants have longer
lateral roots in response to H2O2 compared with the wild type
seems contradictory to the conclusion that mpk6 mutants have
reduced NR and NO synthesis (Figure 6C), which is not the case.
As mentioned above, a threshold concentration of NO must be
reached to initiate the growth response. Beyond the optimum
concentration, NO becomes inhibitory. By this logic, the NO
concentration inmpk6mutants should be below optimal for lateral
root growth, since lowconcentrationsof SNPcauseda stimulation
of lateral root growth (Figure 6B). Consistent with this, external
nitrate stimulates the elongation of lateral roots (Zhang et al., 1999,
2007b; Linkohr et al., 2002). In the case of mpk6 mutants, lateral
root growth appears to bemediated in part by nitrate. On the other
hand, NO levels in the wild type after application H2O2 is;2-fold
higher than that in mpk6 mutants (Figures 1B and 2A). Thus,
accumulation of supraoptimal NO levels was caused not only
through activation of MPK6 by H2O2 but also through the positive
feedback loop of NO (Figure 2D). However, the precise threshold
that NO stimulates or inhibits lateral root growth and its exact
mechanisms require further investigation.
Another possibility is that the retardation of lateral root growth
in the wild type is attributed mainly to the cell death and cell
damage under H2O2 treatment. Previous reports indicated that
ethylene biosynthesis was positively regulated by MPK6 through
rate-limiting ACC synthase isoforms, ACS2 and ACS6 (Liu and
Zhang, 2004; Joo et al., 2008). Exogenous ethylene increased
superoxide anion (O2–)-dependent cell death,whereas impairment
of ethylene perception and plant development, including smaller,
more erect rosettes, altered leaf shape, and short roots in ozone
oversensitive radical-induced cell death 1 (rcd1) (Overmyer et al.,
2000; Ahlfors et al., 2004). Early accumulation of ethylene stimu-
lates spreading of cell death (Tuominen et al., 2004). On the other
hand, it has been reported in tobacco plants that NO and per-
oxynitrite inhibit the activities of CAT and ascorbate peroxidase,
the two major H2O2-scavenging enzymes of plants (Clark et al.,
2000). This breakdown of the antioxidant systemwould lead to an
overproduction of H2O2 and toxicity in the plant cells. Therefore,
both ethylene and ROS seem to exert their effects by mediating
cell death and toxicity to cells, resulting in a consequently stronger
growth retardation of lateral roots in wild-type plants.
Consistently, the evidence that c-PTIO obliterated the H2O2-
induced difference in themorphology and number of lateral roots
of root system between wild-type and mpk6 mutants suggests
that the phenotype differences between wild-type and mpk6
mutants resulted from the NO generation. However, under these
conditions, both wild-type andmpk6mutants still display severe
growth retardation phenotypes (Figures 6B and 6C). A major
reason for the pleiotropic phenotypes of plants in response to
Figure 8. Model for the Putative Pathway of NO Biosynthesis and Signal
Transduction Mediated by H2O2-Activated MPK6 in Arabidopsis.
The solid and dotted arrows indicate the positive regulation based on our
data and the previous results, respectively. Please see the text for a
detailed description of this model.
2992 The Plant Cell
H2O2 is the result of ABA, auxin, and ethylene signaling and/or an
indirect effect of cell damage or photoinhibition (Rao et al., 1997;
Meinhard et al., 2002; De Cnodder et al., 2005; Tarantino et al.,
2005; Nakagami et al., 2006).
Previous evidence has demonstrated that mutation of MEKK1,
the kinase upstream of MPK6 (Asai et al., 2002), results in fewer
lateral roots and shorter root hairs inArabidopsis (Nakagami et al.,
2006) and that NR serves as a source of NO for the induction of
lateral root development by indole-3-butyric acid (Lombardo et al.,
2006; Kolbert et al., 2008). Therefore, it is likely that NO derived
fromMPK6-mediated NR activity is themain original source of NO
that affects lateral root development. Together, these data give
solid support to the hypothesis that MPK6 stimulates the NR-
associated production of NO that regulates lateral root develop-
ment. This also shows that an H2O2 signaling event lies upstream
of NO biosynthesis. However, our results do not rule out the
possibility that MPK6 upregulates NR activity indirectly through
another mechanism that is as yet unidentified.
Therefore, we propose a pathway for the regulation of NO
biosynthesis that involves the modulation of NIA2 by MPK6
(Figure 8).When the intracellular levels of H2O2 increase,MPK6 is
activated, which in turn leads to the phosphorylation of NIA2 at
Ser-627. Phosphorylation of NIA2 by MPK6 dramatically in-
creases the activity of NIA2 and the production of NO and also
results in morphological changes in the root system. Both from
data reported previously (Clarke et al., 2000; Kumar and Klessig,
2000; Zhang et al., 2007a) and transiently activation of MPK6 by
SNP, we infer that a feedback loop exists in which NO could
activate the MAPKs. The alternative way for the regulation of
NIA2 activity is that H2O2 could directly modify the surface-
exposedMet-538 inNRand inhibit the phosphorylation of nearby
sites (Ser-534) (Hardin et al., 2009). Our data suggest that the
increase in H2O2-induced NO production that is dependent on
the MAPK cascade could represent an amplification loop in NO
signaling during root development.
METHODS
Selection of T-DNA Insertion Mutants and Generation of
Double Mutants
The T-DNA insertion lines for MAPK used in this study were mpk6-2
(Salk_073907) andmpk6-3 (Salk_127507), which were the same as those
used previously (Liu and Zhang, 2004). The T-DNA insertion lines for NIA
used in this study were nia1-3 (Salk_004164), nia1-4 (Salk_071547), nia2-1
(Salk_138297), and nia2-2 (Salk_088070). The double mutant of NIA used
in this study was nia1-2 nia2-5 (Wang et al., 2004). The primers to check
the T-DNA insertions were designed by the SIGnAL iSect tool (http://
singal.salk.edu/tdnaprimers.2.html), and PCR was performed using ge-
nomic DNA from seedlings (see Supplemental Table 1 online). To gen-
erate double mutants, the allelesmpk6-2 andmpk6-3were both crossed
with nia2-1, creating nia2-1 mpk6-2 and nia2-1 mpk6-3. Because similar
results were obtained with these double mutants, we present data only
from the analysis of nia2-1 mpk6-3.
PCR
PCR amplifications were performed according to standard protocols in
thermocyclers using Taq DNA polymerase or Pyrobest DNA polymerase
(TaKaRa). For cDNA synthesis, total RNA from Arabidopsis thaliana
seedlings was extracted by the TRIzol method, and reverse transcription
was performed using 5 mg of total RNA and SuperScript II reverse
transcriptase (Invitrogen). Full-length or fragment coding regions ofNIA1,
NIA2,MPK3, orMPK6were amplified and inserted into the corresponding
vector. The primers and vectors used for constructions are listed in
Supplemental Table 1 online.
Transgenic Plants
The coding region of NIA2 was amplified from Arabidopsis ecotype
Columbia-0 using primers that contained KpnI and SalI sites. PCR frag-
ments were cloned together with an N-terminal Flag epitope into the
vector pCAMBIA1205 under the control of the cauliflower mosaic virus
35S promoter. The constructs were introduced into Agrobacterium
tumefaciens strain LBA4404 and transformed by floral infiltration into
wild-type Arabidopsis and the mpk6-3 mutant.
To create transgenic plants mutated at Ser627, the wild-type and
mutant coding regions of NIA2 were cloned into an N-terminal Flag-
epitope vector pTA7002, which contains a DEX-inducible promoter (Ren
et al., 2008).
Detection of Endogenous NO Production
Seedlings were incubated in the cell-permeable fluorescent probe DAF-2
DA (Calbiochem) at a concentration of 5 mM in incubation buffer (50 mM
KCl and 10 mM MES-KOH, pH 7.2) for 30 min. For inhibitor assays,
seedlings were preincubated with the NO scavenger c-PTIO or the
hydrogen peroxide scavenger CAT. Roots were then washed three times
with fresh buffer and examined by epifluorescence (DAF-2 DA excitation
488 nm and emission 500 to 560 nm) and bright-field microscopy using a
Fluoview FV1000 microscope (Olympus). To extract quantitative data,
pixel values were measured over root regions, which were located
manually on confocal images and calculated using ImageJ 1.41 software
(http://rsbweb.nih.gov/ij).
GST Pull-Down Assays
For GST pull-down assays, the coding regions of NIA1 and NIA2 were
amplified by PCR with primers that contained appropriate restriction
sites, and the amplified fragments were inserted into the plasmid pGEX-
6P1 (Amersham Biosciences). The recombinant NIA1-GST and NIA2-
GST fusion proteins were expressed using the pGEX-6P1-NIA1 and
-NIA2 vectors. The coding regions ofMPK6 andMPK3were amplified by
PCR with primers that contained appropriate restriction sites, and the
amplified fragments were inserted into the plasmids pCITE-4a-c(+)
(Novagen). MPK3 and MPK6 proteins that were biotinylated on Lys
residues were produced from pCITE-MPK3 and pCITE-MPK6 using an in
vitro transcription and translation assay kit (TNT Quick Coupled Tran-
scription/Translation system; Promega) with incorporation and detection
of Transcend Biotinylated Lys tRNA (Promega) according to the manu-
facturer’s instructions.
Yeast Two-Hybrid System
For yeast two-hybrid assays, the coding regions of MPK3, MPK6, and
MPK4 were amplified by PCR with primers that contained appropriate
restriction sites. The amplified fragments were inserted into the plasmid
pAS2 (Clontech), which contains the GAL4 DNA binding domain, pro-
ducing the constructs pAS-MPK3, pAS-MPK6, and pAS-MPK4, which
encoded the bait constructs. The full-length coding regions and different
fragments of NIA1 and NIA2 (Figure 4) were cloned in frame between
corresponding restriction sites into the pACT2 vector, which contains
the GAL4 activation domain, creating the prey plasmids pACT2-NIA1,
MPK6 and Nitric Oxide Signaling 2993
pACT2-NIA2, pACT2-NIA2-a (amino acids 1 to 334), pACT2-NIA2-b
(amino acids 335 to 917), pACT2-NIA2-c (amino acids 335 to 620),
pACT2-NIA2-d (amino acids 621 to 917), pACT2-NIA2-e (amino acids 621
to 780), and pACT2-NIA2-f (amino acids 781 to 917). Yeast two-hybrid
interaction assays were performed as described (Song et al., 2005; Miao
et al., 2006). Competent cells of Saccharomyces cerevisiae strain Y190
(Clontech) were transformed simultaneously with pAS-MPK6/3/4 and
pACT2-NIA1/2 or pACT2-NIA2-a to pACT2-NIA2-f. Yeast cells that had
been cotransformed with pAS2 and pACT2 with no inserts were used as
negative controls.
BiFC Assay
To measure in vivo interactions, the coding regions of MPK6, NIA1, and
NIA2 were amplified by PCR with primers that contained appropriate
restriction sites, and the amplified fragments were inserted into the
plasmids pSPYNE and pSPYCE, which contain DNA encoding the
N-terminal and C-terminal region of YFP, respectively (Walter et al.,
2004), to form pSPYNE-MPK6, pSPYCE-NIA1, and pSPYCE-NIA2, re-
spectively. Protoplasts isolated from Arabidopsis leaves were trans-
formed with the following combinations of plasmids: pSPYNE-MPK6
and pSPYCE, pSPYNE and pSPYCE-NIA1/2, or pSPYNE-MPK6 and
pSPYCE-NIA1/2, according to the previous protocols (Walter et al.,
2004). For the GFP constructs, the coding regions of MPK6, NIA1, and
NIA2 were amplified by PCR and inserted into the modified plasmid
pHBT-GFP-NOS (Sheen, 2001) to form pMPK6-GFP, pNIA1-GFP, and
pNIA2-GFP, respectively. The protoplast transient expression assay was
performed as described (Sheen, 2001). After incubation for 16 to 20 h, the
fluorescence of the protoplasts was measured with a confocal laser
scanning microscope FV1000 (Olympus). All figures show representative
images from three independent experiments.
Recombinant Protein Expression in Escherichia coli
For in vitro protein expression, the DNA regions containing the coding
region of MPK6 and NIA1/2 were amplified and inserted in frame into the
plasmid pET-28a (Novagen). Mutations of NIA2d were introduced by
QuickChange site-directed mutagenesis (Stratagene). pET-MPK6 and a
construct that expressed Flag-tagged MKK5DD (Ren et al., 2002) were
introduced into E. coli BL21(DE3) cells. The recombinant His-tagged
proteins were purified using Ni-NTA agarose (Qiagen) according to the
manufacturer’s protocol.
Immunoprecipitation for Full-Length NIA2
To obtain the native NIA2 proteins, 2-week-old seedlings of NIA2WT and
NIA2A were incubated in 15 mM DEX for 24 h, and total protein was
extracted from seedlings by grinding in IP buffer (20 mM Tris-HCl, pH 7.5,
150mMNaCl, 1mMEDTA, 1mMEGTA, 1mMNa3VO4, 1mMNaF, 10mM
b-glycerophosphate, 1 mg/mL leupeptin, 1 mg/mL aprotinin, and 0.1%
Triton X-100). Two hundred micrograms of total protein was add to 20 mL
anti-FLAGM2 affinity gel (Sigma-Aldrich) and incubated at 48C for 4 h on a
rocker. Afterwashingwith TBS (20mMTris-HCl, pH 7.5, and 150mMNaCl)
three times, agarose bead-protein complexeswere used for kinase assays.
Expression of NIA2 Protein in Pichia pastoris
Expression of NIA2 protein in P. pastoris was performed as described
previously (Su et al., 1996). Wild-type and mutant forms of NIA2 cDNA
with a C-terminal MYC-epitope were directly inserted into the BamHI/
EcoRI site of the P. pastoris expression vector pPIC9K (Invitrogen). The
expression clones were transformed into P. pastoris strain SMD1168 by
electroporation. Growth media and conditions for selection of transform-
ants were as described by the manufacturer. HIS+ and G418 transform-
ants were selected. NIA2 proteins were induced in MM medium (0.35%
yeast nitrogen base with 1% ammonium sulfate, 0.5%methanol, and 43
10 to 5%biotin). Two grams of yeast cells were collected in 2mL IP buffer,
and an equal volume of glass beads was added. The mixture was vor-
texed for 10 min and centrifuged, and the supernatants were used
for immunoprecipitation with anti-c-MYC agarose affinity gel (Sigma-
Aldrich). After washingwith TBS three times, and thenwashedwith kinase
buffer or NR extraction buffer one more time, NIA2 proteins were eluted
with c-Myc peptide and used for kinase assays or NR activity assays.
Phosphorylation Assay
The in vitro kinase assay was performed as described previously (Liu and
Zhang, 2004). Recombinant His-tagged MPK6 (10 mg) was activated by
incubation with recombinant MKK5DD (1 mg) in the presence of 50 mM
ATP in 50mL of reaction buffer (20mMHEPES, pH 7.5, 10mMMgCl2, and
1 mM DTT) at 258C for 30 min. Activated MPK6 was used to phosphor-
ylate recombinant protein purified either from E. coli or P. pastoris (1:10
enzyme:substrate ratio) or 10 mL anti-Flag beads-protein complex in
the same reaction buffer, with 50 mM ATP and [g-32P]ATP (0.1 mCi per
reaction). The reactions were stopped by the addition of SDS loading
buffer after 60min. The phosphorylated NIA2c and NIA2d were visualized
by autoradiography after separation on a 10% SDS polyacrylamide gel.
After electrophoresis, the gel was exposed to a Kodak X-Omat film for 60
min. For enzymes used in the immunoblot assay, phosphorylation was
performed without the addition of [g-32P]ATP.
In-Gel Kinase Assay and Immunobloting
Total protein was extracted from seedlings by grinding in extraction buffer
(100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM Na3VO4, 10
mM NaF, 50 mM b-glycerophosphate, 10 mM DTT, 1 mM phenyl-
methylsulfonyl fluoride, 5 mg/mL leupeptin, 5 mg/mL aprotinin, and 5%
glycerol). After centrifugation at 18,000g for 40 min, supernatants were
transferred into clean tubes, quickly frozen in liquid nitrogen, and stored
at 2808C until analyzed. The concentration of protein extracts was de-
termined according to the method of Bradford with BSA as the standard
(Bradford, 1976).
The in-gel kinase assay was performed as described previously (Zhang
and Klessig, 1997). Aliquots of extract that contained 10 mg protein were
electrophoresed on 10% SDS-polyacrylamide gels that contained 2 mg/
mLmyelin basic protein (MBP) in the separating gel as a substrate for the
kinases. After electrophoresis, the gel was washed three times for 20 min
at room temperature with washing buffer (25 mM Tris, pH 7.5, 0.5 mM
DTT, 0.1mMNa3VO4, 5mMNaF, 0.5mg/mLBSA, and 0.1%Triton X-100)
and incubated at 48C overnight, with three changes of renaturing buffer
(25 mM Tris, pH 7.5, 1 mMDTT, 0.1 mMNa3VO4, and 5 mMNaF). The gel
was incubated at room temperature in 30 mL reaction buffer (25 mM Tris,
pH 7.5, 2 mM EGTA, 12 mMMgCl2, 1 mMDTT, and 0.1 mMNa3VO4) with
200 nM ATP plus 50 mCi of [g-32P]ATP for 60 min, after which the reaction
was stopped by transferring the gel into 5% (w/v) trichloroacetic acid
and 1% (w/v) sodium pyrophosphate. The gel was washed in the same
solution for at least 6 h with five changes of buffer. The gel was dried and
exposed to Kodak X-Omat film for 10 h.
For immunoblotting, 10 mg total proteins was separated by electro-
phoresis on 10% SDS-polyacrylamide gels, and the proteins were
transferred to polyvinylidene difluoride membranes (Roche) by elec-
trophoretic transfer. After blocking with blocking buffer (20 mM Tris, pH
7.5, 30 mM NaCl, and 0.05% Tween 20) at room temperature, the
membranes were incubated with monoclonal anti-Flag M2 antibody,
monoclonal antipolyhistidine antibody (Sigma-Aldrich), or phospho-
P44/42 MAPK antibody (Cell Signaling Technology). After washing
three times, the membranes were incubated with horseradish perox-
idase–conjugated secondary antibody and visualized using lumi-light
2994 The Plant Cell
protein gel blotting substrate (Roche) following the manufacturer’s
instructions.
Analysis of Root Architecture
Seeds of wild-type Arabidopsis and the mpk6 mutant were germinated
and grown onMS plates (Murashige and Skoog, 1962) that contained 2%
sucrose and 0.8%phytogel (Sigma-Aldrich). Five-day-old seedlings were
transferred to MS plates that contained different concentrations of H2O2,
SNP, or tungstate. For root morphology assays of NIA2A, NIA2D, and
NIA2WT transgenic plants, 5-d-old seedlings were transferred to MS
plates that contained 0.02 or 0.1 mM DEX and 15 mM SNP. Photographs
were taken and the total length of lateral roots of each seedling were
determined at the indicated times after transfer.
Measurement of NR Activity
Wild-type and mutant plants were grown on MS medium at 228C with a
16-h-light photoperiod for 2 weeks. Seedlings were harvested 30 min
after being sprayed with 10 mM H2O2 for 30 min. Samples of 0.2 g of
control and H2O2-treated seedlings were ground to a fine powder in liquid
nitrogen and homogenized in 1 mL NR extraction buffer (50 mM MOPS-
NaOH, pH 7.5, 10mMMgCl2, 1mMEDTA, 5mMDTT, 0.1%Triton X-100,
0.4 mg/mL leupeptin, and 1 mg/mL pepstatin). The mixture was vortexed
and then centrifuged at 14,000 rpm for 10 min at 48C. The supernatant
was used directly for NR assays as described previously (Huber et al.,
1992; Su et al., 1996). The concentration of protein in the extracts was
determined by Bradford assay with BSA as the standard, and 20 mg
protein extract was used for the assay of NR activity.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative databases under the following accession numbers: MKK5,
At3g21220; MPK3, At3g45640; MPK4, At4g01370; MPK6, At2g43790;
NIA1, At1g77760; and NIA2, At1g37130. T-DNA insertion lines used here
are mpk6-2 (Salk_073907, germplasm 4676495), mpk6-3 (Salk_127507,
germplasm 4845066), nia1-3 (Salk_004164, germplasm 4514312), nia1-4
(Salk_071547, germplasm 4674135), nia2-1 (Salk_138297, germplasm
4855856), nia2-2 (Salk_088070, germplasm 4690658), and nia1-2 nia2-5
(germplasm CS6512).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. NO Biosynthesis and MPK6 Activity after
Application of H2O2.
Supplemental Figure 2. Expression of NIA1, NIA2, and MPK6 in
T-DNA Insertion Mutants and Double Mutants.
Supplemental Figure 3. Expression of NIA2 in Wild-Type and mpk6-3
in Response to Exogenous H2O2.
Supplemental Figure 4. Effects of Tungstate and H2O2 on NR
Activity in Wild-Type Plants.
Supplemental Figure 5. Effects of Tungstate on H2O2-Induced
Lateral Root Growth.
Supplemental Figure 6. Phosphorylation of Full-Length NIA2 Protein
by MPK6.
Supplemental Figure 7. The Length of Lateral roots in Wild-Type and
NIA2 Transgenic Plants in Response to H2O2.
Supplemental Table 1. Primers Used in This Study.
Supplemental Methods. Quantitative RT-PCR.
ACKNOWLEDGMENTS
We thank Y. Guo for technical help and discussion and Sophie Song for
reading of the manuscript. This work was supported by the National
Natural Science Foundation of China (30625005, 90817106, and
30600344).
Received November 19, 2009; revised August 26, 2010; accepted Sep-
tember 10, 2010; published September 24, 2010.
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2998 The Plant Cell
DOI 10.1105/tpc.109.072959; originally published online September 24, 2010; 2010;22;2981-2998Plant Cell
Pengcheng Wang, Yanyan Du, Yuan Li, Dongtao Ren and Chun-Peng SongArabidopsisand Signal Transduction in
Mediated Activation of MAP Kinase 6 Modulates Nitric Oxide Biosynthesis−Hydrogen Peroxide
This information is current as of May 10, 2020
Supplemental Data /content/suppl/2010/09/13/tpc.109.072959.DC1.html
References /content/22/9/2981.full.html#ref-list-1
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