Mol. Cells 29, 1-10, January 31, 2010
Identification of an Arabidopsis Nodulin-Related
Protein in Heat Stress
Qiantang Fu1,2, Shujia Li1, and Diqiu Yu1,*
We identified a Nodulin-related protein 1 (NRP1) encoded
by At2g03440, which was previously reported to be RPS2
interacting protein in yeast-two-hybrid assay. Northern
blotting showed that AtNRP1 expression was suppressed
by heat stress (42°C) and induced by low temperature
(4°C) treatment. Strong GUS staining was observed in the
sites of meristematic tissues of pAtNRP1:: GUS transgenic
plants, such as shoot apex and root tips, young leaf veins,
stamens and stigmas of flowers, and abscission layers of
young siliques. To study AtNRP1 biological functions, we
have characterized both loss-of-function T-DNA insertion
and transgenic overexpression plants for AtNRP1 in
Arabidopsis. The T-DNA insertion mutants displayed no
obvious difference as compared to wild-type Arabidopsis
under heat stress, but the significant enhanced suscepti-
bility to heat stress was revealed in two independent
AtNRP1-overexpressing transgenic lines. Further study
found that the decreased thermtolerance in AtNRP1-
overexpressing lines accompanied significantly decreased
accumulation of ABA after heat treatment, which was
probably due to AtNRP1 playing a role in negative-feedback
regulation of the ABA synthesis pathway. These results
support the viewpoint that the application of ABA inhibits
nodulation and nodulin-related gene expression and threaten
adverse ambient temperature can impact the nodulin-related
Plants are exposed to various environmental stresses during
their growth and development. Among these stresses, high
temperature is one of the major problems that limit the growth
and distribution of plants (Boyer, 1982). On the other hand,
plants have evolved various mechanisms for adapting to the
effects of heat shock (HS). Accumulation of heat shock proteins
(HSPs), membrane compositional changes necessary for main-
tenance of functional integrity, and activation of oxidative de-
fensive systems are involved in improving plant thermotoler-
ance (Kaplan et al., 2004; Kotak et al., 2007b; Locato et al.,
2008; Queitsch et al., 2000; Sung et al., 2001). Transcription
activation of HSPs genes is regulated by heat shock transcrip-
tion factors (HSFs) through binding to heat shock promoter
elements (HSEs) in the promoter regions of HSPs genes during
heat stress (Baniwal et al., 2004; Yamamoto et al., 2005). In
addition, the induction of abscisic acid (ABA), salicylic acid (SA),
and calcium-based signaling pathways were reported to be
involved in heat-stress adaptation (Clarke et al., 2004; Larkin-
dale and Knight, 2002; Larkindale et al., 2005; Liu et al., 2008).
Exogenous application of these signaling agents to plants can
also result in some degree of enhanced thermotolerance
(Larkindale and Knight, 2002). These multiple responses sug-
gest that many alternative processes are involved in thermotol-
erance. Plants at various growth stages respond differently to
heat stress, suggesting a link between development and
thermotolerance (Hong et al., 2003).
The infection of the plant by rhizobia bacteria is a complex
process which a number of plant genes take part in. Legumes
form a specialized organ termed the nodule via the expression
of their genes that encode proteins named ‘nodulins’ when the
leguminous plants were elicited by the secretion of bacterial
effectors called nod factors in their root hairs (Stougaard, 2000).
For non-nodule plants, Arabidopsis, when the plant roots have
been colonized by several specific strains of Pseudomonas
spp., they would develop a protective defense response that is
called rhizobacteria-mediated induced systemic resistance
(Cartieaux et al., 2003). Recent study has indicated that
abscisic acid (ABA) can not only inhibit Nod factor signal trans-
duction and promote or suppress resistance against various
pathogens (de Torres-Zabala et al., 2007; Ding et al., 2008;
Mohr and Cahill, 2007), but also can break a new signaling
pathway to heat stress (Larkindale and Huang, 2004; Larkin-
dale et al., 2005). A transient peak in ABA levels was reported
in response to HS in pea plants (Liu et al., 2006) and during
recovery from HS treatments in creeping bentgrass (Larkindale
and Huang, 2004). It has been reported that ABA induces
thermotolerance in cell-suspension cultures of Bromus inermis
Leyss. (Robertson et al., 1994), and ABA pretreatment can
increase cell viability and growth upon HS (Zhang and Fe-
vereiro, 2007). In addition, the overexpression of transcription
factor ABF3 in ABA signaling induced high-temperature toler-
ance in transgenic Arabidopsis (Kim et al., 2004). These results
suggested that ABA could also be involved in the response to
heat stress. Moreover, genotypes with a putative high ABA
1Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming 650223, People’s
Republic of China, 2Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
Received August 14, 2009; revised September 24, 2009; accepted September 25, 2009; published online December 7, 2009
Keywords: abscisic acid (ABA), heat stress, nodulin-related protein 1, thermotolerance
2 A Nodulin-Related Protein in Heat Stress
level may be more tolerant to stresses (Xiong and Zhu, 2003).
The Arabidopsis ABA signaling mutants abscisic acid insensi-
tive 1 (abi1) and abi2 showed reduced survival after HS, but the
accumulation of HSPs was not affected in these mutants, and
ABA does not appear to be required for HSP synthesis during
HS (Larkindale et al., 2005). So it seems that ABA related heat
tolerance may be independent on heat shock proteins synthe-
Nodulin-related protein 1, encoded by At2g03440, is a gene
of unknown function encoding a putative mature protein of 187
amino acids. Bioinformatics analysis revealed that the amino acid
sequence of the nodulin-related protein has some similarity to
early nodulin 12B precursor (Bauer, 1994), and there are 41.3%
identity (63.0% similarity) in proline-rich domain of the nodulin-
related protein 1 and alfalfa early nodulin 12B precursor (see
Supplementary Fig. 1), we designated the protein ‘AtNRP1’. It
has been demonstrated that AtNRP1 was an especially strong
interactor of RPS2 (recognition of P. syringae strains) in yeast-
two-hybrid assay (Quirino et al., 2004), and a recent study
showed that AtNRP1 was strongly up-regulated in Capsicum
annuum hypersensitive induced reaction gene (CaHIR1)-
overexpressing transgenic Arabidopsis, and was also differen-
tially induced at the transcriptional level by infection of Pseu-
domonas syringae pv. tomato DC3000 (Jung et al., 2008).
However, the specific roles of AtNRP1 in Arabidopsis have not
been determined so far.
In this study, we cloned AtNRP1 cDNA using a temperature
screen. AtNRP1 expression was significantly suppressed by heat
stress and induced by low temperature treatment. Histochemical
analysis in pAtNRP1::GUS transgenic plants showed that
AtNRP1 expressesed mainly in the apical meristematic tissues.
The AtNRP1-overexpressing transgenic plants showed de-
creased thermotolerance, but the expression of some known
heat-induced genes, such as HsfA2, MBF1c, Hsp101 and Hsp70,
had no significant difference compared with wild-type plants un-
der heat stress. Further study found that AtNRP1 was an ABA
regulated gene, and the ABA accumulation in AtNRP1-over-
expressing transgenic plants was lower than that of WT and nrp1
under heat stress. These results showed AtNRP1 might play a
role in thermotolerance through the ABA synthesis pathway.
MATERIALS AND METHODS
Plant materials and growth conditions
The Arabidopsis thaliana ecotype Columbia was used as the
WT for experiments in this study. The Arabidopsis seeds (WT,
nrp1, and AtNRP1-overexpressing transgenic plants) were
surface sterilized for 15 min in 20% (v/v) bleach, and rinsed
twice with sterile distilled water. Seeds were grown on 1/2 Mu-
rashige and Skoog (MS) solid medium with 0.7% (w/v) agar
and 1.5% sucrose. After 3 days stratification in darkness at 4°C,
seeds were transferred to plant incubators with a 12 h-light (100
μE m-2 s-1) / 12 h-dark photoperiod at 22°C. The seedlings were
either grown in axenic culture on 1/2 MS medium or transferred
into soil in the greenhouse (22°C with a 12 h photoperiod) 7 d
after germination on 1/2 MS medium.
Isolation of T-DNA insertion mutants
The AtNRP1 T-DNA insertion line (SALK_111768), in the Col
background, was obtained from the Arabidopsis Biological Re-
source Center (ABRC; Ohio State University, USA). The T-
DNA insertion sites were confirmed by PCR using a T-DNA
border primer (5′-AAACGTCCGCAATGTGTTAT-3′) and the
gene-specific primer (5′-ACGAGTCAGACAAGCTTGACA-3′).
Plants homozygous for the T-DNA insertion were identified by
another PCR using the above gene-specific primer and gene-
specific reverse primer (5′-TTTGCCAAAACCTAAAAATTA-
TCA-3′) and Northern blot analysis.
Construction of AtNRP1-overexpressing transgenic plants
The full-length cDNA of AtNRP1 (U22696) was obtained from
ABRC and confirmed by sequence analysis. To generate the
35S::AtNRP1 construction, the full-length cDNA fragment of
AtNRP1 was cloned into the BamHI and SalI sites of pOCA30
(Chen and Chen, 2002). The recombinant plasmid was intro-
duced into Agrobacterium tumefaciens strain GV3101 and
followed by transformation into wild-type Arabidopsis plants by
the floral dip method (Clough and Bent, 1998). The seeds were
collected from the A. tumefaciens-infected plants and selected
on 1/2MS agar medium containing 50 μg ml-1 kanamycin.
Northern blot analysis was performed to further identify trans-
genic plants. T3 homozygous AtNRP1-overexpressing trans-
genic plants were selected for further experiments.
Heat treatments assays
Thermotolerance assays of seven-day-old normal-grown and
2.5-day-old dark-grown seedlings were performed as described
by Larkindale et al. (2005) with minor modifications. Seven-day-
old seedlings were exposed to a 45°C HS for 260 min after a
pretreatment for 90 min at 38°C and 120 min at 22°C, and then
returned to 22°C to recover for 7 d. Seedlings were photo-
graphed to record viability, and the seedlings that were still
green and producing new leaves were scored as surviving. The
survival rate was the ratio of surviving seedlings to total treated
seedlings. For the hypocotyl elongation assay, 2.5-day-old
dark-grown seedlings on vertical plates were heat-treated at
45°C for 180 or 220 min after a pretreatment for 90 min at 38°C
and 120 min at 22°C. After an additional 2.5 days growing in
the dark, seedlings were measured and photographed. In these
assays, nrp1 and AtNRP1-overexpressing transgenic plants
were compared with WT plants growing on the same plate to
ensure the compared plants received identical stress conditions.
Three-week-old WT, nrp1 and AtNRP1-overexpressing trans-
genic plants cultured in soil were heat treated at 42°C for 0 min,
30 min, or 90 min for northern blot analysis.
Electrolyte leakage assays
To explore the difference in thermotolerance of adult plants,
leaves subjected to heat stress were assayed for electrolyte
leakage according to methods of Clark et al. (2004). The plants
were cultured in soil for 3 weeks, and the 3rd to 5th leaves were
used for electrolyte leakage tests. The leaves were placed in
glass tubes containing 10 ml distilled water and were incubated
in the water bath at 42°C in the dark. Water conductance was
measured at intervals during heat treatment and calculated per
Quantification of ABA content
The ABA content was assayed using enzyme-linked immu-
nosorbent assays (ELISA) as previously described (Yang et al.,
2001) Three-week-old seedlings were treated at 38°C for 0.5, 1
and 2 h. Approximately 0.5 g tissue samples were frozen in
liquid nitrogen and ground to a fine powder. Endogenous ABA
was extracted and the content analyzed by ELISA.
RNA isolation and gel blotting
Total RNA was extracted from three-week-old Arabidopsis
plants using the TRIZOL reagent (Invitrogen). For RNA gel blot
analysis, total RNA (10 μg) was separated on 1.2% agarose-
formaldehyde gels and blotted onto nylon membranes. Blots
Qiantang Fu et al. 3
Fig. 1. Expression analysis of AtNRP1. Expression of AtNRP1 in
response to (A) 42°C and (B) 4°C. Each lane was loaded with 10
µg total RNA prepared from 3-week-old plants treated with the
various stresses as described in experimental procedures. Equal
loadings were confirmed by Ribosomal RNA prestained gels with
ethidium bromide. The 400 bp specific fragment of AtNRP1 cDNA
was used for the probe. The experiments were repeated at least
three times with similar results. h, hours post-treatment.
were hybridized with α-32P-dATP-labeled gene-specific probes
corresponding to the following genes: AtNRP1, HsfA2, MBF1c,
Hsp70 and Hsp101, respectively, following standard procedures.
Hybridization was performed in PerfectHyb plus hybridization
buffer (Sigma-Aldrich) overnight at 68°C. The membrane was
then washed according to the procedure described by Yu et al.
PAtNRP1::GUS construction and GUS staining
To clone the promoter of AtNRP1, a 1866-bp AtNRP1 promoter
region was amplified from Arabidopsis genomic DNA by PCR
using the specific forward primer 5′-TTGCGGCCGCAATG-
AAAGATCCGGAAAGTGTAATTT-3′ and the specific reverse
which contain a NotI and a XbaI restriction sites (underlined),
respectively. The PCR fragment was digested with NotI and XbaI,
and was firstly inserted in frame upstream of the glucuronidase
(GUS) reporter gene in pJS131B vector. The recombinant plas-
mids were digested with SmaI and BamHI, and the resulting
AtNRP1 promoter-GUS fusion fragments were cloned into trans-
formation vector pOCA28 (Du and Chen, 2000).
GUS staining was performed using the method of Sieburth and
Meyerowitz (1997). Leaves or tissues were gently fixed by incu-
bation in 90% acetone on ice for 20 min and were then trans-
ferred into staining solution that contains 50 mM sodium phos-
phate (pH 7.0), 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6 , 0.1% v/v
Triton X-100 and 2 mM X-gluc (Sigma-Aldrich), vacuum infiltrated
for 5 min, and incubated in darkness at 37°C overnight. After
staining, tissues were washed with 70% ethanol.
Expression level of AtNRP1 is regulated by temperature
In order to determine the biological function of AtNRP1, we inves-
tigated its expression pattern in response to different stress with
northern blot analysis. Three-week-old seedlings were treated
with either 42°C, 4°C, 300 mM NaCl or natural drought at indi-
cated time. As shown in Fig. 1, expression of the AtNRP1 gene
was readily affected by temperature stress. The expression of
AtNRP1 decreased significantly and a minimum expression level
was reached at 4 h after 42°C treatment, followed by a gradual
recovery of transcription to the background level at 6 h (Fig. 1A).
On the other hand, AtNRP1 transcription was induced by low-
temperature stress. A rapid accumulation of AtNRP1 transcripts
was observed 2 h after initiation of cold treatment, and these
accumulated over a period of 12 h with a maximum level of tran-
scripts attained after 4-6 hours (Fig. 1B). Expression of the
AtNRP1 gene was down-regulated slightly under NaCl stress
and did not respond to dehydration stress (data not shown).
These results suggest that AtNRP1 is very sensitive to environ-
mental stimuli, and it appears to be positively regulated by cold
stress but negatively by heat stress. Collectively, these data indi-
cate that AtNRP1 may behave as a stress-responsive gene.
Developmental expression pattern directed by
To study the expression pattern of AtNRP1 in different devel-
opmental stages and different tissues, we analyzed transgenic
Arabidopsis plants expressing a β-glucuronidase (GUS) re-
porter gene driven by AtNRP1 promoter. GUS staining was
observed in root tips of 1- and 2-day-old pAtNRP1::GUS trans-
genic seedlings after germination, whole plant of 3- and 5-day-
old pAtNRP1::GUS transgenic seedlings grown in normal con-
dition, root tips and junctions of roots and hypocotyls of 5-day-
old transgenic seedlings grown in darkness (Figs. 2A-2E). With
the rapid growth, GUS staining enhanced greatly in 10-day-old
transgenic seedlings (Fig. 2F). While the expression of AtNRP1
dramatically declined with further growth of three-week-old
transgenic seedlings, GUS staining was only found in vascula-
ture, hydathodes of adult leaves, the sites of meristematic tis-
sues, such as vegetative shoot apex and root tips (Fig. 2G).
AtNRP1 also expressed in developing organs such as stamens
and stigmas of flowers, and abscission layers of young siliques
(Fig. 2H). Interestingly, the expression level of the AtNRP1
gene in apical meristematic regions was much higher than that
in other regions.
Overexpression of AtNRP1 results in altered response to
In order to further determine the function of AtNRP1, we identi-
fied a T-DNA insertion mutant for AtNRP1. The mutant (Salk_
111768) contains a T-DNA insertion in the extron region of
AtNRP1 (Fig. 3A), and the homozygous mutant plants were
confirmed by PCR with gene-specific primers. Northern blot
analysis used a DNA fragment that corresponds to the region
downstream of the T-DNA insertion site in AtNRP1 as the
probe. The AtNRP1 transcripts were detected in WT plants but
not in nrp1 under normal conditions (Fig. 3B). Mutant plants
grow and develop normally, with no obvious differences in mor-
phological or growth phenotype from that of WT plants (data
To characterize the effect of AtNRP1 overexpression, Arabi-
dopsis WT plants were transformed with a full-length AtNRP1
cDNA driven by the CaMV 35S promoter. Overexpression of
AtNRP1 was confirmed by RNA gel blot analysis in the inde-
pendent lines. Besides the level of AtNRP1 expression, no
other notably different morphological or growth phenotypes were
observed between WT plants and the 35S::AtNRP1 transgenic
lines (data not shown). Two transgenic lines, line 3 and line 8,
4 A Nodulin-Related Protein in Heat Stress
C D E F
Fig. 2. Developmentally-regulated GUS expression driven by
AtNRP1 promoters in transgenic Arabidopsis. (A) GUS staining of
pAtNRP1::GUS transgenic seedling 1 DAG. (B-D) GUS staining of
transgenic seedlings 2, 3 and 5 DAG, respectively. (E) GUS stain-
ing of dark-grown transgenic seedling 5 DAG. (F) GUS staining of
transgenic seedling 10 DAG. (G) GUS staining of transgenic seed-
ling 21 DAG. (H) GUS staining in inflorescence of transgenic seed-
ling. DAG, days after germination.
which constitutively expressed AtNRP1 at elevated levels (Fig.
3C), and an AtNRP1 mutant line were chosen for further study.
T3 homozygous 35S::AtNRP1 transgenic, WT and nrp1 plants
were then tested for differences in their response to heat stress.
The seeds of the tested materials were grown on 1/2 MS me-
dium plates. The nrp1 and WT seedlings exhibited similar ther-
motolerance with survival rates of 73% and 68%, respectively
(Figs. 4A and 4B). However, the 35S::AtNRP1 transgenic lines
showed severely impaired thermotolerance compared with the
WT. After HS at 45°C, only 18% or 23% of the AtNRP1-over-
expressing lines survived after 7 d recovery at 22°C, and thus
about 80% of the AtNRP1-overexpressing seedlings died (Figs.
4A and 4B).
The 2.5-day-old dark-grown seedlings displayed a severe re-
duction in hypocotyl elongation under HS compared with the
control treatment. Hypocotyl elongation of AtNRP1-overexpress-
ing seedlings was nearly 70% lower compared with that recorded
in the WT and nrp1 seedlings (Figs. 4C and 4D). The WT and
nrp1 seedlings exhibited a marked delay in hypocotyl elongation
to some extent after HS, but most of the seedlings continued to
grow (Figs. 4C and 4D).
We then tested electrolyte leakage (EL) of 21-day-old plants
during 42°C stress. No significant difference in relative electro-
lyte leakage was recorded among the tested leaves bathed at
42°C for 0 h or 1 h. However, the AtNRP1-overexpressing
transgenic leaves showed almost 2-fold greater relative electro-
lyte leakage than that of the nrp1 and WT leaves after bathing at
42°C for 3 h or 6 h (Fig. 4E). This result indicates that the re-
duced thermotolerance in adult AtNRP1-overexpressing plants
is associated with increased relative electrolyte leakage. Com-
pared with WT and nrp1, the AtNRP1-overexpressing lines
exhibited thermosensitivity at different developmental stages.
AtNRP1-mediated thermosensitivity is independent of
HS-related regulon expression
Many HSFs and HSPs have been shown to be important in re-
sistance to heat stress (Baniwal et al., 2004). The different ther-
motolerance among AtNRP1-overexpressing lines, WT and nrp1
led us to hypothesize that the biological function of AtNRP1 may
be dependent on heat-inducible genes in response to changing
thermotolerance. To explore the molecular basis of the altered
responses of the AtNRP1-overexpressing transgenic lines to
heat stress, we compared the expression of HsfA2, MBF1c,
Hsp70 and Hsp101 in these plants after heat stress.
First, expression of two heat stress-induced transcriptional
regulator factors, HsfA2 and MBF1c, were monitored. The tran-
script abundance for the two genes was similar in the AtNRP1-
overexpressing transgenic lines compared with those of WT
and nrp1 in response to heat stress (Fig. 5), indicating that
AtNRP1 was not regulated by these two genes. Moreover, the
expression of Hsp70 and Hsp101 were also not altered in WT,
nrp1 and AtNRP1-overexpressing plants after heat shock
treatment (Fig. 5). These results indicate that the functions of
AtNRP1 in thermal adaptation may be independent of the
from leaves of wild-type plants (Col-0) or eight independent trans genic 35S::AtNRP1 lines. Lines 3 and 8 expressed elevated levels of AtNRP1.
T3 homozygous progeny plants of the two lines were used for further analyses.
Fig. 3. Characterization of the AtNRP1 T-DNA
insertion mutant and overexpressing trans-
genic plants. (A) Diagram of AtNRP1 and its
T-DNA insertion mutant (SALK_111768). (B)
Northern blot analysis of the nrp1. Each lane
was loaded with 20 µg RNA prepared from
three-week-old wild-type plants (Col-0) or
nrp1. The blot was probed with an AtNRP1
DNA fragment corresponding to the region
downstream of the T-DNA insertion in the
mutant. Ribosomal RNA was used as a load-
ing control. (C) AtNRP1 expression in trans-
genic plants constitutively overexpressing
AtNRP1. RNA samples (5 µg) were prepared
Qiantang Fu et al. 5
ABA content analysis
ABA can inhibit Nod factor signal transduction of resistance to
various pathogens (de Torres-Zabala et al., 2007; Ding et al.,
2008), and some studies have indicated that the ABA content
was increased in response to heat stress (Kim et al., 2004;
Zhang and Fevereiro, 2007). So we propose AtNRP1 may
function in thermotolerance through the ABA signaling pathway.
One assumption is that the decreased heat tolerance of
AtNRP1-overexpressing lines under heat stress resulted from
alteration of endogenous ABA homeostasis. Quantification of
ABA content in AtNRP1-overexpressing lines, WT and nrp1
plants under heat stress showed that ABA content rapidly ac-
cumulated in all seedlings under heat stress after 30 min, and
increased continuously for the duration of the heat-stress treat-
ment, however, AtNRP1-overexpressing transgenic lines ac-
cumulated substantially lower amounts of ABA than WT and
nrp1 (Fig. 6A). We also analyzed AtNRP1 expression in re-
sponse to ABA treatment. As shown in Fig. 6B, AtNRP1 ex-
pression decreased gradually after treatment with 100 μM ABA.
In contrast, no significant difference was observed in AtNRP1
expression after treatment with water control. Northern blot
analysis of AtNRP1 expression in the ABA biosynthesis mutant
aba2 under heat stress showed that heat-stress-induced re-
pression of AtNRP1 transcription was abolished, and the aba2
have higher level of AtNRP1 compared to WT under heat
stress (Fig. 6C). These data indicate that AtNRP1 gene may be
regulated by the ABA synthesis pathway during heat stress.
AtNRP1 was an interacting protein of RPS2, and took part in
response in restricting growth of DC3000 (avrRpt2+) (Quirino et
al., 2004). AtNRP1 was a nodulin-related protein, and regulated
by temperature stress and ABA treatment (Figs. 1 and 6B).
AtNRP1 may play a role in tolerance to pathogens and the
In the present study, we analyzed preliminarily the expression
profile and GUS staining-histochemical location of AtNRP1.
Northern blot showed that AtNRP1 expression showed that
AtNRP1 was suppressed by heat treatment, however rapidly
increased during cold stress (Fig. 1), suggesting it was ready to
be affected by adverse ambient temperature. Extreme tempera-
ture is one of major factor which affect plant growth and devel-
opment, and many genes expression had similar patterns in
response to temperature factor, that is to say, the genes are in-
duced or suppressed by high or low temperature stress (Fu et al.,
Fig. 4. Altered responses of 35S::
AtNRP1 plants to heat stress. (A) Seven-
day-old seedlings of wild-type, nrp1 and
35S::AtNRP1 lines grown on 1/2 MS solid
medium were treated at 45°C for 260 min
after a pretreatment for 90 min at 38°C
and 120 min at 22°C. Seedlings were
scored and photographed after recovery
for an additional 7 days. (B) Percentage
survival after heat treatment in the ex-
periment summarized in (A). Data are
presented as the mean ± SD (n = 3).
Each replicate consisted of 50 seedlings.
Date were analyzed using the Mann-
Whitney U test; significant at *P < 0.05.
(C) Hypocotyl elongation of 2.5-day-old
seedlings. Seedlings were grown on ver-
tical plates in the dark for 2.5 d and, fol-
lowing a pretreatment for 90 min at 38°C
and 120 min at room temperature, and
220 min heat stress at 45°C, received a
further 2.5 d recovery. (D) Hypocotyl
length recorded after the final 2.5 days
recovery in the experiment summarized
in (C). Data are presented as the mean ±
SD (n = 3). Each replicate consisted of 40
seedlings. Data were analyzed using the
Mann-Whitney U test; significant at *P <
0.05. (E) Electrolyte leakage assay per-
formed on 21-day-old seedlings grown in
soil. The 3rd-5th leaves of WT, nrp1 and
AtNRP1-over-expressing lines were incu-
bated in a 42°C water bath for 1, 3 or 6 h,
and transferred to 22°C, when the bathing
solution conductivity was monitored. Data
are presented as the means of three
experiments each with 10 plants per
treatment. Bars represent the SD (n =
20). Data were analyzed using the Mann-
Whitney U test; significant at * P < 0.05.
6 A Nodulin-Related Protein in Heat Stress
Fig. 5. Expression of heat-induced genes in three-week old WT,
nrp1 and 35S::AtNRP1 plants during heat treatment. Total RNA (10
µg) was loaded into each lane and Ribosomal RNA was used as a
control for RNA loading. The gene-specific fragments for HsfA2,
MBF1c, Hsp70, and Hsp101 were used for the probe. The experi-
ments were repeated three times with similar results.
1998; Knight and Knight, 2001), however, AtNRP1 was sup-
pressed with heat treatment and induced during cold stress. Re-
sponse mechanism of AtNRP1 in high and low temperature
stress may be different. Further characterization is still needed to
determine the specific functions of AtNRP1 in temperature stress.
It is clear that genes affected by multiple types of stress are gen-
eral and potentially confer tolerance to environmental stress
(Aviezer- Hagai et al., 2007; Hazen et al., 2003). AtNRP1 ex-
pression is affected by temperature factor (Fig. 1), suggesting
that AtNRP1 may play a role in resistance to environmental
stress. GUS staining demonstrated that AtNRP1 expressed in
the sites of meristematic tissues such as the vegetative shoot
apex and root tips, developing organs such as floral buds, sta-
men and pistils of young flowers, abscission layers of immature
siliques and junctions of pedicels (Fig. 2). Considering the speci-
ficity of the expression, AtNRP1 may play a certain function in
development of Arabidopsis seedlings and floral organs. In addi-
tion, these tender tissues are sensitive to environmental factors
and are readily to affect by stresses (Cazalé et al., 2009).
To elucidate the biological function of AtNRP1, AtNRP1 null
mutants and AtNRP1-overexpressing transgenic plants were
generated (Fig. 3). Transgenic plants overexpressing AtNRP1
exhibited less tolerance to high temperature stress than WT
and nrp1. This reduced tolerance was illustrated by both the
lower survival rate of seven-day-old seedlings and shorter hy-
pocotyl elongation of 2.5-day-old dark-grown seedlings under
heat stress (Figs. 4A and 4C). Electrolyte leakage assays also
showed that the three-week-old overexpressing transgenic
plants were thermosensitive compared with WT (Fig. 4E). Hy-
pocotyl elongation, survival rate of seedling, and electrolyte
leakage assay are effective parameters in analysis of heat
tolerance (Clark et al., 2004; Hong and Vierling, 2000; Li et al.,
2009). The AtNRP1-overexpressing transgenic plants showed
decreased thermotolerance at different growth stages and the
null mutation at the AtNRP1 gene was the same as WT under
heat stress, which indicated that the single-gene mutation of
AtNRP1 was insufficient to alter heat tolerance. There are many
cases where a T-DNA KO line does not show any phenotype
because of functional complementation of related genes (Cutler
and McCourt, 2005; Higashi et al., 2008; Lee, et al., 2008).
AtNRP1 may have functional overlap with other heat-related
genes, and these functional-overlap genes may compensate for
Fig. 6. Analysis of the molecular mechanisms of decreased thermo-
tolerance in 35S::AtNRP1 plants. (A) ABA content in WT, nrp1, and
35S::AtNRP1 transgenic plants after heat treatment at 38°C for 0.5,
1 or 2 h. Three-week-old WT, nrp1, and 35S::AtNRP1 transgenic
plants grown in soil were harvested after heat treatment for the
indicated time periods. ABA content was determined by ELISA
analysis. The results showed 35S::AtNRP1 overexpressing plants
accumulated low levels of ABA with three independent experiments
in the same condition. Data are presented as the mean ± SD (n =
10). Date were analyzed using the Mann-Whitney U test; significant
at *P < 0.05. (B) Expression of AtNRP1 in response to application
ABA. (C) Expression of AtNRP1 in WT and aba2-3 during heat
treatment. Total RNA (10 µg) was loaded into each lane and Ribo-
somal RNA was used as the control for RNA loading. The gene-
specific fragment of AtNRP1 was used for the probe. The experi-
ments were repeated three times with similar results.
the lack of function of AtNRP1, so there are no obvious pheno-
type differences between nrp1 and WT under nomal conditions
or heat stress. Thus, we deduced that AtNRP1 might play a
negative role in thermotolerance.
This decreased thermotolerance exhibited by AtNRP1-
overexpressing transgenic plants may be due to the decreased
expression of heat-stress-inducible HSFs and HSPs genes.
AtHsfA2 is an important regulator of thermotolerance, and is
required for extension of acquired thermotolerance in Arabi-
dopsis (Charng et al., 2007). AtHsp101 and AtHsp70 are be-
lieved to play a pivotal role in thermotolerance in Arabidopsis
Qiantang Fu et al. 7
(Queitsch et al., 2000; Sung et al., 2001). The transcriptional
coactivator MBF1c, is a key regulator of thermotolerance in
Arabidopsis thaliana, and MBF1c is not required for the expres-
sion of transcripts encoding HsfA2 and different heat shock pro-
teins in response to heat stress (Suzuki et al., 2008). Expression
of HsfA2, MBF1c, Hsp70 and Hsp101 were characterized in
among AtNRP1-overexpressing plants, nrp1 and WT under heat
stress. Northern blot analyses indicated that over-expressing of
AtNRP1 did not alter the expression of these known heat induc-
ible genes. Expression levels of HSFA2, MBF1c, HSP101 and
HSP70 are same among the over-expression, nrp1 and WT
under heat stress (Fig. 5). These results suggest that the heat
sensitivity of AtNRP1-overexpressing plants is not conferred by
these heat-induced genes, and other signaling pathways may be
involved in the heat stress response of AtNRP1-overexpressing
ABA acts through complex signalling networks to induce
changes in multiple physiological processes. ABA levels in-
crease when plants encounter adverse environmental condi-
tions, and increased ABA content is beneficial for plants under
environmental stress (Xiong and Zhu, 2003; Zhu, 2002). The
accumulation of substantially amounts of ABA in nrp1 and WT
plants was higher than in AtNRP1-overexpressing transgenic
lines (Fig. 6A), which was consistent with their relative thermo-
tolerance phenotypes. The signals of environmental stresses
trigger ABA synthesis, which in turn induces expression of vari-
ous responsive genes. These downstream genes also appear
to involve extensive feedback regulation of ABA accumulation
(Uno et al., 2001; Verslues and Bray, 2006). The expression
level of AtNRP1 was higher in aba2 than in wild-type under
heat stress (Fig. 6C). Heat stress-induced inhibition of AtNRP1
expression is likely to be mediated through the ABA synthesis
pathway, and AtNRP1 may affect feedback regulation of ABA
accumulation in AtNRP1-overexpressing lines during heat
stress. These results suggest that AtNRP1 takes part in heat
stress response in plants via regulation of the ABA synthesis
Although the molecular mechanisms underlying the roles of
AtNRP1 under heat stress conditions are not clearly under-
stood, the alteration of ABA accumulation in 35S::AtNRP1
plants compared with wild-type plants suggests a possible func-
tion of AtNRP1 under heat-stress conditions. It appears that the
change in ABA accumulation in AtNRP1-overexpressing plants
partly contributes to the decreased heat-stress resistance of
AtNRP1 transgenic plants.
In conclusion, this paper presents these novel experimental
data that increase our knowledge of the roles of AtNRP1 in
response to environmental stresses. Phenotypic analysis of
AtNRP1-overexpressing transgenic plants and knockout mu-
tants implies that AtNRP1 may play a role as a negative regula-
tor of seedling growth under heat stress. Whether the AtNRP1-
overexpressing plants and/or the loss-of-function mutant plants
exhibit altered responses to other abiotic stress conditions has
not been determined. Future studies in this direction may reveal
not only additional roles for AtNRP1 in plant stress responses,
but also possible interactions between their respective signaling
Note: Supplementary information is available on the Molecules
and Cells website (www.molcells.org).
We thank the ABRC at the Ohio State University (Columbus,
OH) for seeds of the Arabidopsis mutants. This work was sup-
ported by the Natural Science Foundation of China (grant no.
90817003), the Ministry of Science and Technology of China
(grant no. 2006AA02Z129), and the Hundred Talents Program
of the Chinese Academy of Sciences.
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