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Nitric oxide causes root apical meristem defects and
growth inhibition while reducing PIN-FORMED 1
(PIN1)-dependent acropetal auxin transport
María Fernández-Marcos
a,1
, Luis Sanz
a,1
, Daniel R. Lewis
b
, Gloria K. Muday
b
, and Oscar Lorenzo
a,2
a
Department of Fisiología Vegetal, Centro Hispano-Luso de Investigaciones Agrarias, Facultad de Biología, Universidad de Salamanca, 37185 Salamanca,
Spain; and
b
Department of Biology, Wake Forest University, Winston-Salem, NC 27106
Edited* by Luis Herrera Estrella, Centro de Investigacion y de Estudios Avanzados, Irapuato, Mexico, and approved September 27, 2011 (received for review
May 31, 2011)
Nitric oxide (NO) is considered a key regulator of plant develop-
mental processes and defense, although the mechanism and direct
targets of NO action remain largely unknown. We used phenotypic,
cellular, and genetic analyses in Arabidopsis thaliana to explore the
role of NO in regulating primary root growth and auxin transport.
Treatment with the NO donors S-nitroso-N-acetylpenicillamine, so-
dium nitroprusside, and S-nitrosoglutathione reduces cell division,
affecting the distribution of mitotic cells and meristem size by re-
ducing cell size and number compared with NO depletion by 2-(4-
carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO).
Interestingly, genetic backgrounds in which the endogenous NO lev-
els are enhanced [chlorophyll a/b binding protein underexpressed
1/NO overproducer 1 (cue1/nox1) mirror this response, together with
an increased cell differentiation phenotype. Because of the impor-
tance of auxin distribution in regulating primary root growth, we
analyzed auxin-dependent response after altering NO levels. Both
elevated NO supply and the NO-overproducing Arabidopsis mutant
cue1/nox1 exhibit reduced expression of the auxin reporter markers
DR5pro:GUS/GFP. These effects were accompanied by a reduction in
auxin transport in primary roots. NO application and the cue1/nox1
mutation caused decreased PIN-FORMED 1 (PIN1)-GFP fluorescence
in a proteasome-independent manner. Remarkably, the cue1/nox1-
mutant root phenotypes resemble those of pin1 mutants. The use of
both chemical treatments and mutants with altered NO levels dem-
onstrates thathigh levels of NO reduce auxin transport and response
by a PIN1-dependent mechanism, and root meristem activity is re-
duced concomitantly.
cell division and elongation
|
plant growth regulator
|
root development
Nitric oxide (NO) is a signaling molecule involved in a variety of
physiological processes during plant growth and development
and also is an important modulator of disease resistance. Extensive
research has shown that NO is involved in the promotion of seed
germination, photomorphogenesis, mitochondrial activity, leaf ex-
pansion, root growth, stomatal closure, fruit maturation, senescence,
and iron metabolism (as reviewed in ref. 1). NO also is important for
defense response, playing key roles in the activation of defense genes
(e.g., pathogenesis-related protein 1), in phytoalexin production,
andinmodulationofprogrammedcelldeath(1–3). The mechanism
for NO signal transduction, plant resistance to pathogens and cell
death, cellular transport, basic metabolism, and photosynthesis fre-
quently occurs through an NO-induced change in transcription (4).
Additionally, NO is produced in plant tissues by two major
pathways, one enzymatic and the other nonenzymatic (5). The
enzymatic pathway of NO production is being studied thoroughly,
and much information about the type and subcellular localization
of the enzymes involved is available. Different enzymes have been
identified that catalyze the synthesis of NO from two different
substrates, nitrate and arginine. The first enzyme identified was
nitrate reductase, which usually reduces nitrate to nitrite but also
is able to reduce nitrite to NO using NADPH as a cofactor.
Another key enzyme in NO biosynthesis is Arabidopsis thaliana
NO-associated (AtNOA1), previously described as catalyzing the
conversion of L-arginine to L-citrulline (6). Recent biochemical
reports demonstrate a GTPase activity for this enzyme (7, 8).
AtNOA1 is localized in the plastids and has a putative role in ri-
bosome assembly (9). Other enzymes, including xanthine oxidase/
dehydrogenase and cytochrome P450, have been suggested oc-
casionally as sources for NO (10). Experimental evidence also
suggests a nonenzymatic pathway to produce NO based on the
reduction of nitrite to NO at acid pH, mainly in the apoplast of the
aleurone cell layer during seed germination (11).
One mechanism of NO action in plant tissues may be the redox-
based posttranslational modification of target proteins through
S-nitrosylation. NO is able to modify thiol groups of specific cys-
teine residues in target proteins reversibly and thereby alter
protein function. Previous proteomic profiling in plants has
identified a number of S-nitrosylated proteins (12). Recent results
support the S-nitrosylation of key proteins such as nonexpressor
of pathogenesis-related genes 1 (13) and the Arabidopsis thaliana
salicylic acid (SA)-binding protein 3 (14), both of which are in-
volved in SA-dependent defense responses. The stability of per-
oxiredoxin II (15) and iron regulatory protein 2 is regulated by
S-nitrosylation via the ubiquitin–proteasome pathway (16). In
animals, this posttranslational modification also has been shown
to cause protein degradation via the ubiquitin-dependent pro-
teasome pathway.
Despite its relevance as a plant growth and stress regulator, our
current knowledge about the mechanism of NO action is still
limited. Therefore, the identification and characterization of NO
targets at the molecular level is essential for deeper insight into this
pathway. Here we uncovered a role for NO on primary root growth
in Arabidopsis thaliana. We found that NO treatment affects
meristem size in the primary root mainlyby decreasing cell-division
rates and promoting cell differentiation. A reduction in meristem
size also is observed in the NO overaccumulating mutant chloro-
phyll a/b binding protein underexpressed 1/NO overproducer 1
(cue1/nox1). Interestingly, the root apical auxin maximum is al-
tered after NO addition. Auxin transport and the level of the auxin
efflux protein PIN-FORMED 1 (PIN1) are reduced significantly in
the cue1/nox1 background. Consistently, the distorted organiza-
tion of the quiescent center and surrounding cells of cue1/nox1
mutants mimics to some extent the phenotype of pin1-mutant
roots, suggesting a link between NO and auxin signaling in main-
taining the size and activity of the root apical meristem.
Author contributio ns: O.L. designed resear ch; M.F.-M., L.S., and D. R.L. performed re-
search; L.S., G.K.M., and O.L. analyzed data; and O.L. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1
M.F.-M. and L.S. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail: oslo@usal.es.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1108644108/-/DCSupplemental.
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Results
Effect of NO on Arabidopsis Primary Root Growth. Exogenous ap-
plication of NO donors in tomato (17) and genetic mutants with
altered endogenous NO levels in Arabidopsis (18) indicated that NO
affects root architecture, reducing overall primary root growth.
However, our knowledge of the molecular mechanisms by which NO
regulates growth and development in Arabidopsis is still fragmentary.
To investigate the role of NO in the regulation of primary root
growth in Arabidopsis,WT(Arabidopsis thaliana ecotype Colum-
bia-0, Col-0) plants were germinated on plates containing different
concentrations of NO released by the specific NO donor S-nitroso-
N-acetyl-DL-penicillamine (SNAP). As shown in Fig. 1A, the in-
hibition was dose dependent because a gradual decrease in the
length of the primary root (from 0.8 ±0.1 to 0.3 ±0.1 cm) was
observed as SNAP levels increased from 0–1mM.TheNOscav-
enger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-
oxide (cPTIO) partially blocked this NO effect, clearly establi-
shing that NO is the major contributor to the effect of SNAP on
root growth (Fig. 1A). To validate our results by using other NO
donors, we tested sodium nitroprusside (SNP), S-nitrosoglutathione
(GSNO), delivery of NO gas, and mutants with high levels of
endogenous NO (cue1/nox1). The observed inhibition of primary
root growth seemed to be independent of the NO source, because
different NO donors and the cue1/nox1 mutant had similar effects
(44.3% inhibition in the cue1/nox1 mutant and 90.2%, 60.2%, and
38.3% inhibition under 100 μM SNP, 1 mM SNAP, and 1 mM
GSNO treatments, respectively) (Fig. 1B).
Determination of Endogenous NO Abundance and Distribution in
Arabidopsis Roots. Sites of NO and other reactive oxygen species
production in plant tissues can be identified by using the fluores-
cence indicator 4,5-diaminofluorescein diacetate (DAF-2DA)
(17, 19). DAF-2DA is a cell-permeable compound hydrolyzed in-
side the cells that emits fluorescence when nitrosylated by endog-
enous NO. By examining the endogenous NO levels in multiple
2-d-old WT roots with DAF-2DA, we identified NO-dependent
fluorescence in the basal meristem and rapid elongation zone in
young primary roots (Fig. 1C). DAF-2DA staining of 7-d-old WT
roots revealed that NO production was localized mainly in the root
apex, as described previously (17). In addition, the maximum NO
level was restricted mainly to epidermal cell files closest to the
basal meristem, lateral root cap, and cortex/endodermal initial
cells (Fig. 1D). Application of the NO scavenger cPTIO reduced
NO-dependent DAF-2DA fluorescence (Fig. 1Dand Fig. S1). In
the cue1/nox1 background NO accumulation in these tissues was
increased threefold or more.
Increases in NO Concentration Reduce the Number of Dividing Cells
and Promote Early Differentiation in the Primary Root Meristem. To
determine whether the inhibition of primary root growth after NO
treatment might be related to differences in the number of cells
and/or cell size in the root meristem, we measured the size and
number of root cells in a cortical cell file, determining the length of
cells from the initials adjacent to the quiescent center (QC) to the
rapid elongation/differentiation zone. We found that the total
number of cells between the QC and the start of the rapid elon-
gation zone in the cortex layer is affected significantly by altered
NO levels. The inflection point on the cell-length curve marking
the transition to the rapid elongation zone occurs around cell
numbers 17, 33, and 28 in SNP-treated, cPTIO-treated, and con-
trol plants, respectively (Fig. 2 Aand B). Analysis of the cue1/nox1
mutant revealed results similar to those obtained after SNP
treatment with an inflection point around cell numbers 17–18 (Fig.
2Aand B), suggesting that NO levels are correlated inversely with
the number of cells in the meristematic zone. Interestingly, sig-
nificant differences in cell sizes in the root apical meristem also
were detected at this stage (cells 1–10 and cells 11–20 in Fig. 2D).
As shown in Fig. 2 Aand C, application of the NO scavenger
cPTIO partially blocked the action of the NO donor SNAP, clearly
establishing that NO levels are correlated positively with cell
elongation (Fig. 2A). Hence, in the initial phases of root growth
after germination, increases in NO concentration reduce the size
of the primary root meristem (309.2 ±15.6 μm in control plants vs.
318 ±9.1 μm in cPTIO-treated, 171.3 ±17.4 μm in SNP-treated,
and 247.2 ±22.9 μm in SNAP-treated plants, and 329.0 ±6.55 μm
in plants treated with SNAP plus cPTIO; Fig. 2E) by promoting
cell elongation in the root meristem and concurrently decreasing
the number of dividing cells. Remarkably, long-term treatment
(up to 5 d) with the NO donor SNP almost abolished the pool of
dividing cells and enhanced cell elongation in all cell types of the
root meristem (Fig. S2).
Early-differentiation phenotypes also are present in the cue1/
nox1-mutant background. In agreement with the reduced size of
the primary root meristem, development of epidermal root hairs
and premature vacuolization commence much closer to the ini-
tials in cue1/nox1 than in WT (Col-0) roots (Fig. 2 F–Hand Fig.
S3). A distorted organization of QC and columella stem cells
(CSC) is clearly visible in the cue1/nox1 mutant along with a dif-
ferent pattern of starch granule accumulation, suggesting that
the increase in NO levels causes abnormal differentiation in this
genetic background as compared with WT (Col-0) (Fig. 2I).
High Levels of NO Reduce the Overall Distribution of Mitotic Cells in
Arabidopsis Primary Roots. Because NO affects primary root
growth by reducing the pool of dividing cells, we marked cells in
Fig. 1. Effect of NO in the regulation of Arabidopsis primary root growth.
(A)(Upper) Photograph showing the length of the primary root of WT (Col-
0) seedlings grown for 7 d on Murashige and Skoog (MS) agar plates that
were untreated (Control) or supplemented with 10 μM, 100 μM, 200 μM, 500
μM, or 1 mM of the NO donor SNAP or with 1 mM SNAP plus 1 mM of the NO
scavenger cPTIO. (Lower) Measurements were obtained 4 d after the treat-
ment of 3-d-old seedlings. Values represent the mean of 30 measurements ±
SD. Asterisks indicate significant differences compared with the untreated
control (P<0.05) (a) and with 1 mM SNAP (P<0.05) (b). (B) Inhibition of
primary root growth after delivery of NO gas (300 ppm), by treatment with
the NO donors SNP (100 mM), SNAP (1 mM), or GSNO (1 mM) and in mutants
with high levels of endogenous NO (cue1/nox1). Measurements were taken 4
d after the treatment of 3-d-old seedlings (n= 25). (C) Detection of en-
dogenous NO production using DAF-2DA. Plants were grown for 2 d (Upper
and Lower Left)or7d(Right) on agar plates and then subjected to DAF-2DA
incubation. (D)(Upper) Detection of endogenous NO production using DAF-
2DA in WT (Col-0) and cue1/nox1 seedlings in control conditions and after
NO scavenging by cPTIO. (Lower) Measurement of NO levels in WT (Col-0)
seedlings and the cue1/nox1 mutant. Asterisk indicates a statistically signif-
icant difference from the WT.
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the G2 stage of the cell cycle with the reporter CycB1;1
pro
:GUS-
DB (β-glucuronidase-destruction box) to analyze further the ef-
fect of NO on cell division. We observed the effect of modulating
NO accumulation on the expression of this reporter. SNP and
SNAP treatments reduced the number of cells expressing the
CycB1;1
pro
:GUS-DB reporter (Fig. 3A). However, the overall
distribution of mitotic cells was not affected significantly by cPTIO
treatment. In contrast, cPTIO partially rescued the effect of NO
donors on the zone of CycB1;1
pro
:GUS-DB expression (Fig. 3A).
Furthermore, we observed a decrease in CycB1;1
pro
:GUS-DB ex-
pression in the cue1/nox1 background, where endogenous NO
levels are enhanced (Fig. 3Band Fig. S4). To determine whether
an increase in NO level was able to diminish the normal rate of cell
division in the root apical meristem, we measured the number of
mitotic events in the root apical meristem in cue1/nox1-mutant
and WT plants. DAPI staining followed by microscopic analysis
revealed a significant reduction in the mitotic activity of cue1/nox1
(Fig. S4). Taken together, these results led us to conclude that
high levels of applied or endogenous NO cause a reduction in cell-
division rates and an increase in cell lengths consistent with NO
positively regulating the exit of cells from the primary root meri-
stem into the elongation and differentiation zones (Fig. 2).
Increasing NO Levels Affects Auxin Response and Reduces Auxin
Transport. Spatial patterns of auxin response based on auxin
gradients are important factors in the regulation of many plant
developmental processes, including cell division, elongation,
and differentiation during primary root growth. Using the auxin
response reporter DR5
pro
:GUS/GFP,weobservedthatin-
creasing NO levels after application of the NO donor SNP
attenuated DR5 activity in the QC and CSC at 3, 24, and 48 h
after treatment (Fig. 4A). The NO scavenger cPTIO partially
rescued the depletion of DR5
pro
:GUS expression in roots treated
with SNP (Fig. 4B). Similar to the expression pattern of DR5
pro
:
GUS in the pin1 mutant (Fig. 4B), the DR5
pro
:GUS/GFP spatial
pattern was altered in cue1/nox1 mutants, where endogenous
NO levels are enhanced (Fig. 4Cand Fig. S5), clearly estab-
lishing that increasing NO accumulation depletes auxin-de-
pendent reporter expression in the apical auxin maximum. This
alteration of auxin-dependent response in the meristematic zone
is different from that produced by application of the auxin
transport inhibitor napthylphthalamic acid (NPA) (Fig. 4C).
To determine whether the effect of high levels of NO on the
auxin distribution and root meristem activity could be related
to NO regulation of polar auxin transport, we tested acropetal
Fig. 2. Effect of NO on the Arabidopsis
root meristem. (A) Confocal images of roots
from seedlings grown for 5 d on unsup-
plemented MS agar plates (Control), cue1-
mutant seedlings, or WT seedlings supple-
mented with 100 μM of the NO donor SNP,
1 mM of the NO scavenger cPTIO, 1 mM
SNAP, and 1 mM SNAP plus 1 mM cPTIO.
Vertical lines indicate apical (AM) and basal
regions (BM) of the primary meristem re-
gion (PM). Cells 1 and 15 from the QC are
highlighted in green. Note that meriste-
matic cells are enlarged in the presence of
NO and that the start of net elongation is
further from the QC in cPTIO-treated
seedlings than in untreated controls. (Band
C) Cell sizes in the cortical layer of the root.
(B) Average cell size in the cortical cell layer
(cells 1–40 from QC) of untreated WT
seedlings, WT seedlings treated with SNP or
cPTIO, and untreated cue1-mutant seed-
lings. (C) Average cell size in the cortical
layer (cells 1–40 from QC) of WT (Col-0)
seedlings grown as described above and
seedlings that were untreated (control) or
treated with SNAP or with SNAP plus cPTIO.
Measurements were taken 2 d after the
treatment of 3-d-old seedlings. (D) Average
cortical cell sizes are shown for cells 1–10
and 11–20 (counted from the QC) of the
seedlings in A.(E) Size of root meristem in
WT (Col-0) seedlings grown for 5 d on
unsupplemented MS agar plates (Control)
or on medium supplemented with 1 mM of
the NO scavenger cPTIO, 100 μM of the NO
donor SNP, 1 mM of the NO donor SNAP, or
1 mM SNAP plus 1 mM of the NO scavenger
cPTIO. A minimum of five roots per treat-
ment was analyzed. Asterisks indicate sig-
nificant differences compared with
untreated control (P<0.05). (F) Quantifi-
cation of the distance between the root tip
and the first root hair formed in WT (Col-0)
and cue1-mutant seedlings. A minimum of
8–10 roots per genotype was analyzed.
Asterisks indicate significant differences
compared with untreated WT (P<0.05). (G)
Representative images of the root tip and the first root hair formed in WT Col-0) and cue1-mutant seedlings. (Hand I) Representative images of the root
meristem of 7-d-old WT (Col-0) and cue1-mutant seedlings stained with modified pseudo-Schiff propidium iodide (mPS-PI) (Upper) or Lugol’s solution (Lower)
highlighting vacuolization (H) and QC/CSC disorganization and starch accumulation (I). Red and green arrowheads indicate QCs and CSCs, respectively.
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auxin transport in roots of WT (Col-0) and cue1/nox1 mutants
(Fig. 4D). A drastic reduction in auxin movement was detected
by examining transport of radiolabeled auxin in cue1/nox1,
supporting the hypothesis that enhanced NO levels in this
mutant background cause a defect in acropetal indoleacetic
acid (IAA) transport capacity.
We also examined fluorescence of GFP fusions to the auxin
efflux carriers PIN1 and PIN2 in the presence of the NO
donors SNP, SNAP, and GSNO and the NO scavenger cPTIO
(Fig. 5Aand Fig. S6). Confocal time-course analysis showed
that PIN1-GFP fluorescence was decreased clearly in the stele
and primary root meristem upon treatment with the NO donors
SNP, SNAP, and GSNO. Using immunoblot analysis with anti-
GFP antiserum, we also detected a reduction of GFP protein in
PIN1
pro
:GFP-PIN1 seedlings after treatment with NO donors
(Fig. 5B). In agreement with these results, PIN1
pro
:GFP-PIN1
fluorescence clearly was reduced in genetic backgrounds where
endogenous NO levels are enhanced (Fig. 5C), consistent with
the conclusion that increases of NO levels reduce PIN1 levels.
Interestingly, PIN1 levels were not altered significantly in lat-
eral root primordia after treatment with cPTIO or SNP or in
the cue1/nox1-mutant background, suggesting that regulation
of PIN1 levels by NO is restricted exclusively to the primary
root (Fig. S6). PIN2 levels in the PIN2
pro
:GFP-PIN2 line were
not altered significantly in any of the previous treatments (Fig.
S6), suggesting that NO regulation of auxin transport in the
primary root is specific to the acropetal transport stream and is
mediated by changes in PIN1 protein levels.
NO-Dependent Reductions in PIN1 Protein Level Do Not Require
Proteasome Activity. We conducted quantitative RT-PCR (qRT-
PCR) and proteasome inhibitor experiments to understand the
mechanism of NO-dependent reductions in the levels of PIN1
protein. qRT-PCR analysis showed that PIN1 transcript levels
were not altered significantly after treatment with the NO donor
SNP or by mutations in CUE1/NOX1 (Fig. S7). To determine if
PIN1 protein was degraded by the proteasome after NO treat-
ment, we treated PIN1
pro
:GFP-PIN1 seedlings with the known
proteasome inhibitor MG132, both in the presence and absence
of NO (Fig. 5 Aand B). PIN1 and PIN2 levels in MG132-treated
plants did not differ significantly from that in untreated plants or
in plants treated with the NO scavenger cPTIO (Fig. 5 Aand B
and Fig. S6). Interestingly, PIN1
pro
:GFP-PIN1 plants treated both
with an NO donor (SNP or SNAP) and with MG132 displayed
a low GFP signal not different from that in plants treated only
with the NO donor. Furthermore, using immunoblot analysis with
anti-GFP antiserum, we could not detect changes in the accu-
mulation of PIN1 in NO-treated PIN1
pro
:GFP-PIN1 seedlings
after the 26S proteasome was inhibited by MG132 (Fig. 5B).
Therefore, we propose that the PIN1 level is regulated by NO and
that high levels of endogenous or applied NO promote reductions
in PIN1 protein levels by a proteasome-independent mechanism.
cue1/nox1 Mutant Root Phenotypes Resemble Those of pin1 Mutants.
To analyze further the relevance of PIN1 reductions in root
responses to NO, we analyzed the root phenotype of pin1 mutants
under standard growth conditions and in response to NO donors
(Fig. 5 D–Fand Fig. S8). The distorted organization of the QC,
surrounding cells, and CSC of the NO-overproducing cue1/nox1
mutant is similar to that of pin1 mutants (Fig. 5D). Additionally,
the root meristem size (Fig. 5E) and total primary root length
(Fig. 5F)ofpin1 mutants resembles plants treated with NO do-
nors and the cue1/nox mutant. Interestingly, pin1 mutants do not
display a visible NO-resistant phenotype, instead exhibiting a hy-
persensitive phenotype under low concentrations of SNAP (200
μM) (Fig. S8). Conversely, supplying exogenous auxin (IAA or
Fig. 3. The effect of NO on cell-division activity was monitored using the
CycB1;1
pro
:GUS-DB reporter marking cells in the G2 stage of the cell cycle. (A)
Five-day-old untreated seedlings (Control) and seedlings treated with 1 mM
cPTIO, 100 μM SNP, 1 mM SNAP, 100 μM SNP plus 1 mM cPTIO, or 1 mM SNAP
plus 1 mM cPTIO are shown. Pictures were taken 2 d after the treatment of
3-d-old seedlings. (B) Expression level and localization of CycB1;1
pro
:GUS-DB
in the cue1/nox1 background.
Fig. 4. Pattern of DR5
pro
:GUS/GFP expression in root tip tissue. (A) Confocal
images of the DR5
pro
:GFP reporter line in untreated (Control) seedlings and
in seedlings treated with 1 mM cPTIO or with 100 μM SNP for 3, 24, and 48 h.
(B) Representative close-up views of GUS staining in DR5
pro
:GUS 5-d-old
untreated (Control) seedlings and in seedlings treated with 100 μM SNP,
1 mM cPTIO, or 100 μM SNP plus 1 mM cPTIO. Seedlings were grown on MS
plates for 3 d and then were subjected to donor/scavenger treatments for
2d.(C) Confocal images of the DR5
pro
:GFP reporter line in the cue1 back-
ground and after treatment with 1 μM NPA. (D) Acropetal auxin transport
measured in roots of WT (Col-0) and cue1-mutant 7-d-old seedlings. A
minimum of 15 roots per genotype was analyzed. Asterisk indicates signif-
icant difference compared with untreated WT seedlings (P<0.05).
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1-naphthaleneacetic acid) or the auxin transport inhibitor NPA
to the cue1/nox1 mutant does not normalize the primary root
growth defect present in this genetic background (Fig. S9).
Discussion
The understanding of the importance of NO as a regulator of
plant growth and response to environmental stress has increased
considerably despite limited information on its signaling. A phys-
iological role for NO in the regulation of root growth has been
described (17). NO diminishes primary root growth and promotes
lateral root development in tomato (17) and Arabidopsis (20).
Furthermore, a requirement for NO in auxin-induced adventi-
tious (21) and lateral root (17) development has been reported.
NO-hypersensitive mutants isolated in genetic screens let the
identification of the NO-overproducing mutant nox1 and CUE1 as
the mutated gene (18). However, the mechanisms of the precise
cellular responses to NO are not yet well understood.
Using different NO donors (SNAP, SNP, GSNO, and NO gas)
as well as NO-specific scavengers (cPTIO), we confirmed that high
levels of NO inhibit primary root growth in Arabidopsis (Fig. 1).
The elevation of NO using releasing compounds can cause second-
ary effects (22, 23), prompting us to test NO-overaccumulating
mutants also. Analysis of mutants with high levels of endogenous
NO in roots (cue1/nox1) showed results similar to those obtained
after exogenous NO application (Fig. 2). Thus, exogenously ap-
plied NO phenocopies mutants with increased endogenous NO
levels (18, 24), mirroring the response of these mutants after
specific stress stimuli (25, 26).
To elucidate the cellular modifications and molecular basis of
the NO response in Arabidopsis roots, we observed cell organiza-
tion within the root apex. Microscopic analysis of 5-d-old seedlings
confirmed that the organization of primary root meristem is very
sensitive to changes in NO levels (Fig. 2). Our results suggest that
increases in NO concentration decrease primary root growth by
reducing the number of dividing cells in the meristem. This re-
duction in number causes fewer division cycles to take place within
the root apical meristem (Fig. 3 and Fig. S4). As the seedlings age,
the number of cells in the root apical meristem decreases until,
after 7 d, the root apical meristem becomes practically exhausted,
exhibiting enlargement of all meristematic cells (Fig. S2). Primary
root meristem activity is reduced concomitantly with the occur-
rence of differentiation phenotypes (i.e., development of epider-
mal root hairs and vacuolization) that are much closer to the
initials in cue1/nox1 than in WT (Col-0) roots (Fig. 2 and Fig S3).
Our data also demonstrate that during early seedling root de-
velopment, endogenous NO accumulates mainly in a zone situ-
ated between the apical meristem and the elongation zone (also
called “basal meristem”) (Fig. 2). Interestingly, sites of NO syn-
thesis also have been described in roots (19) where three local
centers of NO production were detected: one at the root cap
statocytes, another one at the QC and distal portion of the mer-
istem, and the third, the most prominent, at the distal part of the
transition zone. The different localization patterns of NO may
mirror the diverse effects of NO on plant growth and devel-
opment. The use of the DAF-2DA fluorescence indicator has the
advantage of following NO production in live cells undergoing
development or in response to a stimulus and allowed us to lo-
calize the sites of NO accumulation in roots (Fig. 2A). Our results
define synthesis of NO in the sites where a physiological effect is
observed (i.e., the elongation zone of primary roots).
Interestingly, scavenging endogenous NO reduces cell elon-
gation in the basal meristem without significantly affecting the
number of dividing cells in the apical meristem (Figs. 2 and 3).
Remarkably, in 7-d-old treated seedlings, we found no significant
differences between cPTIO-treated plants and controls in the
total number of cells between the QC and the start of the rapid
elongation in the cortex. This location marking the transition
into the rapid elongation zone makes the inflection point on the
cell length curve occur around cell number 33 in both control
and cPTIO (Fig. S2). During root meristem establishment, NO
might affect this transition between the basal meristem and the
rapid elongation zone by modifying the number of cells in the
apical meristem and changing its axial position relative to the QC.
Cells in the basal meristem are important in the response of roots
to a variety of environmental signals, such as gravity and thig-
mostimulation, electrotropic stimulation, water stress, and re-
sponses to auxin (27).
Previous studies have described the growth-modulating prop-
erties of NO and its interaction with auxin in modulating root
growth and developmental processes (28 and references herein).
Using Arabidopsis lines containing the DR5 auxin-responsive
promoter linked to GFP or β-GUS, we observed that increases in
NO concentration [either by application of the NO donor (SNP)
or the use of cue1/nox1-mutant background] attenuates auxin-
dependent reporter expression in the QC and CSC. The auxin-
induced maximum gene expression in the root apex, which is
necessary for meristem maintenance, is normally observed in
these cells, but this maximum is diminished in the presence of
elevated NO (Fig. 4 and Fig. S5) (29). Additionally, we found that
NO reduces the frequency of cell division in the root apex as
judged by expression of the mitotic marker CycB1;1
pro
:GUS-DB
(Fig. S4) and that chronically high NO levels eventually cause
meristem collapse. Taken together with the several lines of evi-
dence that support the hypothesis that perturbing auxin distribu-
tion reduces mitotic activity in the root apical meristem, with loss
of the QC (30, 31), our results suggest that NO influences the
maintenance of the apical auxin maximum, presumably by
changing auxin sensitivity, transport, or both.
The process of root organogenesis is controlled by auxin (30, 32,
33). Our data confirm that high levels of endogenous or applied
NO specifically attenuate auxin response (Fig. 4 and Fig. S5). In-
Fig. 5. Disappearance of PIN1 after NO treatment and comparison of cue1
and pin1 root phenotypes. (A) Distribution of PIN1
pro
:GFP-PIN1 protein is
shown in untreated control plants (C), in plants treated with the NO scav-
enger cPTIO (1 mM; 8 h), and in plants treated with the NO donors SNP (100
μM; 3, 8, and 24 h), SNAP (1 mM; 24 h), or GSNO (1 mM; 24 h), with or without
the proteasome inhibitor MG132 (100 μM; 24 h). Root tissues were stained
with propidium iodide. (B) Immunoblot analysis with anti-GFP antiserum of
in vivo levels of PIN1 protein in root extracts of PIN1
pro
:GFP-PIN1 seedlings, in
the absence or presence of NO donors and scavengers together with MG132.
Actin protein levels also were determined as a loading control. (C) Confocal
images of the PIN1
pro
:GFP-PIN1 line in the cue1 background. (D) Confocal
images after mPS-PI staining. (Eand F) Root meristem size (E) and primary
root length (n= 25) (F) of roots from WT (Col-0) and cue1- and pin1
−/−
-mu-
tant seedlings grown for 7 d on MS agar plates. A minimum of 8–10 roots per
genotype was analyzed. Asterisks indicate significant differences compared
with WT (P<0.05).
18510
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www.pnas.org/cgi/doi/10.1073/pnas.1108644108 Fernández-Marcos et al.
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terestingly, conditional loss of function of ABP1, a key regulator
for auxin-mediated responses (34), and NO show similar effects on
the root meristem, exhibiting arrest of cell division and elongated
cells next to partially collapsed root meristems. Similarly, the
phenotypes of the pin1 mutant (30), impaired in the regulation
of polar auxin transport in vascular tissue (35), resemble those of
the NO overaccumulator cue1/nox1 mutant in regards to the or-
ganization of the QC/CSC and starch accumulation. Our results
suggest that polar auxin transport is impacted negatively by over-
accumulation of NO because PIN1 protein levels are reduced
dramatically after delivery of exogenous NO and in the cue1/nox1
background, which produces increased endogenous NO levels
(Fig. 5). Consistent with the PIN1 disappearance with NO expo-
sure, the pin1 mutant is not resistant to NO. Because qRT-PCR
analysis revealed that PIN1 expression is not influenced by NO
(Fig. S7), we hypothesized that the disappearance of PIN1 protein
may be regulated posttranslationally. The MG132 proteasome-
specific inhibitor was used to determine if the decrease the level of
PIN1 protein was caused by proteasome-dependent degradation,
but it did not affect PIN1-GFP fluorescence. Our observations
indicate that NO regulates PIN1 levels posttranscriptionally,
and we propose that reductions in the levels of PIN1 protein are
caused either directly or indirectly by a proteasome-independent
mechanism. This result stands in contrast to PIN2, which is de-
graded in a proteasome-dependent manner during the gravitropic
response (36).
Recently, the intracellular redox homeostasis regulated by thi-
oredoxin (TRX) and glutathione has been shown to modulate
auxin signaling and thus affect key plant developmental processes
(37). Thus, auxin transport is impaired after specific inhibition of
glutathione synthesis, in the triple mutant of the TRX reductases
(ntra;ntrb), and the cadmium sensitive 2 (cad2) glutathione bio-
synthetic mutant. This reduced auxin transport may result from
a decrease in PIN1 (and other PIN auxin efflux proteins). Addi-
tional factors other than PIN mRNA down-regulation (i.e., post-
transcriptional or protein localization modifications) may affect
the regulation of auxin transport (37). These findings also are in
agreement with the precise effect of NO on auxin transport, fur-
ther supporting our conclusions.
We have provided evidence that the disturbance of auxin
transport (PIN1 reduction) and auxin response (alteration of DR5
expression pattern) by high levels of NO in the primary root
meristem leads to an initial reduction of root meristem length and
a progressive disturbance of root apical meristem maintenance.
One of the biggest challenges in investigating NO as a signaling
molecule is identifying the targets of NO (4). Results presented in
this work suggest that PIN1 is a target of NO signaling and that
NO-dependent changes in PIN1 protein levels cause the observed
auxin transport, DR5
pro
:GUS localization, meristem collapse, and
differentiation phenotypes.
Materials and Methods
Plant materials, treatments, measurements of primary root length, cell size,
root hair initiation and acropetal auxin transport in Arabidopsis roots and
additional details about detection of endogenous NO, GUS staining and
other procedures are fully described in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Roberto Solano, Crisanto Gutierrez, Gre-
gorio Nicolás, and Dolores Rodriguez for critical reading of the manuscript
and stimulating discussions. We also thank Centro de Investigación del
Cáncer-Universidad de Salamanca for technical fluorescence microscopy as-
sistance. This work was financed by Grants BIO2008-04698, and CSD2007-
00057 (TRANSPLANTA) from the Ministerio de Educación y Ciencia (Spain)
and by Grant SA048A10-2 from Junta de Castilla y León (to O.L.). L.S. is sup-
ported by Marie Curie European Reintegration Grant FP7-PEOPLE-ERG-2008.
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