Nitrate-responsive miR393/AFB3 regulatory module controls root system architecture in Arabidopsis thaliana.
ABSTRACT One of the most striking examples of plant developmental plasticity to changing environmental conditions is the modulation of root system architecture (RSA) in response to nitrate supply. Despite the fundamental and applied significance of understanding this process, the molecular mechanisms behind nitrate-regulated changes in developmental programs are still largely unknown. Small RNAs (sRNAs) have emerged as master regulators of gene expression in plants and other organisms. To evaluate the role of sRNAs in the nitrate response, we sequenced sRNAs from control and nitrate-treated Arabidopsis seedlings using the 454 sequencing technology. miR393 was induced by nitrate in these experiments. miR393 targets transcripts that code for a basic helix-loop-helix (bHLH) transcription factor and for the auxin receptors TIR1, AFB1, AFB2, and AFB3. However, only AFB3 was regulated by nitrate in roots under our experimental conditions. Analysis of the expression of this miR393/AFB3 module, revealed an incoherent feed-forward mechanism that is induced by nitrate and repressed by N metabolites generated by nitrate reduction and assimilation. To understand the functional role of this N-regulatory module for plant development, we analyzed the RSA response to nitrate in AFB3 insertional mutant plants and in miR393 overexpressors. RSA analysis in these plants revealed that both primary and lateral root growth responses to nitrate were altered. Interestingly, regulation of RSA by nitrate was specifically mediated by AFB3, indicating that miR393/AFB3 is a unique N-responsive module that controls root system architecture in response to external and internal N availability in Arabidopsis.
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ABSTRACT: A precursor of miR156 (MsmiR156d) was cloned and overexpressed in alfalfa (Medicago sativa L.) as a means to enhance alfalfa biomass yield. Of the five predicted SPL genes encoded by the alfalfa genome, three (SPL6, SPL12 and SPL13) contain miR156 cleavage sites and their expression was down-regulated in transgenic alfalfa plants overexpressing miR156. These transgenic plants had reduced internode length and stem thickness, enhanced shoot branching, increased trichome density, a delay in flowering time and elevated biomass production. Minor effects on sugar, starch, lignin and cellulose contents were also observed. Moreover, transgenic alfalfa plants had increased root length, while nodulation was maintained. The multitude of traits affected by miR156 may be due to the network of genes regulated by the three target SPLs. Our results show that the miR156/SPL system has strong potential as a tool to substantially improve quality and yield traits in alfalfa.Plant Biotechnology Journal 12/2014; · 6.28 Impact Factor
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ABSTRACT: Plant microRNAs (miRNAs) play important roles in regulating plant growth, development, and responses to abiotic stresses. In this study, 38 miRNAs (TaMIRs) from wheat (Triticum aestivum L.), 36 from the miRBase database, and two from our previous work were characterized and subjected to an expression pattern analysis under normal conditions and a drought stress. A semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time quantitative PCR (qPCR), and small RNA blot analyses revealed that two TaMIRs (TaMIR1120 and TaMIR1123) were root-predominant and two TaMIRs (TaMIR1121 and TaMIR1134) were leaf-predominant. Seven TaMIR precursors showed altered expressions after the drought; of these, TaMIR1136 was upregulated, whereas TaMIR156, TaMIR408, TaMIR1119, TaMIR1129, TaMIR1133, and TaMIR1139 were downregulated. These seven drought-responsive TaMIRs showed dose-dependent and typical temporal expression patterns during drought induction, and they gradually returned back under the normal growth conditions. The drought-responsive and the tissue-predominant TaMIRs had varying numbers of target genes. Randomly selected target genes exhibited opposite expression patterns to their corresponding TaMIRs suggesting that they were regulated by distinct TaMIRs through a post-transcriptional cleavage. The target genes regulated by drought-responsive and tissue-predominant TaMIRs are involved in various cellular processes, such as signal transduction, transcriptional regulation, primary and secondary metabolisms, development, and defense responses. These results provide a novel insight into the miRNA-mediated responses of wheat to drought stress.Biologia Plantarum 01/2014; 59(1):37-46. · 1.74 Impact Factor
- Journal of Forestry Research 03/2015; 26(1):23-32.
Nitrate-responsive miR393/AFB3 regulatory module
controls root system architecture in
Elena A. Vidala, Viviana Arausa, Cheng Lub, Geraint Parryc, Pamela J. Greenb, Gloria M. Coruzzid,
and Rodrigo A. Gutiérreza,d,1
aDepartamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile, Santiago 8331010, Chile;bDepartment of Plant and Soil
Sciences, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711;cDepartment of Biology, Indiana University, Bloomington, IN 47405;
anddDepartment of Biology, New York University, New York, NY 10003
Edited by Mark Estelle, University of California San Diego, La Jolla, CA, and approved January 4, 2010 (received for review August 24, 2009)
to changing environmental conditions is the modulation of root
system architecture (RSA) in response to nitrate supply. Despite the
fundamental and applied significance of understanding this proc-
ess, the molecular mechanisms behind nitrate-regulated changes in
developmental programs are still largely unknown. Small RNAs
(sRNAs) have emerged as master regulators of gene expression in
plants and other organisms. To evaluate the role of sRNAs in the
nitrate response, we sequenced sRNAs from control and nitrate-
ever, only AFB3 was regulated by nitrate in roots under our exper-
imental conditions. Analysis of the expression of this miR393/AFB3
module, revealed an incoherent feed-forward mechanism that is
induced by nitrate and repressed by N metabolites generated by
nitrate reduction and assimilation. To understand the functional
role of this N-regulatory module for plant development, we ana-
lyzed the RSA response to nitrate in AFB3 insertional mutant plants
altered. Interestingly, regulation of RSA by nitrate was specifically
mediated by AFB3, indicating that miR393/AFB3 is a unique N-
responsive module that controls root system architecture in
response to external and internal N availability in Arabidopsis.
for plant growth and agricultural productivity. Besides its role as
a nutrient, nitrate (as well as other N metabolites) has been
shown to act as a signal that regulates global gene expression (1–
5). Although genomic data show that the nitrate response is
global, little is known about the molecular basis of nitrate
sensing and signaling and how the transcriptomic changes can
result in developmental responses. In the last years, microRNAs
(miRNAs) have emerged as master regulators of gene expres-
sion in plants and other systems (6–8). miRNAs are small ∼21–
22 nt molecules, that play critical roles in various developmental,
stress, and signaling responses (reviewed in refs. 9, 10). Micro-
array analysis showed that target transcripts of miRNAs are
regulated by nitrate and/or sucrose treatments in Arabidopsis
roots (4), suggesting that posttranscriptional gene expression
control by miRNAs can be a general mechanism integrating
nitrate signals into developmental changes. miRNAs have been
known for years to be important for phosphate and sulfate
deprivation responses in plants (11–13). More recently, miR167
and its target ARF8 were shown to be part of an organic N-
responsive regulatory network that controls lateral root ini-
tiation in Arabidopsis (14) and N- and P-limitation regulated
itrate is one of the major forms of inorganic nitrogen in the
biosphere and nitrate availability is the most limiting factor
miRNAs have been identified in Arabidopsis seedlings (15). In
this work, we used 454 sequencing to detect N-regulated miR-
NAs and we identified a nitrate responsive miRNA/target reg-
ulatory module that integrates N and auxin signaling to control
root system architecture (RSA) in response to changes in
Identification of Nitrate-Responsive sRNAs in Arabidopsis Roots. As a
first approximation to evaluate the contribution of sRNAs to the
nitrate response in Arabidopsis, we used the 454 sequencing
technology to identify nitrate responsive sRNAs (16). Sequenc-
ing approaches have been shown to provide accurate estimates of
transcript levels (17, 18) and to allow for the discovery of miR-
NAs [or other sRNAs such as small interfering RNAs (siRNAs)
not previously identified (19–24)]. Previous genomic analyses of
the nitrate and sucrose response in Arabidopsis roots have shown
that most of the previously identified nitrate-responding genes
are in fact regulated by some type of carbon (C)/nitrogen (N)
interaction (4). Therefore, we chose to perform a combined
nitrate/sucrose treatment to maximize N-responding sRNA dis-
covery. Arabidopsis plants were grown hydroponically in basal
MS media without N, supplemented with 1 mM ammonium as
sole N source and 3 mM sucrose for 2 weeks, and were then
treated with 5 mM KNO3and 30 mM sucrose (treatment) or
with 5 mM KCl and 30 mM mannitol (control) for 20 min and for
2 h. These experimental conditions have been shown to elicit a
robust transcriptomic response in previous studies (1, 4). Total
RNA was extracted from seedlings and the small RNA fraction
was isolated for 454 sequencing (16, 25). In a pilot experiment,
we pooled the two time points and obtained ∼16,000 sequences
from the treatment and control samples. The raw sequence data
were processed with custom made PERL scripts, mapped to the
Arabidopsis genome, and a list of sRNAs was generated with the
normalized frequency of occurrence in the control and treated
samples. Using a fourfold difference cutoff between the nor-
malized frequency in the treatment and control samples, we
identified miR393 as the only sRNA induced by the treatment in
these experiments. miR393 targets the transcripts that code for a
basic helix-loop-helix (bHLH) transcription factor (bHLH77, ref.
26) and the auxin receptors TIR1, AFB1, AFB2, and AFB3 (13,
27, 28). Auxin is a key phytohormone, mediating growth and
Author contributions: E.A.V., V.A., and R.A.G. designed research; E.A.V., V.A., C.L., and P.J.
G. performed research; C.L., G.P., P.J.G., G.M.C. contributed new reagents/analytic tools; E.
A.V., V.A., and R.A.G. analyzed data; and E.A.V. and R.A.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| March 2, 2010
| vol. 107
| no. 9
developmental responses in plants (29). Auxin has been pro-
posed as a long-range signal from shoot to root mediating root
developmental responses to nitrate (30, 31) and is clearly
important based on network analysis of nitrate-regulated genes
(4). Thus miR393 was an attractive candidate to mediate
developmental plant responses to nitrate. According to our
sequencing results, miR393 was induced by the treatment with a
expression) of 3.3. To corroborate our results, and to better
define the timing and organ regulation of miR393, we used a
modified Northern blot procedure to analyze the regulation of
this miRNA in shoot and root tissue after 20 min and and 2 h of
nitrate plus sucrose treatment. We found miR393 to be induced
by the treatment specifically in root tissue, after 2 h of treatment
(Fig. S1). To define whether miR393 was responding to nitrate
or to a combined nitrate/sucrose effect, we subjected the plants
to an N-only treatment (5 mM KNO3or 5 mM KCl). miR393
was regulated similarly by nitrate treatments in the absence of
sucrose, indicating that miR393 responds to nitrate independ-
ently of external sucrose levels (Fig. 1). These results prompted
the hypothesis that nitrate regulation of miR393 in roots controls
root auxin receptor levels, which is important for root morpho-
logical changes in response to nitrate.
The miR393/AFB3 Module Is a Unique N-Regulatory Network
Integrating External and Internal N Availability. To evaluate the
effect of miR393 regulation over the transcript levels of its tar-
gets, we analyzed the expression of bHLH77 and of the auxin
receptors in Arabidopsis roots after nitrate treatments. Plants
were grown for 2 weeks in ammonium as sole N source and were
treated with 5 mM KNO3or 5 mM KCl as control for 1, 2, and 4
h. Transcript levels for bHLH77, TIR1, AFB1, AFB2, and AFB3
were analyzed using real-time quantitative reverse transcription
PCR (qRT-PCR). As shown in Fig. 1, nitrate treatment did not
affect bHLH77, TIR1, AFB1, or AFB2 transcript levels in roots.
However, AFB3 was induced in root organs with a peak 1 h after
exposure to nitrate (Fig. 1). Interestingly, we found that AFB3
mRNA levels decreased rapidly over time, suggesting active
transcript degradation. As expected, miR393 shows a peak of
accumulation 2 hours after nitrate treatment, just as the tran-
script levels of AFB3 begin to decrease (Fig. 1). Analysis of AFB3
expression in a miR393 overexpressor line (28) showed that
AFB3 levels are diminished in comparison with wild-type plants
(Fig. 2A). In addition, we found a fragment corresponding to
a miR393 AFB3 cleavage product (Fig. 2B) 2 h after KNO3
treatment using a modified RNA ligase-mediated 5′ rapid
amplification of cDNA ends (RLM-RACE) procedure (28, 32).
Moreover, we compared the kinetics of mRNA accumulation in
afb3 mutant plants expressing a miR393-resistant version of
AFB3 under the control of the endogenous AFB3 promoter
(pAFB3:mAFB3-GUS) (33) (Fig. 2C). In contrast to the rapid
decrease of AFB3 mRNA levels seen in wild-type plants, AFB3
levels did not decrease over time in pAFB3:mAFB3-GUS plants
after nitrate induction (Fig. 2C). These results indicate that
miR393 specifically cleaves AFB3 transcripts under our exper-
imental conditions, controlling AFB3 mRNA accumulation in
roots in response to nitrate exposure.
Nitrate in roots can be converted to other inorganic and
organic N metabolites such as ammonium and the amino acids
glutamate and glutamine. To test whether miR393 or AFB3 were
responding directly to nitrate or to N metabolites produced by
nitrate reduction and/or assimilation, we used a nitrate reductase
(NR)-null mutant of Arabidopsis thaliana (34). This mutant plant
is unable to reduce nitrate, therefore genes responding to nitrate
treatments in the NR-null mutant are controlled by nitrate and
not by N metabolites produced after nitrate reduction. In the
NR-null mutant, AFB3 was induced by nitrate treatments with a
peak of mRNA accumulation 1 h after the treatment (Fig. 3),
similar to what was observed in wild-type plants (Fig. 1). How-
ever, AFB3 levels did not decline over time in the NR-null
mutant (Fig. 3) as compared to wild type (Fig. 1). As expected,
this lack of repression over time, correlated with the lack of
induction of miR393 in the NR-null mutant (Fig. 3). These
results indicate that the induction of AFB3 gene expression is
caused by a nitrate signal (likely acting at the transcriptional
level) but the downregulation seen at later times is caused by
miR393 induction by an N metabolite downstream of nitrate
reduction. To determine possible N signals controlling miR393,
we tested the regulation of miR393 by ammonium and gluta-
mate, N sources downstream of nitrate reduction and assim-
ilation. Both N sources caused an increase in mature miR393
levels after 2 h of treatment (Fig. S2 A and B). These results
indicated. Root transcript levels for bHLH77, AFB1, AFB2, AFB3, and mature
miR393 were analyzed by real-time qPCR. We show the mean and standard
error for three biological replicates. The asterisk indicates means that sig-
nificantly differ between the control and treatment conditions (P < 0.01).
Nitrate consistently regulates miR393 and its target AFB3 in Arabi-
type Col-0 plants and miR393 overexpressor plants (28) were grown in 0.5×
MS salts supplemented with 30 mM sucrose in Petri dishes for 14 days. AFB3
levels were analyzed in seedlings using qPCR. We show the mean and
standard error for three biological replicates. (B) Plants were grown hydro-
ponically for 14 days with ammonium as the sole N source and were treated
with 5 mM KNO3for 2 h. Poly(A)+RNA was extracted from roots and a
modified RLM-RACE procedure was used to amplify a miR393 cleavage
product from AFB3 (28). NTC, no template control. (C) pAFB3:mAFB3-GUS
plants (33) were grown as described in B and were treated with 5 mM KNO3
for the times indicated. AFB3 transcript levels in roots were analyzed by real-
time qPCR. Values are presented as the log2ratio between the treatment
level and the time 0 levels. As a reference, we also present the AFB3 tran-
script levels in wild-type plants from Fig. 1.
AFB3 transcript is cleaved by miR393 in response to nitrate. (A) Wild-
| www.pnas.org/cgi/doi/10.1073/pnas.0909571107 Vidal et al.
indicate that miR393 responds to N signals produced after
nitrate reduction and assimilation and acts as a negative feed-
back loop regulating AFB3 levels over time according to external
and internal N availability.
Nitrate Regulates Primary Root Growth by a Pathway Involving the
AFB3 Auxin Receptor. To understand the function of the miR393/
AFB3 regulatory module in the nitrate response, we first ana-
lyzed the expression of this auxin receptor in roots after nitrate
treatments. We used a previously described reporter line
expressing the β-glucuronidase (GUS) reporter gene fused to a
1,800-bp sequence upstream of the AFB3 transcription initiation
site, pAFB3::GUS (35). We treated the reporter lines with 5 mM
KNO3or 5 mM KCl for 1 h and we stained for GUS activity. We
found that the AFB3 promoter is able to drive expression of
GUS throughout the root in both KNO3- and KCl-treated roots,
indicating root expression of the AFB3 gene as previously
described (35). However, the nitrate treatment increased GUS
activity preferentially in the root tip area (Fig. 4A), indicating
that AFB3 mRNA accumulation by nitrate is due to transcrip-
tional activation. To confirm these qualitative GUS results, we
analyzed AFB3 RNA levels in the root tip using qRT-PCR. AFB3
was induced after 1 h of treatment in root tips (Fig. 4B). To
evaluate whether this AFB3 induction correlated with increased
auxin activity in the root tip, we analyzed GUS activity in the
DR5::GUS reporter line (36). We found that GUS activity was
also increased in the DR5::GUS line, indicating increased auxin
response in the root tip in the nitrate-treated plants (Fig. 4C).
Consistent with this result, we found that nitrate is able to reg-
ulate auxin-responsive genes in wild-type roots in our exper-
imental conditions (Fig. S3A). In addition, we also found
regulation of auxin-related genes that are not reported to be
auxin responsive, such as the auxin response factors ARF9 and
ARF18 and an auxin efflux carrier (At2g17500) (Fig. S3B). These
results suggest that nitrate is able to modulate auxin signaling
and responses at multiple levels as previously described (4).
Auxin is known to control primary root growth in a concen-
tration-dependent manner (37). Thus, the observed nitrate-
induced auxin activity through AFB3 may lead to a repression in
primary root growth in the nitrate condition. To test this
hypothesis, we subjected wild-type plants and the AFB3 T-DNA
insertional mutant afb3-1 (35) to a 3-day KNO3or KCl treat-
ment. We measured the primary root length of plants grown for
2 weeks on ammonium as sole N source and after 3 days of 5 mM
KNO3or KCl treatment. At the end of the 3-day treatment, we
found that nitrate-treated wild-type plants have shorter primary
roots as compared with control-treated plants (Fig. 4D), indi-
cating that nitrate availability inhibits primary root elongation.
However, the primary roots of afb3-1 plants were not inhibited
by nitrate as observed in wild-type plants or in the other indi-
vidual auxin receptor mutants tir1-1, afb1-1, and afb2-1 (Fig. 4D
and Fig. S4 A and B). These results suggest that AFB3 plays a
specific role in modulating primary root growth in response to
nitrate. We also analyzed the response of the primary root to
KNO3treatments in the miR393 overexpressor line that shows
reduced levels of AFB3 (Fig. 2A). Consistent with the results in
the afb3-1 mutant line, the miR393 overexpressor line is com-
pletely insensitive to the KNO3treatments as compared with the
wild-type plants (Fig. 4E).
Our results show that AFB3 is involved in primary root growth
inhibition in response to nitrate and that this effect is likely
mediated by nitrate regulation of AFB3 levels in root tips.
Nitrate Regulates Lateral Root Growth by a Pathway Involving the
Auxin Receptor AFB3. Analysis of GUS activity in the AFB3::GUS
reporter lines indicated that AFB3 can be also induced by nitrate
in the central vascular area, including the pericycle area (Fig.
5A). It is known that lateral roots are initiated from pericycle
founder cells opposite to the xylem poles (38, 39). Therefore
nitrate regulation of AFB3 in the pericycle may lead to changes
regulation by N metabolites produced after nitrate reduction by a pathway
involving miR393. Nitrate reductase-null mutant plants (34) were grown
hydroponically as described before and were treated with 5 mM KNO3or 5
mM KCl for the times indicated. AFB3 transcript levels and mature miR393
levels were analyzed by real-time qPCR in roots. We show the mean and
standard error for three biological replicates. The asterisk indicates means
that significantly differ between control and treatment conditions (P < 0.01).
AFB3 is directly induced by nitrate and is under posttranscriptional
Plants were grown hydroponically as described before and were treated for
KNO3or KCl and were then stained for GUS activity for 4 h. Qualitative GUS
staining was analyzed using DIC optics. Photographs are representative of at
least 15 stained plants. (Scale bar, 100 μm.) (B) Wild-type plants were treated
for 1 h with 5 mM KNO3or KCl. Root tips were excised from nitrate-treated or
control-treated plants and AFB3 RNA levels were measured using qPCR. Bars
represent SE. The asterisk represents means that significantly differ (P < 0.01).
(C) Auxin reporter DR5::GUS plants (36) were treated for 1 h with 5 mM KNO3
was analyzed using DIC optics. Photographs are representative of at least 15
stained plants. (Scale bar, 100 μm.) (D) Primary root length of Ws wild-type
plants or afb3-1 mutant plants was measured using the ImageJ program after
3 days of 5 mM KNO3or KCl treatment. Bars represent standard errors. Dif-
ferent letters represent significantly different means (P < 0.01). (E) Primary
root length of Col-0 wild-type plants or 35S::miR393 overexpressor plants (28)
was measured using the ImageJ program after 3 days of 5-mM KNO3or KCl
treatment. Bars represent standard errors. Different letters represent stat-
istically different means (P < 0.01).
Nitrate regulates primary root growth by a pathway involving AFB3.
Vidal et al.PNAS
| March 2, 2010
| vol. 107
| no. 9
in lateral root development. To quantify expression of AFB3 in
the pericycle, we isolated KNO3or KCl GFP-tagged pericycle
cells using a fluorescence activated cell sorter (FACS) and
extracted total RNA as described previously (9). KNO3treat-
ment induced AFB3 expression in the pericycle (Fig. 5B), sug-
gesting that nitrate can also regulate auxin signaling in pericycle
cells. Because lateral roots are produced from pericycle cells, we
analyzed the number of initiating (stages I, II, III, IV, Va, Vb,
VIa, VIb, and VII, after ref. 39) and emerging lateral roots using
DIC microscopy in wild-type plants and in the afb3-1 mutant
after 3 days of 5 mM KNO3or 5 mM KCl treatment (Fig. 5C). In
wild-type plants, nitrate treatments increased the density of lat-
eral roots (both initiating and emerging) as compared with the
KCl control condition. Most of the emerging laterals were short
(<0.5 mm; Table S1), which can be related to an inhibitory effect
of KNO3on root elongation as described previously (40). On the
other hand, the increased density of initiating laterals (Table S1)
can be related to a positive KNO3effect on root initiation, as
described previously (14). The lateral root response was altered
in the afb3-1 mutant, which showed decreased density of
emerging and initiating lateral roots as compared to wild type
(Fig. 5C). However, none of the other individual auxin receptor
mutants showed altered lateral root response to nitrate treat-
ments (Fig. S5 A and B). This result shows that, as we have seen
for primary root, AFB3 has also a specific role in lateral root
response to nitrate. We also analyzed lateral root density in the
miR393 overexpressor line (Fig. 5D). Lateral root density after 3
days of KNO3 treatment was no different from lateral root
density after 3 days of control treatment, indicating that this
response was also absent in this line (Fig. 5D). These results
show that nitrate regulates lateral root growth by a pathway
RSA modulation in response to nutrients is a classical example of
plant plasticity to changing environmental conditions (41–43).
Given their importance in controlling growth and developmental
programs, phytohormones have arisen as the missing links
between nutrient availability and plant developmental responses.
Because of its central role in root development (44, 45) a role for
auxin has been proposed in the modulation of RSA in response to
nutrients (46–51). Here, we showed that nitrate treatments mod-
ulate both primary and lateral root growth by a pathway involving
the N-responsive miR393/AFB3 regulatory module. Recently,
identified in Arabidopsis to regulate the ratio between initiating
andemerging lateral roots (14). miR393/AFB3 regulatory module
is unique as nitrate can regulate auxin responses by direct regu-
and thus affecting both primary and lateral root growth. The
regulatory mechanism of miR393/AFB3 regulation by nitrate
involves at least transcriptional and posttranscriptional mecha-
nisms. We showed that nitrate is able to transcriptionally induce
expression of AFB3 in roots (Figs. 1, 4A, and 5A) and that N
(Figs. 1–3). This mechanism (Fig. S6) is consistent with the type I
incoherent feed-forward loop (FFL) motif described for tran-
scriptional networks in yeast, bacteria, and mammals (52–54). In
thismodel, atranscription factorAcanactivateboth atargetgene
Z and also a repressor of Z in response to a signal, leading to a
transient activation of Z. This regulatory design allows for Z to be
rapidly responsive to the input signal (55) and can also produce a
nonmonotonic response, where the output of Z is first increased
The observed regulation of AFB3 expression by nitrate and
metabolites produced downstream of nitrate reduction might
and coherent FFL involving miRNA-target pairs are recurrent
motifs in mammalian gene regulatory networks (54, 59–62).
Because most miRNA targets encode transcription factors in
plants, incoherent FFLs are probably also a common feature of
plant gene networks. Besides the FFL reported here, a coherent
feed-forward loop involving miR164 and its target ORE1/NAC2
has been recently described in Arabidopsis that regulates age-
dependent cell death (63).
AFB3 is part of the ubiquitin protein ligase SCFTIR1/AFBcom-
plex that targets and mediates the polyubiquitination and pro-
teasomal degradation of the Aux/IAA transcriptional repressors
to promote transcription of auxin-responsive genes (64–66). The
finding that both primary and lateral root responses to nitrate are
this response to occur, a functional AFB3 is required. Moreover,
AFB3 is induced by nitrate in primary root tips (Fig. 4C) and in
pericycle cells (Fig. 5B). Therefore, we propose that the increase
in the expression of AFB3 is responsible for the changes observed
in RSA in response to nitrate.
We found that nitrate treatments were able to regulate the
levels of auxin-responsive and auxin-related genes in Arabidopsis
roots (Fig. S3), as previously demonstrated by network analysis
of C- and N-regulated genes (4). However, we did not find
misregulation of any of these genes in the afb3-1 mutant, sug-
gesting that nitrate can modulate auxin signaling and responses
at multiple levels and in an AFB3-dependent and AFB3-
RSA modulation by auxin can depend on three main factors:
changes in auxin homeostasis, auxin transport, and auxin sig-
AFB3. Plants were grown hydroponically as described before and were
treated for the times indicated. (A) pAFB3::GUS plants (35) were treated for
1 h with 5 mM KNO3or KCl and were then stained for GUS activity for 4 h.
Qualitative GUS staining was analyzed using DIC optics. Photographs are
representative of at least 15 stained plants. (Scale bar, 100 μm.) (B) Pericycle
marker line plants were treated for 1.5 h with 5 mM KNO3or KCl. Protoplast
were prepared from roots and pericycle cells expressing GFP were sorted by
FACS. RNA levels for AFB3 were measured using qPCR. Bars represent
standard errors. The asterisk represents statistically different means (P <
0.05). (C) The number of initiating and emerging lateral roots of afb3-1
mutants (35) or Ws wild-type plants treated for 3 days with 5 mM KNO3or
KCl was counted using DIC optics. Bars represent standard errors. Different
letters indicate statistically different means (P < 0.01). (D) The number of
initiating and emerging lateral roots of 35S::miR393 overexpressor plants
(28) or Col-0 wild-type plants treated for 3 days with 5 mM KNO3or KCl was
counted using DIC optics. Bars represent SE. Different letters indicate stat-
istically different means (P < 0.01).
Nitrate regulates lateral root growth by a pathway mediated by
| www.pnas.org/cgi/doi/10.1073/pnas.0909571107Vidal et al.
naling. Here we show that N regulation of a hormone receptor
can lead to changes in hormonal signaling causing RSA changes.
Recently, the auxin receptor TIR1 has been involved in lateral
root formation in response to phosphate starvation in Arabi-
dopsis; however, the other family members might also play a role
in this response (49). Our evidence shows that RSA modulation
in response to nitrate is a specific function of AFB3. Moreover,
AFB3 has a dual role in primary and in lateral root development
in response to nitrate whereas TIR1 seems to have a role only in
lateral root formation (49).
the N source and concentration available for plants as well as on
other environmental conditions. In Arabidopsis, these effects can
include changes in primary root growth (67), lateral root initiation
(14, 68, 69), and elongation (40, 70). Under our experimental
conditions, we found that supplementing 5 mM KNO3to wild-type
ammonium-fed plants causes an inhibition of primary root growth
(Fig. 4 D and E) and an increase in initiating and emerging lateral
root density as compared with the control KCl condition (Fig. 5 C
and D). This observation contrasts the previous observation by
Walch-Liu et al. (67), where it was seen that a 5-mM KNO3treat-
ment stimulatedprimary rootgrowthwhencomparedwitha 5-mM
KCl control. This discrepancy may arise from a higher apparent
nitrate availability in our hydroponic condition, in which roots are
surrounded by media, compared to the vertical plate condition in
assumption is supported by findings by Tian et al. (71) where
or more caused inhibition of root elongation. The inhibitory effect
of KNO3on primary root length was also seen after 18 days of
continuous growth in vertical plates containing high nitrate con-
centrations in Arabidopsis (72). Our results are also similar to the
ones reported by Gifford et al. (14), where the same KNO3treat-
ment caused an increase in the number of initiating lateral roots
is no mention of the effect of nitrate on primary root growth.
Our results are consistent with a model in which a high nitrate
enhance N acquisition) by an interaction with the auxin signaling
pathway mediated by miR393/AFB3, adjusting plant growth and
development to external and internal N availability (Fig. S6).
Materials and Methods
Plant Material. Arabidopsis (A. thaliana) plants were of Columbia (Col-0)
ecotype or Wassilewskija (Ws) ecotype as indicated. tir1-1, afb1-1, afb2-1,
afb3-1, pAFB3::GUS, and pAFB3:mAFB3-GUS lines were kindly donated by
Mark Estelle, University of California San Diego, La Jolla, CA (33, 35). miR393
overexpressor lines were kindly donated by Jonathan Jones, the Sainsbury
Laboratory, John Innes Centre, Norwich, UK (28). Nitrate reductase-NULL
mutant lines were kindly provided by Nigel Crawford, University of Cal-
ifornia San Diego, La Jolla, CA (34). The GFP line that marks the pericycle
(E374) was obtained from http://enhancertraps.bio.upenn.edu.
Growth and Treatment Conditions. Approximately 1,500 Arabidopsis seedlings
were grown hydroponically on Phytatrays on MS-modified basal salt media
without N (Phytotechnology Laboratories, M531) supplemented with 0.5
mM ammonium succinate and 3 mM sucrose under a photoperiod of 16 h of
light and 8 h of darkness and a temperature of 22 °C using a plant growth
incubator (Percival Scientific, Inc.). After 2 weeks, plants were treated with 5
mM KNO3with or without 30 mM sucrose or 5 mM KCl with or without 30
mM mannitol as control for different time periods as indicated. For the
phenotypic analysis of the root response to nitrate treatment, seedlings
were grown as described above and were treated with 5 mM KNO3or 5 mM
KCl for 3 days.
Histochemical Analysis of GUS Activity. For histochemical analysis of GUS
activity, Arabidopsis seedlings were incubated at 37 °C in a GUS reaction
buffer (100 mM sodium phosphate buffer, pH 7.0, 0.5 mM potassium ferri-
cyanide, 0.5 mM potassium ferrocyanide, 0.1% (vol/vol) Triton X-100, 0.1%
(wt/vol) sodium lauroyl sarcosine) plus 1 mM 5-bromo-4-chloro-3-indolyl-β-D-
glucuronide (X-Gluc). The seedlings were cleared according to the protocol
described in ref. 39 and were imaged using DIC optics on a Nikon Eclipse 80i
microscope. For each marker line and treatment, at least 15 plants
Analysis of Root Architecture Traits. Initiating and emerging lateral roots
(stages I, II, III, IV, Va, Vb, VIa, VIb, and VII, according to ref. 39) were counted
using DIC optics on a Nikon Eclipse 80i microscope. For primary root meas-
ures, plants were scanned using an Epson Perfection V700 Photo scanner,
and roots were measured using the National Institutes of Health program
ImageJ. The data were statistically analyzed in the Graph Pad Prism 5 Pro-
ACKNOWLEDGMENTS. We thank Dr. Mark Estelle for kindly providing
the tir1-1, afb1-1, afb2-1, afb3-1, pAFB3::GUS, and pAFB3:mAFB3-GUS
lines; Dr. Nigel Crawford for providing the NR-null mutant line; and Dr.
Jonathan Jones for providing the miR393 overexpressor lines. We thank
Dr. Miriam L. Gifford for sharing her experience in cell-specific analysis.
This work was funded by the Fondo Nacional de Desarrollo Científico y
Tecnológico (1060457), National Institutes of Health-Fogarty International
Research Collaboration Award (F6414-01), International Centre for Genetic
Engineering and Biotechnology (CRPCHI0501), and Millennium Nucleus for
Plant Functional Genomics (P06-009-F) to R.A.G and PhD fellowship from
Comisión Nacional de Investigación Científica y Tecnológica (AT-24080114)
1. Wang R, et al. (2004) Genomic analysis of the nitrate response using a nitrate
reductase-null mutant of Arabidopsis. Plant Physiol 136:2512–2522.
2. Wang R, Okamoto M, Xing X, Crawford NM (2003) Microarray analysis of the nitrate
response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes
and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism.
Plant Physiol 132:556–567.
3. Scheible WR, et al. (2004) Genome-wide reprogramming of primary and secondary
metabolism, protein synthesis, cellular growth processes, and the regulatory infra-
structure of Arabidopsis in response to nitrogen. Plant Physiol 136:2483–2499.
4. Gutierrez RA, et al. (2007) Qualitative network models and genome-wide expression
data define carbon/nitrogen-responsive molecular machines in Arabidopsis. Genome
5. Palenchar PM, Kouranov A, Lejay LV, Coruzzi GM (2004) Genome-wide patterns of
carbon and nitrogen regulation of gene expression validate the combined carbon and
nitrogen (CN)-signaling hypothesis in plants. Genome Biol 5:R91.
6. Bartel DP (2004) MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell
7. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in
plants. Annu Rev Plant Biol 57:19–53.
8. Voinnet O (2009) Origin, biogenesis, and activity of plant micrornas. Cell 136:
9. Kidner CA, Martienssen RA (2005) The developmental role of microRNA in plants. Curr
Opin Plant Biol 8:38–44.
10. Sunkar R, Chinnusamy V, Zhu J, Zhu J-K (2007) Small RNAs as big players in plant
abiotic stress responses and nutrient deprivation. Trends Plant Sci 12:301–309.
11. Fujii H, Chiou TJ, Lin SI, Aung K, Zhu J-K (2005) A miRNA involved in phosphate-
starvation response in Arabidopsis. Curr Biol 15:2038–2043.
12. Bari R, Pant BD, Stitt M, Scheible W-R (2006) PHO2, micro RNA399 and PHR1 define a
phosphate signaling pathway in plants. Plant Physiol 141:988–999.
13. Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs
and their targets, including a stress-induced miRNA. Mol Cell 14:787–799.
14. Gifford ML, Dean A, Gutierrez RA, Coruzzi GM, Birnbaum KD (2008) Cell-specific
nitrogen responses mediate developmental plasticity. Proc Natl Acad Sci USA 105:
15. Pant BD, et al. (2009) Identification of nutrient-responsive Arabidopsis and rapeseed
microRNAs by comprehensive real-time PCR profiling and small RNA sequencing.
Plant Physiol 150:1541–1555.
16. Margulies M, et al. (2005) Genome sequencing in microfabricated high-density
picolitre reactors. Nature 437:376–380.
17. Bloom J, Khan Z, Kruglyak L, Singh M, Caudy A (2009) Measuring differential gene
expression by short read sequencing: quantitative comparison to 2-channel gene
expression microarrays. BMC Genomics 10:221.
18. Fu X, et al. (2009) Estimating accuracy of RNA-Seq and microarrays with proteomics.
BMC Genomics 10:161.
19. Lu C, et al. (2005) Elucidation of the small RNA component of the transcriptome.
20. Henderson IR, et al. (2006) Dissecting Arabidopsis thaliana DICER function in small
RNA processing, gene silencing and DNA methylation patterning. Nat Genet 38:
21. Lu C, et al. (2006) MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-
dependent RNA polymerase-2 mutant. Genome Res 16:1276–1288.
22. Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily
fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20:3407–3425.
Vidal et al.PNAS
| March 2, 2010
| vol. 107
| no. 9
23. Kasschau KD, et al. (2007) Genome-wide profiling and analysis of Arabidopsis siRNAs.
PLoS Biol 5:e57.
24. Fahlgren N, et al. (2007) High-throughput sequencing of Arabidopsis microRNAs:
Evidence for frequent birth and death of MIRNA genes. PLoS One 2:e219.
25. Lu C, Meyers BC, Green PJ (2007) Construction of small RNA cDNA libraries for deep
sequencing. Methods 43:110–117.
26. Heim MA, et al. (2003) The basic helix-loop-helix transcription factor family in plants:
a genome-wide study of protein structure and functional diversity. Mol Biol Evol 20:
27. Sunkar R, Zhu J-K (2004) Novel and stress-regulated microRNAs and other small RNAs
from Arabidopsis. Plant Cell 16:2001–2019.
28. Navarro L, et al. (2006) A plant miRNA contributes to antibacterial resistance by
repressing auxin signaling. Science 312:436–439.
29. Benjamins R, Scheres B (2008) Auxin: The looping star in plant development. Annu
Rev Plant Biol 59:443–465.
30. Walch-Liu P, et al. (2006) Nitrogen regulation of root branching. Ann Bot (Lond) 97:
31. Forde BG (2002) Local and long-range signaling pathways regulating plant responses
to nitrate. Annu Rev Plant Biol 53:203–224.
32. Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrow-like mRNA
targets directed by a class of Arabidopsis miRNA. Science 297:2053–2056.
33. Parry G, et al. (2009) Complex regulation of the TIR1/AFB family of auxin receptors.
Proc Natl Acad Sci USA 106:22540–22545.
34. Wang R, Xing X, Crawford N (2007) Nitrite acts as transcriptome signal at micromolar
concentrations in Arabidopsis roots. Plant Physiol 145:1735–1745.
35. Dharmasiri N, et al. (2005) Plant development is regulated by a family of auxin
receptor F-box proteins. Dev Cell 9:109–119.
36. Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ (1997) Aux/IAA proteins repress
expression of reporter genes containing natural and highly active synthetic auxin
response elements. Plant Cell 9:1963–1971.
37. Evans ML, Ishikawa H, Estelle MA (1994) Responses of Arabidopsis roots to auxin
studied with high temporal resolution: comparison of wild type and auxin-response
mutants. Planta 194:215–222.
38. Dolan L, et al. (1993) Cellular organisation of the Arabidopsis thaliana root.
39. Malamy JE, Benfey PN (1997) Organization and cell differentiation in lateral roots of
Arabidopsis thaliana. Development 124:33–44.
40. Zhang H, Jennings A, Barlow PW, Forde BG (1999) Dual pathways for regulation of
root branching by nitrate. Proc Natl Acad Sci USA 96:6529–6534.
41. Osmont KS, Sibout R, Hardtke CS (2007) Hidden branches: Developments in root
system architecture. Annu Rev Plant Biol 58:93–113.
42. Forde BG, Walch-Liu P (2009) Nitrate and glutamate as environmental cues for
behavioural responses in plant roots. Plant Cell Environ 32:682–693.
43. Desnos T (2008) Root branching responses to phosphate and nitrate. Curr Opin Plant
44. Teale WD, Paponov IA, Palme K (2006) Auxin in action: Signalling, transport and the
control of plant growth and development. Nat Rev Mol Cell Biol 7:847–859.
45. Benkova E, et al. (2003) Local, efflux-dependent auxin gradients as a common module
for plant organ formation. Cell 115:591–602.
46. Mishra BS, Singh M, Aggrawal P, Laxmi A (2009) Glucose and auxin signaling
interactionin controlling Arabidopsis
development. PLoS One 4:e4502.
47. Sanchez-Calderon L, et al. (2005) Phosphate starvation induces a determinate
developmental program in the roots of Arabidopsis thaliana. Plant Cell Physiol 46:
48. Nacry P, et al. (2005) A role for auxin redistribution in the responses of the root
system architecture to phosphate starvation in Arabidopsis. Plant Physiol 138:
thaliana seedlingsroot growth and
49. Perez-Torres CA, et al. (2008) Phosphate availability alters lateral root development in
Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR1 auxin
receptor. Plant Cell 20:3258–3272.
50. Lopez-Bucio J, et al. (2005) An auxin transport independent pathway is involved in
phosphate stress-induced root architectural alterations in Arabidopsis. Identification
of BIG as a mediator of auxin in pericycle cell activation. Plant Physiol 137:681–691.
51. Franco-Zorrilla JM, Martin AC, Leyva A, Paz-Ares J (2005) Interaction between
phosphate-starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of
cytokinin receptors CRE1/AHK4 and AHK3. Plant Physiol 138:847–857.
52. Shen-Orr SS, Milo R, Mangan S, Alon U (2002) Network motifs in the transcriptional
regulation network of Escherichia coli. Nat Genet 31:64–68.
53. Mangan S, Alon U (2003) Structure and function of the feed-forward loop network
motif. Proc Natl Acad Sci USA 100:11980–11985.
54. Tsang J, Zhu J, van Oudenaarden A (2007) MicroRNA-mediated feedback and
feedforward loops are recurrent network motifs in mammals. Mol Cell 26:753–767.
55. Mangan S, Itzkovitz S, Zaslaver A, Alon U (2006) The incoherent feed-forward loop
accelerates the response-time of the gal system of Escherichia coli. J Mol Biol 356:
56. Basu S, Gerchman Y, Collins CH, Arnold FH, Weiss R (2005) A synthetic multicellular
system for programmed pattern formation. Nature 434:1130–1134.
57. Entus R, Aufderheide B, Sauro H (2007) Design and implementation of three
incoherent feed-forward motif based biological concentration sensors. Syst Synth Biol
58. Kaplan S, Bren A, Dekel E, Alon U (2008) The incoherent feed-forward loop can
generate non-monotonic input functions for genes. Mol Syst Biol 4:203.
59. Shalgi R, Lieber D, Oren M, Pilpel Y (2007) Global and local architecture of the
mammalian microRNA-transcription factor regulatory network. PLOS Comput Biol 3:
60. Zhou Y, Ferguson J, Chang J, Kluger Y (2007) Inter- and intra-combinatorial regu-
lation by transcription factors and microRNAs. BMC Genomics 8:396.
61. Cohen EEW, et al. (2009) A feed-forward loop involving protein kinase C-alpha and
microRNAs regulates tumor cell cycle. Cancer Res 69:65–74.
62. Li X, Cassidy JJ, Reinke CA, Fischboeck S, Carthew RW (2009) A microRNA imparts
robustness against environmental fluctuation during development. Cell 137:273–282.
63. Kim JH, et al. (2009) Trifurcate feed-forward regulation of age-dependent cell death
involving miR164 in Arabidopsis. Science 323:1053–1057.
64. Tan X, et al. (2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase.
65. Mockaitis K, Estelle M (2008) Auxin receptors and plant development: A new
signaling paradigm. Annu Rev Cell Dev Biol 24:55–80.
66. dos Santos Maraschin F, Memelink J, Offringa R (2009) Auxin-induced, SCFTIR1-
mediated poly-ubiquitination marks AUX/IAA proteins for degradation. Plant J 59:
67. Walch-Liu P, Forde BG (2008) Nitrate signalling mediated by the NRT1.1 nitrate
transporter antagonises L-glutamate-induced changes in root architecture. Plant J 54:
68. Remans T, et al. (2006) A central role for the nitrate transporter NRT2.1 in the
integrated morphological and physiological responses of the root system to nitrogen
limitation in Arabidopsis. Plant Physiol 140:909–921.
69. Little DY, et al. (2005) The putative high-affinity nitrate transporter NRT2.1 represses
lateral root initiation in response to nutritional cues. Proc Natl Acad Sci USA 102:
70. Zhang H, Forde BG (1998) An Arabidopsis MADS box gene that controls nutrient-
induced changes in root architecture. Science 279:407–409.
71. Tian Q, Chen F, Liu J, Zhang F, Mi G (2008) Inhibition of maize root growth by high
nitrate supply is correlated with reduced IAA levels in roots. J Plant Physiol 165:
72. Linkohr BI, Williamson LC, Fitter AH, Leyser HMO (2002) Nitrate and phosphate
availability and distribution have different effects on root system architecture of
Arabidopsis. Plant J 29:751–760.
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