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2012 Dual involvement of a Medicago truncatula NAC transcription factor in root abiotic stress response and symbiotic nodule senescence .pdf



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The Plant Journal (2012) 70, 220–230

doi: 10.1111/j.1365-313X.2011.04859.x

Dual involvement of a Medicago truncatula NAC transcription
factor in root abiotic stress response and symbiotic nodule
senescence
Axel de Ze´licourt1,2, Anouck Diet1,2, Jessica Marion1, Carole Laffont1, Federico Ariel1, Michae¨l Moison1, Ons Zahaf1,
Martin Crespi1, Ve´ronique Gruber1,2 and Florian Frugier1,*
1
Institut des Sciences du Ve´ge´tal (ISV), Centre National de la Recherche Scientifique, 91198 Gif sur Yvette Cedex, France, and
2
Universite´ Paris Diderot Paris 7, Les Grands Moulins, 16 rue Marguerite Duras, 75205 Paris Cedex 13, France
Received 24 October 2011; accepted 15 November 2011; published online 10 January 2012.
*For correspondence (fax +00 33 1 69 82 36 95; e-mail frugier@isv.cnrs-gif.fr).

SUMMARY
Legume crops related to the model plant Medicago truncatula can adapt their root architecture to
environmental conditions, both by branching and by establishing a symbiosis with rhizobial bacteria to form
nitrogen-fixing nodules. Soil salinity is a major abiotic stress affecting plant yield and root growth. Previous
transcriptomic analyses identified several transcription factors linked to the M. truncatula response to salt
stress in roots, including NAC (NAM/ATAF/CUC)-encoding genes. Over-expression of one of these transcription factors, MtNAC969, induced formation of a shorter and less-branched root system, whereas RNAimediated MtNAC969 inactivation promoted lateral root formation. The altered root system of over-expressing
plants was able to maintain its growth under high salinity, and roots in which MtNAC969 was down-regulated
showed improved growth under salt stress. Accordingly, expression of salt stress markers was decreased or
induced in MtNAC969 over-expressing or RNAi roots, respectively, suggesting a repressive function for this
transcription factor in the salt-stress response. Expression of MtNAC969 in central symbiotic nodule tissues
was induced by nitrate treatment, and antagonistically affected by salt in roots and nodules, similarly to
senescence markers. MtNAC969 RNAi nodules accumulated amyloplasts in the nitrogen-fixing zone, and were
prematurely senescent. Therefore, the MtNAC969 transcription factor, which is differentially affected by
environmental cues in root and nodules, participates in several pathways controlling adaptation of the
M. truncatula root system to the environment.
Keywords: lateral root, nodulation, salt stress, nitrate, Rhizobium, legume.

INTRODUCTION
Plants can adapt their root system architecture to changing
soil environmental conditions through formation of new
lateral organs. In legumes, in addition to root branching,
nitrogen-fixing nodules are formed. Both root lateral organs
develop post-embryonically in front of protoxylem poles by
re-activation of differentiated cells of the parental root,
mainly the pericycle (lateral roots) or cortex (root nodules)
(Gonzalez-Rizzo et al., 2009). Formation of lateral roots
depends on nutrient availability (Lopez-Bucio et al., 2003),
while nodules develop under nitrogen-starvation conditions
in the presence of a specific bacterial microsymbiont: Sinorhizobium meliloti in the case of the model legume Medicago truncatula. The interaction relies on recognition by
legume roots of bacterial Nod factors, which elicit root hair
220

deformations and lead to formation of infection threads,
allowing bacteria to progress towards the cortex (Oldroyd
and Downie, 2008). Simultaneously, nodule organogenesis
proceeds in the inner root cell layers, leading to formation of
a primordium that is colonized by infection threads and
subsequently differentiates into a mature organ (Crespi and
Frugier, 2008). The M. truncatula nodule has indeterminate
growth, and four developmental zones can be defined: a
persistent apical meristem (zone I), a rhizobial infection
region in which cell differentiation is marked by accumulation of amyloplasts (zone II), a functional zone in which
bacteria that have differentiated into bacteroids fix atmospheric nitrogen (zone III), and a senescence zone in which
bacteria and plant cells are degraded (zone IV) (Vasse et al.,
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd

Root stress response and nodule senescence 221
1990). Study of several plant mutants that are unable to form
nodules (reviewed by Oldroyd and Downie, 2008; Kouchi
et al., 2010) led to identification of transcription factors (TFs)
involved in nodulation: two GRAS TFs (NSP1 and NSP2:
Nodulation Signaling Pathway 1 and 2; Kalo et al., 2005; Smit
et al., 2005; Murakami et al., 2006; Heckmann et al., 2006), an
AP2/ERF TF (MtERN1: ERF Required for Nodulation), corresponding to the bit1 (branched infection threads 1) mutant
(Middleton et al., 2007), a putative NIN (nodule inception) TF
(Schauser et al., 1998; Marsh et al., 2007), and a CCAAT TF
(HAP2-1/NF-YA/CBF-B, which affects nodule growth and
differentiation; Combier et al., 2006). In addition, certain
mutants that form more nodules than wild-type plants show
alterations in TFs: the ASTRAY bZIP TF, which controls
nodule number depending on environmental conditions
(Nishimura et al., 2002), and the AP2/ERF TF encoded by efd
(ERF required for nodule formation), which affects nodule
number and differentiation (Vernie et al., 2008). Finally, the
Zpt2-1 C2H2 zinc-finger TF regulates nitrogen-fixing zone
differentiation (Frugier et al., 2000) and the M. truncatula
root response to salt stress (Merchan et al., 2007).
Abiotic stresses such as salinity are major agricultural
constraints affecting crop yield. Plants have developed
various strategies to cope with ionic and osmotic stresses,
including stress avoidance by modifying their root system
architecture to minimize exposure to salt (Malamy, 2005).
Transcriptomic profiling has been widely used in various
plants, including M. truncatula, to identify genes whose
expression levels change in response to saline conditions
(Gruber et al., 2009; Li et al., 2009). Among regulatory genes,
several TFs linked to abiotic stress responses have
been identified, but, to our knowledge, only four have
been functionally analyzed in a homologous legume context. These include the zinc-finger proteins Alfin1 in Medicago sativa (Winicov and Bastola, 1999) the M. truncatula TF
Zpt2-1 mentioned above, the related MtZpt2-2 protein (de
Lorenzo et al., 2007), and the MtHB1 HD-Zip I TF (Ariel et al.,
2010).
Among TFs found to be up-regulated by salt in M. truncatula roots using an extensive real-time RT-PCR profiling
approach, several NAC genes (family named based on its
founding members NAM, ATAF1/2 and CUC2) were identified (Gruber et al., 2009). In non-legumes, certain NAC TFs
are linked to developmental processes, such as those
encoded by the CUC genes (Olsen et al., 2005), whereas
others are related to abiotic stress responses. In Arabidopsis
thaliana, over-expression of ATAF1 (ANAC002) conferred
drought tolerance as well as salt and oxidative stress
hypersensitivity, but the corresponding mutant did not
show an altered phenotype, suggesting functional redundancy of NAC genes in response to abiotic stresses (Wu
et al., 2009). Over-expression of the salt-induced AtNAC2/
AtNAC6/ANAC092/ORE (Oresara1) gene enhanced lateral
root formation under various conditions, including mild salt

stress (4 days of 75 mM NaCl), but no root phenotype was
observed in the corresponding mutant (He et al., 2005).
However, the atnac2 mutant showed a salt-tolerant germination phenotype, whereas plants over-expressing this TF
were salt-hypersensitive (Balazadeh et al., 2010). Related
NAC genes have also been functionally linked to abiotic
stresses in rice (Oryza sativa), as over-expression of the
OsNAC6 gene (closely related to ANAC002/ATAF1 and
ANAC072/RD26 in Arabidopsis) conferred salt tolerance.
Interestingly, up- or down-regulation of OsNAC6 increased
or decreased root length, respectively (Nakashima et al.,
2007; Chung et al., 2009). In addition, over-expression of
OsNAC10 (closely related to AtNAP/ANAC029) induced
drought tolerance, associated with an enlarged root
diameter under control conditions (Jeong et al., 2010). In
legumes, information regarding functional interactions
between NACs, abiotic stresses and the development of
roots or symbiotic nodules is very scarce. As in non-legume
species, expression of several NAC genes is up-regulated in
response to various abiotic stresses (Pinheiro et al., 2009),
but only two NAC TFs have been genetically characterized in
M. truncatula: the nst1 mutant (NAC secondary wall thickening promoting factor 1), which disrupts lignin and polysaccharide biosynthesis pathways and show cell-wall
disorganization (Zhao et al., 2010), and the nac1 mutant,
which, in contrast to mutants of its closest Arabidopsis
homolog, did not show any root phenotype (d’Haeseleer
et al., 2011). Overall, no phenotype linked to roots, nodules
or any specific stress was reported for these mutants
affecting legume NAC TFs.
In this study, we determined that a NAC TF that is rapidly
up-regulated by salt stress (Gruber et al., 2009), MtNAC969,
regulates M. truncatula root architecture and symbiotic
nodulation. In addition, expression of MtNAC969 was
differentially regulated by salt in roots and nodules, and
behaved similarly to previously reported senescence
nodulation markers. Our results suggest that this NAC
TF participates in various pathways required for adaptive
root responses to salt stress and establishment of a
functional symbiotic nodule.
RESULTS
MtNAC969 negatively regulates the salt-stress
root response
By altering the expression of previously identified salt-regulated TFs (Gruber et al., 2009), we developed a screen to
identify salt-response phenotypes in M. truncatula roots
(V.G., F.F. and M.C., unpublished results). Among the
constructs tested, an RNAi down-regulating the MtNAC969
TF (either with or without salt stress; Figure S1A) allowed
maintenance of better root growth under salt stress (as
shown by measuring either root length or root dry weight;
Figure 1a–c). Expression of previously reported Medicago

ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 220–230

222 Axel de Ze´licourt et al.

(a)

(b)

(c)

(d)

Figure 1. Root phenotype in response to salt stress of composite plants in which MtNAC969 had been silenced.
(a) Representative images of 3-week-old GUS RNAi and MtNAC969 RNAi composite plants 7 days after treatment with or without salt (100 mM NaCl).
(b,c) Root length (b) and dry weight (c) of composite plants expressing an MtNAC969 or GUS RNAi construct. Three-week-old composite plants were treated with
salt (100 mM NaCl) or not for 1 week, as shown in (a), and root dry weights and lengths were measured. A representative example of two biological experiments is
shown. Error bars represent the confidence interval (a = 0.05), and asterisks indicate significant differences for a specific condition (treated or not) based on a Mann–
Whitney test (a < 0.05; n > 15).
(d) Expression of CORA, germin and osmotin salt marker genes in GUS RNAi and MtNAC969 RNAi roots under control conditions. Bars show quantification of
specific PCR amplification products for each gene, normalized using the mean value for three constitutive genes (as described in Experimental procedures). GUS
RNAi plant 1 was used to define fold changes (indicated by the dashed line). Plants from a representative example of two biological replicates are shown, and error
bars represent the confidence interval (a = 0.05) of two technical replicates.

salt-stress markers encoding a CORA homolog (coldregulated A; Merchan et al., 2007), a germin and an osmotin
(Gruber et al., 2009) was analyzed in MtNAC969 RNAi roots.
Consistently increased expression was found for two of
these markers: CORA and germin (Figure 1d). When
MtNAC969 was over-expressed, no significant change in
root length or dry weight was detected between salt-treated
and non-treated plants (Figure 2a–c). Under non-treated
conditions, MtNAC969 over-expressing roots showed an
altered development, leading to reduced growth and dry
weight. This phenotype may minimize the root surface
exposed to the stress and explain the lower salt sensitivity of
these roots compared to the wild-type. MtNAC969 overexpression was confirmed by real-time RT-PCR in the
presence or absence of salt (Figure S1B), and expression of
salt-stress markers was reduced in these roots (Figure 2d), in
contrast to the RNAi roots.
The Arabidopsis protein to which MtNAC969 is most
closely related (54% homology) is the AtNAP protein
(‘NAC-like activated by APETALA3/PISTILLATA’; Figure S2),
which has been linked to flower development (Sablowski
and Meyerowitz, 1998). However, MtNAC969 shows a
stronger expression in Medicago roots rather than flowers

(Figure S3A), in which it is up-regulated mainly by salt
among a variety of abiotic stresses (Figure S3B,C; Gruber
et al., 2009). Data available in the Medicago truncatula gene
expression atlas (MtGEA; http://mtgea.noble.org/) indicate
that this gene is also induced in response to a biotic stress
(Phymatotricum sp. infection; Figure S3C).
Overall, these results indicate that MtNAC969 negatively
regulates M. truncatula root growth and molecular
responses under salt stress.
MtNAC969 regulates lateral root formation but do
not affect nodule number
As root growth was affected by over-expression of
MtNAC969 under non-saline conditions, we analyzed the
potential role of MtNAC969 in lateral organ formation.
Using a lateral root-inducing medium (Gonzalez-Rizzo
et al., 2006), we found opposite phenotypes for
MtNAC969 over-expressing and silenced roots, i.e. a
decreased or increased density of emerged lateral roots,
respectively (Figure 3a), suggesting that MtNAC969 negatively regulates lateral root formation. We then tested
whether hormones affecting root architecture can regulate
MtNAC969 expression. This TF is rapidly induced in roots

ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 220–230

Root stress response and nodule senescence 223
Figure 2. Root phenotype in response to salt
stress of composite plants over-expressing
MtNAC969.
(a) Representative images of 3-week-old
35S::GUS and 35S::MtNAC969 composite plants
7 days after treatment with or without salt
(100 mM NaCl).
(b,c) Root length (b) and dry weight (c) of
composite plants over-expressing MtNAC969
or a GUS control grown under salt stress for
1 week, as shown in (a). A representative example of two biological experiments is shown.
Error bars represent the confidence interval
(a = 0.05), and asterisks indicate significant
differences between genotypes for a specific
condition (treated or not) based on a Mann–
Whitney test (*a < 0.05; **a < 0.01; n > 15).
(d) Expression of CORA, germin and osmotin
salt marker genes in 35S::GUS and 35S::MtNAC969
composite plants under control conditions. Bars
show quantification of specific PCR amplification products for each gene, normalized using
the mean value for three constitutive genes (as
described in Experimental procedures).
35S::GUS plant 1 was used to define fold
changes (indicated by the dashed line). Plants
from a representative example of two biological
replicates are shown, and error bars represent
the confidence interval (a = 0.05) of two technical replicates.

(a)

(b)

(c)

(d)

by cytokinins, ethylene and abscisic acid (ABA) (Figure 4a), revealing complex phytohormonal regulations. As
MtNAC969 is expressed throughout the various stages of
nodule development (Figure 4b and Figure S3C), we assessed the nodulation capacity of MtNAC969 overexpressing and RNAi-silenced roots. In contrast to the
lateral root phenotypes previously identified, no significant difference in nodule number was observed (Figure 3b).
MtNAC969 is differentially regulated by salt between roots
and nodules and behaves as a nodule senescence marker
Data from the Medicago truncatula gene expression atlas
(Figure S3C) revealed that MtNAC969 expression is strongly
induced in nodules treated with nitrate for 2 days. Indeed, a
48 h treatment with 10 mM KNO3 resulted in accumulation
of MtNAC969 transcripts (Figure 5a). In situ hybridization
also indicated that MtNAC969 expression in the central tissues of the nodule was enhanced after nitrate treatment
(Figure 4c).
To obtain a more comprehensive picture of MtNAC969
regulation by environmental cues in the two organs, we
comparatively analyzed the effect of a 48 h salt (100 mM
NaCl) or nitrate (10 mM KNO3) treatment in roots and
nodules (Figure 5). Nitrate induced MtNAC969 expression
in both organs (Figure 5a,b, left), but to a lower extent in
roots than in nodules. In addition, MtNAC969 expression
was induced by salt in roots, as expected, but was
surprisingly repressed by salt in nodules (Figure 5a,b,
left).

As nitrate treatment induces nodule senescence (Puppo
et al., 2005), we attempted to correlate the MtNAC969
expression pattern with known salt and nodule senescence
markers. The abiotic stress marker genes CORA, osmotin
and germin were similarly induced by salt stress in roots and
nodules (Figure 5a, right, and Figure 5b, middle), in contrast
to MtNAC969 (Figure 5a,b, left). We then analyzed senescence nodulation markers that are either up-regulated
during both aged and induced senescence (e.g. a putative
S-receptor kinase) or continuously up-regulated during
aged-senescence and transiently induced during darkinduced senescence (e.g. cysteine proteases; Perez Guerra
et al., 2010). Expression of the gene encoding a putative
S-receptor kinase is, as expected, strongly enhanced by
nitrate treatment compared to markers of aged-senescence
(e.g. cysteine proteases; Figure 5b, right). This further supports results showing that exogenous nitrate treatment
induces nodule senescence. In contrast to MtNAC969,
expression of the senescence markers was not detectable
in roots, with or without salt or nitrate treatment (data not
shown). Interestingly, salt treatment in nodules inhibited
expression of these senescence markers, as observed for
MtNAC969 (Figure 5b, left and right).
Altogether, these results show that this TF acts in
different environmentally dependent regulatory pathways
depending on the organ studied. Indeed, the differential
expression of MtNAC969 between roots and nodules
defines a new pattern, similar to that of salt stressresponsive markers in roots and senescence-related markers in nodules.

ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 220–230

224 Axel de Ze´licourt et al.

(a)

(a)

(b)

(b)

(c)
Figure 3. Root lateral organogenesis of composite plants in which MtNAC969
was over-expressed or silenced.
(a) Lateral root density (number of lateral roots per cm parental root) for
composite plants in which MtNAC969 was over-expressed or silenced
(normalized relative to 35S::GUS or GUS RNAi constructs, respectively, as
indicated by the dashed line) measured 5 days after transfer to lateral rootinducing medium.
(b) Nodule number in composite plants in which MtNAC969 was overexpressed or silenced (normalized relative to 35S::GUS or GUS RNAi
constructs, respectively, as indicated by the dashed line) determined 21 days
post-inoculation with S. meliloti Sm1021. In both cases, a representative
example of two biological experiments is shown (n > 20). Error bars represent
the confidence interval (a = 0.05), and asterisks indicate significant differences relative to the GUS control based on a Mann–Whitney test (*a < 0.05;
**a < 0.01; n > 20).

MtNAC969 RNAi nodules show premature senescence
To further assess the potential role for NAC969 in nodule
senescence, we characterized the histology of differentiated
MtNAC969 RNAi nodules. This revealed the presence of
infection threads as well as infected cells, with elongated
bacteroids radiating from a central vacuole as observed in
control nodules (Figure 6a,b). However, cells located in the
nitrogen-fixing zone of the MtNAC969 RNAi nodules accumulated a higher number of amyloplasts than those from
wild-type nodules, as shown by lugol staining (Figure 6c,d)
or electron microscopy (Figure 6e,f), suggesting an altered
nodule metabolism. We then analyzed expression of the
various senescence nodulation markers previously used
(Figure 7). In accordance with the cellular phenotype, two
cysteine protease-encoding markers of nodule aged-senescence accumulate in MtNAC969 RNAi nodules, indicating
the premature senescence of these nodules. These results

Figure 4. Regulation of MtNAC969 expression by phytohormones and in
symbiotic nodules.
(a) MtNAC969 expression in roots after short-term (1 or 3 h) hormonal
treatments: auxin (10)7 M IAA), cytokinin (10)7 M BAP), ethylene (10)7 M ACC)
and abscisic acid (10)7 M ABA).
(b) Expression of MtNAC969 during a nodulation kinetic (from 0 to 40 days
post-inoculation with S. meliloti Sm1021). The non-inoculated root (0 dpi)
was used to define fold changes (indicated by the dashed line).
In (a) and (b), bars show quantification of specific PCR amplification products
for each gene, normalized using the mean value for three constitutive genes
(as described in Experimental procedures). Fold changes are indicated by the
dashed line relative to the non-treated condition. A representative example of
two biological replicates is shown, and error bars represent the confidence
interval (a = 0.05) of two technical replicates.
(c) Analysis of MtNAC969 spatial expression using in situ hybridization in
nodules treated with 10 mM KNO3 or not for 48 h. Left: MtNAC969 sense
probe (negative control); right and middle: MtNAC969 antisense probe in
nodules treated with KNO3 or not, respectively. Scale bars = 500 lm.

therefore indicate that the MtNAC969 TF is required for
establishment of a functional nitrogen-fixing nodule, and
may negatively regulate nodule senescence.

ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 220–230

Root stress response and nodule senescence 225

(a)

(b)

Figure 5. Expression of MtNAC969 in roots or nodules treated with salt or nitrate.
(a) Expression of MtNAC969 (left) and salt response marker genes (CORA, germin and osmotin; right) in roots treated with salt (100 mM NaCl) or nitrate (10 mM
KNO3) for 48 h.
(b) Expression of MtNAC969 (left), salt response marker genes (CORA, germin and osmotin; middle) and senescence marker genes encoding a putative receptor
kinase and cysteine proteases (MtCP2 and MtCP3) (right) in nodules treated with salt (100 mM NaCl) or nitrate (10 mM KNO3) for 48 h.
In (a) and (b), bars show quantification of specific PCR amplification products for each gene, normalized using the mean value for three constitutive genes (as
described in Experimental procedures). Fold changes are indicated by the dashed line relative to non-treated conditions. A representative example of two biological
experiments is shown, and error bars represent the confidence interval (a = 0.05).

DISCUSSION
In this study, we characterized a NAC transcription factor
that is rapidly induced by salt stress and various plant
hormones and plays a role in the regulation of root architecture and symbiotic nodulation in Medicago truncatula.
MtNAC969 ectopic expression and inactivation by a RNAi
approach affected lateral root formation, as well as salt
stress response. Accordingly, expression of salt-stress
markers was regulated in opposite ways in these roots. In
addition, MtNAC969 down-regulation led to formation of
nodules with an altered development, characterized by
aberrant amyloplast accumulation and de-regulation of
senescence markers. These results reveal the dual involvement of this TF in root system adaptation to abiotic stress
and in symbiotic nodule senescence. Nodules and roots
may then use related TFs for various adaptive developmental responses depending on environmental conditions.
Ectopic or down-regulation of MtNAC969 expression led
to changes in root system architecture. MtNAC969 overexpression reduced lateral root numbers, but RNAi roots
showed increased lateral root density (Figure 3a). In addition to the root growth defect (Figure 2b), this finding
probably explains the reduced dry weight of roots overexpressing MtNAC969 compared to control plants
(Figure 2c). MtNAC969 thus acts as a negative regulator of
lateral root formation. Interestingly, MtNAC969 expression

in the roots is rapidly affected by various phytohormone
treatments, being induced by cytokinins, ethylene and
ABA (Figure 4a). In Arabidopsis, these hormones are
predicted to negatively regulate lateral root formation
(Fukaki and Tasaka, 2009; Perilli et al., 2010), in contrast to
auxin, which is linked to the promotion of root growth and
does not up-regulate MtNAC969 expression. Hormonal
regulation of the MtNAC969 expression is therefore consistent with the root phenotypes observed when the gene is
down-regulated. However, hormones that induce
MtNAC969 expression play either negative or positive roles
in nodule initiation (Gonzalez-Rizzo et al., 2009), and interestingly no effect on nodule number was observed in
MtNAC969 RNAi roots. MtNAC969 appears to function at
the crossroads of several hormonal pathways controlling
root growth and architecture.
The closest homolog of MtNAC969 in Arabidopsis is
AtNAP/ANAC029. This gene was initially linked to floral and
stamen development that is dependent on APETALA3 and
PISTILLATA TFs (Sablowski and Meyerowitz, 1998). AtNAP
was later shown to be induced by salt and slightly regulated
by other abiotic stresses (Jensen et al., 2010), as is the case
of MtNAC969. With regard to hormonal induction, AtNAP is
induced by ethylene and ABA (Guo and Gan, 2006), and the
latter hormone also regulates many other NAC genes
(Jensen et al., 2010). However, no NAP function in root
development or any abiotic stress response has been

ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 220–230

226 Axel de Ze´licourt et al.

(a)

(b)

(c)

(d)

(e)

Figure 7. MtNAC969 RNAi nodules accumulate senescence markers. Expression of senescence marker genes encoding a putative receptor kinase and
cysteine proteases (MtCP2 and MtCP3) in GUS RNAi and MtNAC969 RNAi
roots under control conditions. The GUS RNAi nodules sample 1 (Nod 1) was
used to define fold changes (indicated by the dashed line). Bars show
quantification of specific PCR amplification products for each gene, normalized using the mean value for three constitutive genes (as described in
Experimental procedures). A representative example of two biological
experiments is shown, and error bars represent the confidence interval
(a = 0.05).

(f)

Figure 6. Comparison of the ultrastructural organization of GUS RNAi and
MtNAC969 RNAi nodules.
(a,b) Toluidine blue-stained sections of GUS RNAi (a) and MtNAC969 RNAi (b)
nodules 21 days post-inoculation. Zones I (meristem), II (infection/differentiation) and III (nitrogen-fixing) are defined as described by Vasse et al. (1990).
Scale bars = 500 lm.
(c,d) Lugol staining of starch granules in GUS RNAi (c) and MtNAC969 RNAi
(d) nodules 21 days post-inoculation. Starch accumulation, used as a marker
for nodule differentiation, is enhanced in the nitrogen-fixing zone of
MtNAC969 RNAi nodules. Scale bars = 500 lm.
(e,f) Electron micrographs of longitudinal ultrathin sections of GUS RNAi (e)
and MtNAC969 RNAi (f) nodule cells from the nitrogen-fixing zone, as outlined
in red in (a) and (b), 21 days post-inoculation. A high number of amyloplasts
(asterisks) are found in MtNAC969 RNAi cells. Scale bars = 5 lm.

identified in Arabidopsis. Over-expression of the closest rice
MtNAC969 homolog, OsNAC10, induced drought tolerance
and a modified root system under control conditions (Jeong
et al., 2010). Similarly, over-expression of MtNAC969 modified the M. truncatula root system architecture, which may
allow plants to maintain a higher root mass under salt stress.
Additionally, roots over-expressing or down-regulating
MtNAC969 showed decreased or increased expression of
salt-stress markers, respectively. This suggests that
MtNAC969 represses root responses to salt. In Arabidopsis,
a few TFs transcriptionally induced by ABA and abiotic
stresses (such as EARLY RESPONSIVE TO DEHYDRATION 15/ERD15) have been described as acting as negative
regulators of the stress response (Kariola et al., 2006). These
TFs may act in a stress attenuation response, preventing or

modulating the stress response, or in a negative feedback
mechanism to adapt root responses (and their ABA sensitivity) to the intensity or duration of the stress.
By comparing the expression of MtNAC969 in roots and
nodules under salt stress, we identified an unexpected
differential behavior of this TF depending on the organ
studied (Figure 5). This gene thus behaves as a stressresponsive marker in roots and as an environmentally
induced senescence marker in nodules, revealing interactions between nodule senescence and root abiotic stress
responses. Several genes have already been described as
stress-responsive and senescence-related, suggesting certain common pathways (Puppo et al., 2005). However,
recent results obtained in M. truncatula indicate that
developmentally programmed nodule senescence and the
senescence induced in response to environmental signals,
such as dark or nitrate, are more divergent than anticipated,
as approximately 50% of the genes associated with these
processes are differentially regulated (Perez Guerra et al.,
2010). Comparable results were obtained for Arabidopsis
leaf senescence, one of the best documented senescence
processes (Lim et al., 2007), in which genes regulated
during developmental or dark-induced senescence only
partly overlap (Buchanan-Wollaston et al., 2005). NACs
were the largest TF family regulated during leaf senescence
(one-fifth of the family, i.e. 20 members; Guo et al., 2004).
This includes AtNAP, and expression of this TF induced
premature leaf senescence and activated the senescenceassociated marker genes SAG12 and SAG13 (Guo and
Gan, 2006). Conversely, atnap mutants show delayed

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Root stress response and nodule senescence 227
developmental leaf senescence. A similar phenotype was
recently reported for the atnac2 mutants, and delayed darkinduced senescence was observed in detached leaves
(Balazadeh et al., 2010). The AtNAC2/ORE1/ANAC092 TF is
salt-induced, and promotes stress-induced senescence and
activates a large number of SAG genes (i.e. approximately
one-third of the 170 reported SAG genes; Balazadeh et al.,
2010). In addition, using the Leaf Senescence Database
(http://www.eplantsenescence.org/), expression of 30 NAC
genes was found to be associated with leaf senescence,
including AtNAM/anac018/At1g52880, ANAC055/At3g15500,
ANAC019/At1g52890 and AtATAF1/anac002/At1g01720, and
these are included in the phylogenetic tree shown in
Figure S2A. Interestingly, of the 827 and 445 genes
involved in Arabidopsis and Medicago leaf senescence,
respectively, MtNAC969 and its closest homolog AtNAP are
among 150 genes showing an evolutionarily conserved
senescence expression pattern (De Michele et al., 2009).
Surprisingly, however, only one NAC-encoding gene was
identified in transcriptomic studies that aimed to characterize nodule senescence in M. truncatula (Van de Velde
et al., 2006). This TF shows only 35% homology with
MtNAC969, and its closest Arabidopsis homolog FEZ/
ANAC009/At1g26870 has not been linked to senescence
(Willemsen et al., 2008). However, this low representation
of NAC TFs in nodule senescence may be due to the
reduced size of the transcriptome assayed in these studies
(approximately 15 000 gene tags defined using cDNA-AFLP
[amplified fragment length polymorphism]; Van de Velde
et al., 2006).
In MtNAC969 RNAi nodules, a late symbiotic nodulation
phenotype was identified, consisting of aberrant accumulation of amyloplasts and premature expression of senescence markers (Figures 6 and 7). Interestingly, several
studies using bacteria defective in nitrogen fixation previously linked accumulation of amyloplasts to premature
senescence (Lodwig et al., 2003; Harrison et al., 2005).
Functional characterization of genes acting in nodule senescence remains very limited. Antisense plants with disrupted
expression of MtZPT2-1, which encodes a C2H2 zinc finger
TF, developed non-functional nodules that accumulated
amyloplasts (Frugier et al., 2000). This gene is regulated by
salt stress in Medicago roots in a similar way to MtNAC969,
and roots over-expressing this TF showed better growth
under salt stress (de Lorenzo et al., 2007). The MtZPT2-1
pathway therefore also links stress responses to differentiation and functionality of symbiotic nodules.
Overall, this study shows that the MtNAC969 TF characterized in this study regulates both root architecture and
symbiotic nodulation, and links root abiotic stress responses
to symbiotic nodule senescence. The divergent transcriptional regulation of MtNAC969 by abiotic signals in roots
versus nodules further highlights the complexity of environmental responses in the legume below-ground system.

EXPERIMENTAL PROCEDURES
Plant material and treatments
Medicago truncatula Jemalong A17 seeds were sterilized as described by Gonzalez-Rizzo et al. (2006), and stratified at 4 C for
3 days to ensure uniform germination.
For hormonal treatments, 15 germinated seedlings were placed
in a Magenta box with 30 mL of low-nitrogen ‘i’ medium and grown
in a shaking incubator (125 rpm) at 24 C under long-day conditions
(16 h light/8 h dark) as described by Gonzalez-Rizzo et al. (2006).
After 5 days, seedlings were treated with 10)7 M indole-3-acetic acid
(IAA), 10)7 M 1-aminocyclopropane-1-carboxylic acid (ACC), 10)7 M
benzylaminopurine (BAP) or 10)7 M abscisic acid (ABA) (all obtained
from Sigma-Aldrich, http://www.sigmaaldrich.com/), and maintained under the same growth conditions for various incubation
times (0, 1 and 3 h). Bioactive hormonal conditions were defined
based on previous studies (Gonzalez-Rizzo et al., 2006; Ariel et al.,
2010; Plet et al., 2011), and M. truncatula root physiological and
molecular responses were tested for a range of concentrations and
time points.
For abiotic stresses analysis, plants were similarly grown and
then treated for 1 h with 100 mM NaCl or 200 mM mannitol (i.e. an
osmolarity of 200 mOsmol in each case) or kept for 1 h at 4 or 37 C.
For nodulation experiments, plants were transferred to the
greenhouse (16 h light/8 h dark, 22 C, 60–70% relative humidity)
in pots containing perlite/sand (3:1 v/v), and irrigated with ‘i’
medium (Gonzalez-Rizzo et al., 2006). After 5 days, roots were
inoculated with 150 mL of Sinorhizobium meliloti Sm1021 suspension (OD600 nm = 0.05) per pot. Twenty-one days post-inoculation
(dpi), plants were treated with 100 mM NaCl or 10 mM KNO3 for 48 h
(based on results available in the M. truncatula gene expression
atlas; Benedito et al., 2008). Uninoculated plants were submitted to
the same treatments. The early nodulation stages (i.e. 1, 3 and 8 dpi)
of the nodulation kinetics were determined in vitro as described by
Laporte et al. (2010).
In all cases, two or three biological replicates were performed per
treatment.

Agrobacterium rhizogenes root transformation
To knockdown MtNAC969 expression using RNA interference, a
specific region was amplified by PCR using the primers NAC969RNAi-F (5¢-CACCTTTCATCCAACTGATGAGGAAC-3¢) and NAC969RNAi-R (5¢-CTTTGATCCACTTTTGATTGCC-3¢), and cloned into the
pFRN destination vector (derived from pFGC5941; NCBI accession
number AY310901) using Gateway technology (Invitrogen, http://
www.invitrogen.com/). A pFRN vector that includes the GUS
reporter gene was used as a control (Gonzalez-Rizzo et al., 2006).
To ectopically express MtNAC969, a full-length cDNA was
amplified from salt-treated M. truncatula roots using primers
NAC969-FL-F (5¢-GCTCTAGAGAAGGAGATAGAACCATGGAGAGT
AGTGCAAG-3¢) and NAC969-FL-R (5¢-GGGGTACCTATGCTAAAT
AATTATGCATTTGTTTCAC-3¢). The MtNAC969 sequence was submitted to the National Center for Biotechnology Information database (accession number JN833713). After digestion with XbaI and
KpnI (New England Biolabs, http://www.neb.com/; restriction sites
underlined in primers described above), fragments were cloned into
the binary vector pMF2 (containing a 35S CaMV promoter; Merchan
et al., 2007). An empty pMF2 vector expressing the GUS reporter
was used as control (35S:GUS).
All constructs were introduced into Agrobacterium rhizogenes
ARqua1 strain (an SmR-derivative strain of A4T; Quandt et al., 1993)
and used for M. truncatula root transformation as described by
Boisson-Dernier et al. (2001). The ‘composite plants’ obtained

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228 Axel de Ze´licourt et al.
consist of wild-type shoots and transgenic roots, and each plant
therefore corresponds to at least one independent transformation
event. The efficiency of silencing and specificity of the RNAi were
checked for each construct using real-time RT-PCR on several
representative individual clones (n > 3).

Treatment of Agrobacterium rhizogenes composite plants
For salt treatment, composite plants were transferred to the
greenhouse (16 h light/8 h dark, 22 C, 60–70% relative humidity) in
pots containing perlite/sand (3:1 v/v), and irrigated with ‘i’ medium
for 1 week. Plants were then supplemented with or without 100 mM
NaCl for 1 week. Root length was measured using ImageJ software
(http://rsbweb.nih.gov/ij/), and aerial parts and roots were then
individually dried at 60 C to determine their dry weight. Three
independent biological replicates were performed (n > 12 plants for
each construct and condition).
To determine lateral root number, composite plants were
transferred in vitro onto growth papers (Mega International,
http://www.duclosinternational.eu/) on SN/2 medium (Soluplant
18.6.26, Duclos International) with 0.9% Kalys agar (Kalys HP696,
http://en.kalys.com/) for 3 days. Plants were then transferred to
lateral root-inducing medium to induce synchronously lateral root
development (Gonzalez-Rizzo et al., 2006). The position of root
tips was marked (with a pencil) at the time of transfer. Primary
root length and lateral root number were measured from this
mark after 5 days using ImageJ software, and lateral root density
was then calculated (i.e. lateral root number/cm main root length).
Two biological experiments were performed, and a minimum of
20 independent plants per construct and per condition was
measured.
For nodulation assays, transgenic roots were transferred to the
greenhouse (16 h light/8 h dark, 22 C, 60–70% relative humidity) in
pots containing perlite/sand (3:1 v/v) and irrigated with ‘i’ medium
for 5 days. Transgenic roots were inoculated with 150 mL of
Sinorhizobium meliloti Sm1021 suspension (OD600 nm = 0.05). Nodulation was evaluated at 21 dpi, and nodules were collected for
histological analysis or harvested in liquid nitrogen for molecular
assays.

Gene expression analysis
Total RNAs were isolated from belowground organs by phenol/
chloroform extraction of samples ground to a fine powder and
allowed to thaw into five volumes of a buffer containing 0.1 M NaCl,
2% SDS, 50 mM Tris/HCl, pH 9.0, 10 mM EDTA and 20 mM b-mercaptoethanol. Total RNA (1 lg) was then subjected to DNase I
treatment (Fermentas, http://www.fermentas.com/), and first-strand
cDNAs were synthesized using the Superscript II first-strand synthesis system (Invitrogen). Primers were designed using Primer3
software (http://frodo.wi.mit.edu/primer3/). Primer combinations
showing a minimum amplification efficiency of 85% were used in
real-time RT-PCR experiments (Table S1), and specificity was
checked using a dissociation curve. Real-time RT-CRs were performed on a Roche LightCycler480 using Roche reagents (http://
www.roche.com). Two independent biological experiments and
technical replicates (based on two independent cDNA syntheses
derived from the same RNA sample) were performed in all cases.
Three genes were defined as reference genes using GeNorm software (http://medgen.ugent.be/~jvdesomp/genorm/): MtACT (Actin 12), MtH3L (Histone 3-Like) and MtRBP1 (RNA Binding
Protein 1). Gene fold changes were calculated using the mean
expression of the three genes calculated using LightCycler480
software. The experimental control conditions were used to determine expression fold changes.

In situ hybridization
In situ hybridizations were performed as described by Boualem
et al. (2008) on 21 dpi nodules using an Intavis InsituPro automat
(http://www.intavis.com/en/). RNA probes were marked by digoxygenin (DIG) and revealed using an antibody coupled to alkaline
phosphatase. An antisense RNA probe corresponding to a carbonic
anhydrase gene (MtCA1) was included as a positive control (Boualem et al., 2008). An antisense probe designed to target specifically
MtNAC969 transcripts (i.e. with <70% identity with other known
Medicago genes) was designed in addition to a corresponding
sense probe as a negative control. Primers used to generate
MtNAC969 probes are listed in Table S1.

Bioinformatic and phylogenetic analyses
The amino acid sequences of Medicago NAC genes were obtained
based on the M. truncatula Gene Index database (MtGI version 8,
http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=medicago). To construct the phylogenetic tree, the three NAC TFs previously identified as salt-induced in roots (Gruber et al., 2009)
[MtNAC969 (TC96130), MtNAC1081 (TC80936) and MtNAC1126
(NP7267109)] were included, as well as TC108627, the Medicago
sequence most closely related to MtNAC969 (based on a nucleotide
BLAST search on MtGI version 8). Correspondence between various Medicago resources (mainly MtGI, MtGEA, 16K+ arrays and the
Medicago genome) was established using the Legoo ‘nickname’
tool (http://www.legoo.org/). Several A. thaliana NAC proteins
functionally characterized in relation to abiotic stresses or most
closely related to MtNAC969 were also included: AtNAC2 (or ORE1,
Oresara1/anac092/At5g39610), AtNAP (NAC-like activated by
APETALA3/PISTILLATA, anac029/At1g69490), AtATAF1 (anac002/
At1g01720), ATAF2 (anac081/At5g08790), AtNAM (No Apical Meristem, anac018/At1g52880), TIP (TCV-interacting protein; anac091/
At5g24590), ANAC055 (At3g15500), CUC1 (CUP-SHAPED COTYLEDON1; anac054/At3g15170), CUC2 (anac098/At5g53950), CUC3
(anac031/At1g76420), NAC1 (anac022/At1g56010) and NAC2
(anac078/At5g04410). Two rice NAC peptide sequences functionally
characterized in relation to abiotic stress were also used for this
phylogenetic tree: OsNAC6 (onac048/AK068392) and OsNAC10
(AK069257).
Full-length amino acid sequences of selected NAC proteins were
aligned using ClustalW (http://clustalw.genome.ad.jp/) with default
parameter values, and analyzed using MEGA4 software version
4.0.2 (http://www.megasoftware.net/). The evolutionary history was
inferred using the neighbor-joining method. The tree was drawn to
scale, with branch lengths in the same units as those for the
evolutionary distances used to create the phylogenetic tree. The
evolutionary distances were computed using the Poisson correction
method, and are in the units of the number of amino acid
substitutions per site. All positions containing gaps and missing
data were eliminated from the dataset.

Histological analysis of nodules
For amyloplast detection, 21-day-old nodules were collected from
composite plants silenced for MtNAC969 or GUS genes and
embedded in 6% agarose (Eurobio, http://www.eurobio.fr/). Sections (100 lm) of nodules cut using a Vibratome VT1200S (Leica,
http://www.leica.com/) were directly stained with lugol (Fluka, http://
www.sigmaaldrich.com/) and observed using a Reichert Polyvar
microscope equipped (http://www.reichert.com/) with a Nikon digital DXM1200 camera (http://www.nikon.com/).
For transmission electronic microscopy, 14 dpi nodules were
processed as described by Laporte et al. (2010) using conventional

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The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 220–230

Root stress response and nodule senescence 229
methods described by Hawes and Satiat-Jeunemaitre (2001), except
that the osmium post-fixation step was replaced by a mixture of 1%
osmium and 1.5% potassium ferrocyanide (Aubert et al., 2011).
Specimens were embedded in epoxy resin (agar low-viscosity
premix kit medium, Oxford Instruments, http://www.oxinst.com/)
and polymerized for 16 h at 60 C. Thick sections (300 nm), obtained
using a Leica Ultracut UC6 ultramicrotome, were collected on a
glass slide and stained with toluidine blue for 1 min. Samples were
observed using a Nikon AZ100 macroscope. Ultrathin sections
(70–90 nm) were counterstained using aqueous 2% uranyl acetate
and lead citrate (Hawes and Satiat-Jeunemaitre, 2001). Grids were
examined using a JEOL (http://www.jeol.com/) 1400 transmission
electronic microscope operating at 120 kV. Images were acquired
using a post-column high-resolution (11 megapixels) camera
(SC1000 Orius, Gatan Inc., http://www.gatan.com/).

ACCESSION NUMBER
The NCBI accession number for the MtNAC969 sequence is
JN833713.

ACKNOWLEDGMENTS
We thank Julie Plet and Philippe Laporte for providing cDNAs,
Alexis Canette for his technical assistance in electron microscopy,
Emmanuelle Dheilly for help with real-time RT-PCR experiments,
and Fabien Marcel for help with composite plant experiments. This
work used the imaging facilities of the Imagif platform (Cell Biology
Unit, Centre de Recherche de Gif sur Yvette, France; http://
www.imagif.cnrs.fr), supported by the Conseil Ge´ne´ral de l’Essonne. O.Z. was supported by a grant from the Tunisian Government
(Tunis). This work was partly supported by LEGUROOT, a French
Agence Nationale de la Recherche project.

SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Expression analysis of MtNAC969 and related M. truncatula NAC genes in composite plants in which MtNAC969 was overexpressed or down-regulated.
Figure S2. Phylogenetic tree of selected NAC proteins and
MtNAC969/AtNAP sequence comparison.
Figure S3. MtNAC969 gene expression profiles based on the
Medicago truncatula gene expression atlas (MtGEA).
Table S1. List of primers used in this study.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.

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ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 220–230


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