1. Physiological and metabolomic analysis of a knockout
mutant suggests a critical role of MtP5CS3 gene in osmotic
stress tolerance of Medicago truncatula
Minh Luan Nguyen • Goon-Bo Kim • Sun-Hee Hyun •
Seok-Young Lee • Chae-Young Lee • Hong-Kyu Choi •
Hyung-Kyoon Choi • Young-Woo Nam
Received: 18 January 2013 / Accepted: 7 June 2013 / Published online: 25 July 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract In the model legume Medicago truncatu-
la, D1
-pyrroline-5-carboxylate synthetase (P5CS), the
rate-limiting enzyme of proline biosynthesis, is
encoded by three closely related genes, MtP5CS1,
MtP5CS2, and MtP5CS3. While MtP5CS1 is consti-
tutively expressed, MtP5CS2 and MtP5CS3 are
induced by adverse environmental conditions, of
which MtP5CS3 is prevalently expressed during
drought and salinity stresses. Mtp5cs3, a transposon
(Tnt1) insertion mutant of MtP5CS3 that cannot
synthesize a mature protein, showed decreased proline
accumulation and increased sensitivity to salinity,
drought, and low water potential stresses, as evidenced
by decreased seedling growth and chlorophyll content
and increased hydrogen peroxide content. These
defective phenotypes were complemented by exter-
nally supplied proline or ectopically expressed cDNA
to the wild-type gene (MtP5CS3). Gas chromatogra-
phy–mass spectrometry-based analysis of soluble
metabolites revealed that some major metabolites
contributing to osmotolerance, including certain
amino acids, sugars, and polyols, accumulated more
abundantly in the Mtp5cs3 roots than in the wild type,
whereas a few other amino acids accumulated less
during drought and salinity stresses. While such
metabolic reconfiguration apparently fell short of
compensating for proline deficiency in Mtp5cs3,
overexpression of MtP5CS3 significantly increased
tolerance of M. truncatula to salinity and low water
potential stress. Thus, MtP5CS3 plays a crucial role in
proline accumulation and osmotic stress tolerance of
M. truncatula. Manipulation of this predominant
proline biosynthetic gene will facilitate the develop-
ment of environmentally stable legume crops.
Keywords Medicago truncatula Á Proline Á
D1
-Pyrroline-5-carboxylate synthetase 3 gene Á
Knockout mutant Á Metabolomic analysis Á
Drought Á Salinity Á Osmotic stress tolerance Á
Legume improvement
Introduction
Many plants accumulate low-molecular-weight osmo-
lytes such as certain amino acids, sugars, and reduced
Electronic supplementary material The online version of
this article (doi:10.1007/s10681-013-0957-4) contains supple-
mentary material, which is available to authorized users.
M. L. Nguyen Á G.-B. Kim Á Y.-W. Nam (&)
Department of Life Science, Sogang University,
Seoul 121-742, Korea
e-mail: ywnam@sogang.ac.kr
S.-H. Hyun Á S.-Y. Lee Á H.-K. Choi
College of Pharmacy, Chung-Ang University,
Seoul 156-756, Korea
C.-Y. Lee Á H.-K. Choi
Department of Genetic Engineering, College of Natural
Resources and Life Science, Dong-A University,
Pusan 604-714, Korea
123
Euphytica (2013) 193:101–120
DOI 10.1007/s10681-013-0957-4

2. sugars under osmotic stress. These molecules are
considered to adjust intracellular osmotic potential,
maintain turgor pressure, and protect subcellular
structures against possible stress-caused damages
(Hare et al. 1988). Particularly, proline may accumu-
late up to 100 times under stressed conditions caused
by salinity, drought, heavy metal, pathogen infection,
and UV irradiation than under a normal condition
(Hare and Cress 1997; Verbruggen and Hermans
2008). Proline plays not only as a compatible solute
against these stresses (Handa et al. 1986), but as a
signal eliciting other adaptive responses (Maggio et al.
2002), as a molecular stabilizer that maintains the
structure of cellular and cell wall proteins (Szabados
and Savoure´ 2010), as a scavenger of reactive oxygen
species (ROS) (Sharma et al. 2011), and especially in
chloroplasts, as a NADP/NADPH balancer (Sze´kely
et al. 2008). However, external supply of proline can
also be toxic to plants (Hellmann et al. 2000).
Proline is synthesized from glutamate by a three-
step pathway in which glutamate is first converted to
c-glutamyl phosphate and then to glutamate semial-
dehyde, which is spontaneously cyclized to D1
-pyrro-
line-5-carboxylate (P5C), an immediate precursor of
proline. The first two reactions, which are the rate-
limiting steps of proline synthesis, are catalyzed by
P5C synthetase (P5CS), a bifunctional enzyme (Hu
et al. 1992). The final conversion of P5C to proline is
catalyzed by P5C reductase (P5CR) (Delauney and
Verma 1990). The expression of P5CS is regulated at
the transcriptional level (A´ braha´m et al. 2003) and its
enzyme activity is inhibited by feedback mechanism
by proline (Zhang et al. 1995). Proline can also be
synthesized from ornithine by a transamination reac-
tion catalyzed by a mitochondrial ornithine-d-amino-
transferase (OAT), but this pathway is considered to
contribute more to arginine catabolism (Funck et al.
2008). In mitochondria, proline is degraded by proline
dehydrogenase (Kiyosue et al. 1996) and P5C dehy-
drogenase (P5CDH) (Deuschle et al. 2004). Proline
accumulation during osmotic stress is believed to be
mediated by its increased synthesis and reduced
degradation (Szabados and Savoure´ 2010).
A small family of genes, mostly consisting of two,
encodes P5CS enzymes in plants (Fujita et al. 1998;
Ginzberg et al. 1998). In Arabidopsis, AtP5CS1 is
upregulated by dehydration, salinity, and abscisic acid
(ABA), while AtP5CS2 is expressed in dividing cell
cultures and rapidly growing shoot and root meristems
(Strizhov et al. 1997; A´ braha´m et al. 2003). These
differential expression patterns were confirmed by
mutant studies, wherein Atp5cs1, a knockout mutant,
showed a decrease in salinity-induced proline synthe-
sis, while Atp5cs2 resulted in embryo abortion (Sze´k-
ely et al. 2008). In leaf mesophyll cells, AtP5CS1 was
imported into chloroplasts under osmostic stress,
while AtP5CS2 remained in the cytoplasm (Sze´kely
et al. 2008). In M. truncatula, three MtP5CS genes
have been isolated. While MtP5CS1 was constitu-
tively expressed, MtP5CS2 and MtP5CS3 were
induced by salinity and drought (Armengaud et al.
2004; Kim and Nam 2013). The deduced amino acid
sequence of MtP5CS3 was similar to those of
MtP5CS1 and MtP5CS2 except that a long intron
interrupted the first exon encoding a unique 42-amino-
acid amino (N)-terminal peptide segment. Also, a
splice variant of MtP5CS3, originating from its first
intron, was expressed at low levels under normal
condition (Kim and Nam 2013).
Recently, global changes in the composition of low-
molecular-weight metabolites have been investigated
by using analytical techniques such as nuclearmagnetic
resonance spectroscopy, gas chromatography (GC),
and mass spectrometry (MS) (Last et al. 2007; Saito and
Matsuda 2010). Particularly, metabolomic analyses
have been useful for characterizing reconfiguration of
metabolite composition in (a)biotically stressed plants.
Examples include studies showing differences between
Arabidopsis and its halophytic relatives tolerant to
osmotic stress (Gagneul et al. 2007; Lugan et al. 2010),
reconfiguration of global metabolites in an Arabidopsis
knockout mutant lacking a key transcriptional regulator
or defective in the synthesis of a plant growth regulator
in response to dehydration (Cook et al. 2004; Urano
et al. 2009), and metabolic changes associated with
drought acclimation in another model legume Lotus
japonicus (Sanchez et al. 2008a). These studies showed
that global changes in metabolites were responsible for
different levels of dehydration tolerance between
genotypes, indicating that differential regulation of
the major metabolic pathways during adaptation of
plants to abiotic stresses can lead to a profound shift in
metabolome (Sanchez et al. 2008b, 2011).
A knockout mutant of M. truncatula, Mtp5cs3,
which could not synthesize a mature protein due to a
transposon inserted in the first exon, showed dimin-
ished proline accumulation under high salinity (Kim
and Nam 2013). This defect was associated with its
102 Euphytica (2013) 193:101–120
123

3. functional deficiency in supporting nitrogen fixation
during symbiosis between rhizobia and M. truncatula.
To further investigate the roles of MtP5CS3 in abiotic
stress responses, we tried to characterize the Mtp5cs3
mutant thoroughly. In this study, we present results
from physiological analysis of Mtp5cs3 in response to
osmotic stresses. Metabolic profiling of the wild type
and the mutant plants indicates that mutation of this
single gene involved in proline biosynthesis causes an
unexpectedly broad range of reconfiguration of me-
tabolome under osmotically challenged conditions.
The effects of ectopic expression of the wild type gene
on osmotic stress tolerance are also presented and
discussed in relation to the potential usefulness of this
gene in legume crop improvement.
Materials and methods
Plant growth conditions and stress treatment
Seeds of Medicago truncatula (Gaertn) ecotypes Jem-
along A17 and R108-1 were germinated as previously
described (Kim and Nam 2013). One-day-old seed-
lings were transferred to pots (6 3 6 3 7 cm3
) filled
with perlite-Turface mixture (3:1, v/v) and fertilized
with 19 solution of modified Fahraeus medium
(mFM) {0.5 mM MgSO4Á7H2O, 0.7 mM KH2PO4,
0.8 mM Na2HPO4Á2H2O, 0.05 mM Fe–NaEDTA,
0.75 mM Ca(NO3)2, 0.9 mM CaCl2, 1 mM NH4NO3,
1 mL/L micronutrient solution (100 mg/L each of
CuSO4, MnSO4, ZnSO4, H3BO3, and Na2MoO4)}once
per week in a greenhouse. Seedlings were also
transferred to an aerated hydroponic container filled
with 19 hydroponic nutrient solution (HY) (Barker
et al. 2006). For in vitro culture, seedlings were
transferred into pouch paper on top of mFM agar in
square petri dishes (12 3 12 cm2
). Both greenhouse-
grown and in vitro cultured plants were placed under
the conditions of 16-h photoperiod (100–120
lEm-2
s-1
), 22/18 °C (day/night), and 70 % humid-
ity. Four-week-old greenhouse-grown plants were
treated with 50 mL of 150 or 200 mM NaCl solution
or sterile water. To measure root lengths of in vitro
cultured plants, 2-day-old seedlings were transferred
onto pouch paper laid on top of mFM agar medium in
square petri dishes (12 3 12 or 24 3 24 cm2
) that
contained 0, 75, or 100 mM of NaCl. For drought
treatment, watering was suspended for designated
time periods. For low water potential treatment, 2-day-
old seedlings were transferred to a hydroponic
container, incubated for 2 days, and treated with a
medium containing 150 or 200 g/L of polyethylene
glycol (PEG) 6000. The water potential of the
hydroponic or solid mFM agar medium was estimated
to be -1.48 (100 g/L of PEG 6000), -2.95 (150 g/L
of PEG 6000), or -4.91 Bars (200 g/L of PEG 6000),
as calculated by the method of Michel and Kaufmann
(1973). For low temperature treatment, plants were
transferred to 4 °C, and incubated for 7 days before
freezing damage was estimated. Cold-acclimated
plants were subsequently transferred to a freezing
chamber, kept at -8 °C for 10 h, transferred back to
4 °C for 1 day, and finally to the greenhouse for a
12-day recovery.
Plasmid construction
The full coding region of MtP5CS3 (GenBank
Accession JN800725) was PCR amplified from first-
strand cDNA with a pair of primers (50
-AAAAAG
CAGGCTCCATGGAAGTTTTGCAAAATGGCT-30
and 50
-AGAAAGCTGGGTGTGCCTATTATGCTT
CTATTAGCTGC-30
) and cloned into pDONR221
via recombination in the presence of BP Clonase Mix II
(Invitrogen) to result in an entry plasmid, pENTR_
P5CS3. The nucleotide sequence of the MtP5CS3 (756
amino acids)-encoding insert (2,268 bp) was verified
by DNA sequencing. pENTR_P5CS3 was subse-
quently recombined with pKWG2D (Karimi et al.
2002), a destination vector that carried a cauliflower
mosaic virus (CaMV) 35S promoter-controlled
enhanced green fluorescent protein gene (Egfp) and a
neomycin phosphotransferase II (NPTII) cassette
encoding kanamycin resistance, to result in a binary
plasmid, pK_35S-P5CS3.
Agrobacterium rhizogenes-mediated plant
transformation
Hairy root transformation of M. truncatula with A.
rhizogenes strain ARqua1 (Quandt et al. 1993) was
carried out as previously described (Boisson-Dernier
et al. 2001). Transformed roots were selected by
visualizing GFP fluorescence through a Wratten #15
orange filter (Peca Products, USA) under a
BP480DF10 excitation filter (Omega Optical, USA)-
screened blue LED (Devicebay, Korea). The
Euphytica (2013) 193:101–120 103
123

4. transformed plants were further grown for 1–2 weeks
in filter paper sandwich on mFM agar at 24 °C before
abiotic stress treatment.
Real-time reverse transcription polymerase chain
reaction (RT-PCR) and RNA gel blot analyses
For RT-PCR, total RNA was isolated and cDNA
synthesized as previously described (Kim and Nam
2013). MtP5CS3 and its splice variant MtP5CS3a
were amplified with gene-specific primers that were
complementary to the 50
-untranslated region (UTR)
and each respective exon 1. MtP5CS1, MtP5CS2, and
MtCorA1 (Merchan et al. 2007) were amplified with
primers (Supplementary Table S1). Typically, 1 ll of
1/10-diluted cDNA was used as a template in a 20-ll
mixture that contained 250 nM of primers, 1 nM of
reference dye ROX, and 1/2 volume of 29 SYBR
Green Mix (KAPA Biosystems). PCR was carried out
under a program (10 min at 94 °C; 40 cycles of 15 s at
94 °C and 1 min at 60 °C) with the ABI 7500 Real-
Time PCR System (Applied Biosystems). Quantita-
tive real-time PCR analysis was also carried out as
previously described (Kim and Nam 2013) using
primers (Supplementary Table S1).
For RNA gel blot analysis, 20 lg of total RNA was
isolated using the single-step method (Chomczynski
and Sacchi 1987), separated on a 1.2 % agarose gel
containing 19 MOPS buffer and 2.2 M formaldehyde,
and subsequently transferred onto positively charged
nylon membrane (Magna Charge 0.45, Schleicher &
Schuell). The resulting blot was UV cross-linked at
150 mJ, and then hybridized according to the standard
protocol. As probes, 100–200 ng of partial cDNA
fragments amplified from each designated cDNA with
primers (Supplementary Table S1) was labeled with
[a-32
P]dCTP using the Rediprime II Random Labeling
Kit (GE Healthcare Life Sciences). A 0.5-kb MtP5CS2-
specific probe was amplified with primers (50
-GTTG
CAGCACGCGACAGCTC-30
and 50
-CCCTTGAAG
TCACAAGGCCA-30
). A 236-bp MtP5CS3-specific
probe was amplified with primers (50
-TTCTCCTCAT
TATACTAGCTAGC-30
and 50
-CAGCTGTTCCAA
CCTTAACAAT-30
) from the 50
-UTR and the first
exon. A 207-bp MtP5CS3a-specific probe was ampli-
fied with primers (50
-CTCGGGTCTCTCCGTCACT
CAC-30
and 50
-TACCTGCTCACATAGAGCT-30
)
from the 50
-UTR and exon 2. After hybridization at
65 °C overnight, blots were washed in 29 SSC at 25 °C
twice, and then in 19 SSC/0.1 % SDS and 0.59 SSC/
0.1 % SDS at 65 °C twice each for 20 min. The washed
blots were exposed to an X-ray film at -70 °C.
P5CS activity assay
Frozen samples (100 lg)weregroundinliquid nitrogen
and placed in 1 mL of extraction buffer (0.1 M Tris–
HCl, pH 7.2, 10 mM MgCl2, 10 mM 2-mercap-
toethanol, and 1 mM PMSF). The homogenate was
centrifuged at 10,0009g for 20 min at 4 °C twice and
protein quantity estimated (Bradford 1976). P5CS
activity was measured based on the rate of NADPH
consumption. Typically, a 0.5-mL reaction mixture
containing 100 mM Tris–HCl (pH 7.2), 25 mM MgCl2,
75 mM Na-glutamate, 40 lg of enzyme extract, 5 mM
ATP, and 0.4 mM NADPH was incubated at 37 °C for
15 min, and absorbance at 340 nm was measured. The
P5CS activity was then calculated as lmol of NADPH
oxidized/mg protein per min (Garcı´a-Rı´os et al. 1997).
Quantification of proline
Approximately 0.1 g (fresh weight, FW) of pulverized
frozen material was homogenized in a 2-mL eppendorf
tube containing 1.7 mL of 3 % (w/v) aqueous sulfo-
salicylic acid solution by a minishaker for 20 min.
Then the tubes were centrifuged at 12,0009g for
10 min, and 1 mL of supernatant was pipetted into a
10-mL test tube, to which 1 mL of glacial acetic acid
and 1 mL of ninhydrin reagent (prepared by adding
1.25 g of ninhydrin to 30 mL of glacial acetic acid,
4 mL of 14.5 M phosphoric acid, and 16 mL of
distilled water in a 50-mL tube) were added. The test
tube was autoclaved at 105 °C for 30 min. Exactly
2 mL of toluene was added to each tube and vigorously
shaken at least for 2 min. After phase separation at
25 °C for 30 min, three 300-ll aliquots from the upper
toluene phase were transferred into a quartz multi-well
plate. A blank toluene-phase sulfosalicylic acid solu-
tion was used as a control.Absorbance was measured at
520 nm using a microplate photometer (Gemini XPS,
Molecular Devices). The resulting values were cali-
brated as described (Kim and Nam 2013).
Electrolyte leakage measurement
Electrolyte leakage of frozen and low water potential-
exposed tissues was measured as described by
104 Euphytica (2013) 193:101–120
123

5. Verslues et al. (2006). M. truncatula seedlings were
transferred to a tube containing 10 mL of deionized
water, incubated at 25 °C for 30–60 min, and sub-
jected to conductivity measurement using the Trace-
able Digital Conductivity Meter (VWR). In parallel,
each tube, capped and autoclaved, was used for the
measurement. The relative electrolyte content was
calculated by dividing the measured conductivity by
that obtained from autoclaved tissue.
Hydrogen peroxide (H2O2) measurement
H2O2 content was measured as described by Alexieva
et al. (2001). M. truncatula roots and shoots were
ground with 0.1 % trichloroacetic acid and centri-
fuged at 12,0009g at 4 °C for 15 min. To 0.5-mL
supernatant, 0.5 mL of 10 mM potassium phosphate
buffer (pH 7.0) and 1.0 mL of 1 M KI were added and
mixed gently. After 1 h, absorbance was measured at
390 nm, and H2O2 was quantified based on a calibra-
tion curve obtained with solutions of known H2O2
concentrations. Colorimetric detection of H2O2 was
carried out as described by Sze´kely et al. (2008).
Seedlings grown in a hydroponic container with or
without NaCl or PEG were dipped in 1 mg/mL of 3,30
-
diaminobenzidine (DAB) solution (Sigma), incubated
at pH 5.5 at 25 °C for 2 h, and the generation of H2O2
was detected by reddish-brown color that developed.
Subsequently, seedlings were transferred to hot etha-
nol (96 %) for 10 min to bleach chlorophyll, and
stored in 96 % ethanol.
Chlorophyll measurement
Chlorophyll content was measured as described by
Hiscox and Israelstam (1979). M. truncatula leaves
(50 mg) were transferred to glass vials containing
5 mL of dimethyl sulphoxide that had been preheated
at 65 °C. After incubation for 30 min, samples were
subjected to absorbance measurement at 645 and
663 nm. Chlorophyll content was calculated by trans-
forming the estimated quantity of chlorophyll a or b
into a total value as described by Sze´kely et al. (2008).
Extraction and derivatization of metabolites
Four-week-old wild type andMtp5cs3plantsgrown in a
greenhouse under standard condition were treated with
or without 150 mM NaCl or drought for 5 days. Each
M. truncatula sample (10 mg) was separately weighed
and transferred into an Eppendorf tube. To extract
metabolites, 1 mL of 70 % methanol (HPLC grade,
Burdick & Jackson) in water was added to the sample,
and the tube was vortexed for 1 min, sonicated for
40 min, and kept at 25 °C for 10 min to let precipitates
settle down. Subsequently, the supernatant was sepa-
rately collected and filtered through 0.45 lm PVDF
Syringe Filters(Membrane Solutions). Then,100 lL of
filtrate was transferred into GC vials and evaporated
with nitrogen gas at 60 °C. For trimethylsilyl (TMS)
derivatization,30 lL of 20,000 lg/mLmethoxylamine
hydrochloride in pyridine and 50 lL of N,O-bis
(trimethylsilyl) trifluoroacetamide (Alfa Aesar) con-
taining 1 % trimethylchlorosilane were added to the
dried samples. Thereafter, 10 lL of 300 lg/mL 2-chlo-
ronaphthalene (Tokyo Chemical Industry) in pyridine
was applied as an internal standard and incubated in a
water bath at 60 °C for 60 min. After incubation, the
sample was transferred to the insert and 1 lL of the
solution was used for GC analysis.
Gas chromatography/mass spectrometry (GC/MS)
analysis
A gas chromatograph (model 7890, Agilent) interfaced
to the Quadrupole Mass Spectrometer (MS) (model
5975C, Agilent) was used for metabolomic analysis.
The temperature of the MS source and the Quadrupole
were set at 250 and 150 °C, respectively, with the
electron ionization mode set at 70 eV. Mass range was
set at 50–600 Da for global metabolites. The gas
chromatograph was equipped with a split/splitless
injector operated at 250 °C. One microliter of split
(split ratio 1:5) sample was injected using an Agilent
model 7683B autosampler and helium carrier was used
at a constant flow rate of 1 mL/min. A DB-5 column
(Agilent 5 %-phenyl-methylpolysiloxane; 30 m, 0.25
mm ID, 0.25 lm df) was installed in the GC oven. For
global metabolites, the oven temperature was set at
60 °C, and in 5 min, ramped to 130 °C (10 °C/min),
210 °C (4 °C/min), and 300 °C (15 °C/min), each kept
for 2 min. The total run time was 40 min.
Metabolomic data processing and statistical
analysis
For mass spectral data processing, the Automated Mass
Spectral Deconvolution and Identification System
Euphytica (2013) 193:101–120 105
123

6. (AMDIS, http://chemdata.nist.gov/mass-spc/amdis/)
was initially used. To separate peaks from noise and
deconvolute overlapped peaks, parameters were set at
70 (minimum match factor), 12 (component width), 1
(adjacent peaks subtraction), medium resolution, low
sensitivity, and medium shape requirement. The
resulting ELU files were subsequently analyzed by the
SpectConnect online peak filtering algorithm (http://
spectconnect.mit.edu). Identification and relative
quantification of metabolites were performed using the
Whiley-NIST MS library. For multivariate statistical
analysis, the AMDIS/SpectConnect-processed GC/MS
datawereimportedintothe SIMCA-Psoftware(version
12.0, Umetrics, Umea˚, Sweden). Data were mean-cen-
tered and scaled with Pareto (Par) scaling, which was
performed by dividing each variable by the root of
standard deviation. Partial least squares-discriminant
analysis (PLS-DA) was carried out to clearly separate
metabolitesthathighlycontributedtothe differentiation
of the samples derived from the mass spectral data. For
comparative study of total metabolites, all mass spectral
data were used for PLS-DA. To show relative profiles of
quantified metabolites, standard Z-scores were calcu-
lated with an equation, {x – mean (x)}/sd (x), by which
the difference between the relative GC/MS intensity of
metabolite x and the mean value was divided by stan-
dard deviation (sd). The Z-scores were then used to
draw a heat map (see Fig. 6) by using the heatmap
method in R language (version 2.15.2; http://www.r-
project.org/).
Results
The Mtp5cs3 mutant and its phenotypes
under normal condition
A Tnt1 insertion line (NF4970) of M. truncatula D1
-
pyrroline-5-carboxylate synthetase 3 gene (MtP5CS3)
(GenBank Accession JN800725) was isolated as previ-
ously described (Kim and Nam 2013). The insertion at
?97 nucleotide (nt) position in exon 1 (Supplementary
Fig. S1a), was predicted to terminate the synthesis of
MtP5CS3 prematurely. Seedlings of this mutant
(Mtp5cs3), screened for homozygous recessives by
PCR-based genotyping for three generations (Supple-
mentary Fig. S1b), showed nearly indistinguishable
phenotypes from those of the wild type when grown in
soil or in vitro under standard conditions. Not only the
germination rates (95–98 %) were similar between the
two genotypes, but the FW of the Mtp5cs3 seedlings
was only insignificantly lower than that of the wild type
(Supplementary Fig. S1c). Free proline contents were
also similar in roots and shoots of the two genotypes,
reaching a typical level of 0.3–0.5 lmol/g FW (Sup-
plementary Fig. S1d). Mature seeds exhibited generally
higher proline contents in both genotypes. In contrast,
dry weights of ten fruits and 100 seeds of Mtp5cs3 were
noticeably lower than those of the wild type (Supple-
mentary Fig. S1e).
Decreased tolerance of the mutant to drought
and salinity
To assess the effects of Mtp5cs3 mutation on plant
growth under salinity, the wild type and mutant
seedlings were cultivated in vitro with or without
75–100 mM NaCl. The primary root length of Mtp5cs3
under 75 mM NaCl was only 63 % that of the wild type
after 22-day growth (Fig. 1a, b). With 100 mM NaCl,
the difference became smaller, Mtp5cs3 being 77 % of
the wild type, apparently because the wild type itself
began to be affected by salinity (Fig. 1c, d). Addition-
ally, purple or bleached spots, a typical salt-responsive
symptom, were visible only in the young leaves of
Mtp5cs3 (Fig. 1c). However, the total lateral root length
of Mtp5cs3 was only slightly shorter than that of the
wild type with or without NaCl (Supplementary Table
S2). To examine the cause of salt sensitivity, Mtp5cs3
plants were grown in the absence or presence of
externally supplied proline. Addition of proline com-
plemented the defects of Mtp5cs3 phenotypes, signif-
icantly increasing the number of lateral roots and the
total lateral rootlength(Fig. 1e,f;SupplementaryTable
S2). These results indicate that low proline content was
a main cause of salt sensitivity of Mtp5cs3 seedlings.
Under greenhouse conditions, salinity or drought
affected Mtp5cs3 more severely than the wild type
(Fig. 2a). Although the extents of damage appeared
generally minor (Fig. 2a), fresh and dry weights
measured at 8 and 16 days under saline condition
and FWs measured at 4 days under drought condition
were significantly different (Fig. 2b–d). While fresh
and dry weights of the wild type decreased by 21 and
33 %, respectively, those of Mtp5cs3 decreased by 52
and 60 %, respectively, after a 16-day treatment of
150 mM NaCl (Fig. 2b). Under drought for 4 days, 20
and 45 % decreases in FWs were observed in the wild
106 Euphytica (2013) 193:101–120
123

7. type and Mtp5cs3, respectively (Fig. 2d). Under high
salinity, Mtp5cs3 showed fewer lateral roots and more
extensive leaf senescence than the wild type (Fig. 1g).
Such high sensitivity led to much lowered survival
rates of the mutant—only *65 % compared to
*95 % of the wild type—under salinity or drought
(Table 1). These results indicate that Mtp5cs3 was
more sensitive to water loss than the wild type.
In response to 200 mM NaCl, the Mtp5cs3 mutant
accumulated proline in shoots and roots by 5- and
25-fold, respectively, while the wild type by 26- and
70-fold after 3-day treatments (Fig. 2e). After 6-day
treatments, the proline content of Mtp5cs3 still
remained at 2/3 (root) and 1/4 (shoot) those of the wild
type. Under drought, Mtp5cs3 accumulated proline
only up to 75 % (root) and 65 % (shoot) of those of the
wild-type (Fig. 2e). Notably, drought-induced 56-fold
(wild type) and 43-fold (Mtp5cs3) increases in proline
content (*60 lmol/g FW) were the highest values
measured in this study. The increases in proline content
were correlated with the increases in the steady-state
transcript level of MtP5CS3 in the wild type, as
monitored by RT-PCR (Fig. 2f). In Mtp5cs3, the
MtP5CS3 transcript was undetectable, although that
(A) (B)
WT p3 WT p3
Control 75 mM NaCl
2 cm
WT
(C) (D)
s100 mM NaCl
WT p3 p3
(E) (F)
WT p3 WT p3
+ Pro NaCl + Pro
(G)
WT p5cs3 WT p5cs3
150 mM NaClUntreated
Fig. 1 Phenotypes of the Mtp5cs3 mutant under salinity and the
effects of proline supplementation. a–d Plant responses to salt
stress. Two-day-old wild-type (WT) and mutant (p3) seedlings
were transferred onto pouch paper laid on top of mFM agar
medium with or without 75 mM (a, b) or 100 mM (c, d) of
NaCl. Primary root lengths were measured at 2-day intervals (b,
d). Close-up views of the shoots are shown (c, right). e–f Effects
of supplementing proline (1 mM) to the plants in the absence or
presence of NaCl (75 mM). In b, d, and f, bars indicate standard
errors. g Greenhouse-grown 4-week-old wild type and mutant
plants were treated with or without 150 mM NaCl for 16 days
and removed from soil for the whole plants to be photographed
Euphytica (2013) 193:101–120 107
123

8. of MtP5CS3a, a splice variant (Supplementary Fig.
S1a), accumulated at low levels regardless of stresses.
These results indicate that Mtp5cs3 was less efficient in
early accumulation of proline in response to drought or
salinity than the wild type.
To further assess the effects of Mtp5cs3 mutation,
plants were grown with or without PEG. Under
150–200 g/L of PEG, Mtp5cs3 showed a slower root
elongation rate than the wild type (Fig. 2g), accumu-
lated much less (2-fold) proline than the wild type (7-
fold) (Fig. 2g, right), and exhibited 2 to 3-fold
decreases in the survival rate (Table 1). Moreover,
under 150 g/L of PEG, Mtp5cs3 suffered from elec-
trolyte leakage slightly more severely than the wild
WT p3
Control 100 g/L PEG 100 mM NaCl
WT p3 WT p3
2 cm
(J)
(H) (I)
(A)
Untreated
150 mM NaCl
10 cm
WT Mtp5cs3
Control PEG
p5cs3WT p5cs3WT
p3WT
5 cm
(G)
(B) (D)(C)
C S3 S6 D C S3 S6 D
0
20
40
60
∗∗∗
∗∗∗
∗
∗∗∗
∗∗∗
∗ns ns
Root Shoot
Proline(µmoles/gfw)
C PEG
0
2
4
WT
p5cs3
∗∗∗
ns
Greenhouse Hydroponic
0
10
20
30
40
50
WT
p5cs3
∗∗∗
Control 150 g/L PEG
Root Shoot Root Shoot
H2O2(ng/gfw)
(F)(E)
Fig. 2 Sensitivity of the Mtp5cs3 mutant to salt, drought, or
low water potential stress. a Four-week-old wild type and
mutant plants grown in a greenhouse were treated with 150 mM
NaCl for 16 days or not watered for 7 days. b–d Time course of
changes in fresh weight (b) and dry weight (c) of plants under
salinity and fresh weight of plants under drought stress (d).
e Proline contents in roots (R) and shoots (Sh) of salt (200 mM
NaCl)-, drought-, or PEG-treated plants. For drought treatment,
8-week-old plants were unwatered for 5 days (left). For PEG
treatment, 2-day-old seedlings were hydroponically grown in
the presence of 200 g/L of PEG for 13 days (right). f RT-PCR
analysis of MtP5CS3 and MtP5CS3a transcript levels in
drought-treated plants. Amplified and agarose gel-separated
154-bp (MtP5CS3), 145-bp (MtP5CS3a), 181-bp (DR-A17; a
dehydrin gene of A17), and 262-bp (MtACTIN; b-ACTIN2 of M.
truncatula) DNA fragments are shown (Supplementary Table
S1). g Plant responses to low water potential stress. Two-day-old
seedlings were transferred onto mFM agar medium with or
without 150 g/L PEG, and grown for 1 month. Shoot tips after
treatment are shown in the insets. h Electrolyte leakage
measured from the leaves in g. i H2O2 contents measured from
the plants (p3, Mtp5cs3) in g. j Detection of H2O2 by DAB
staining of seedlings treated with PEG or NaCl for 4 days. In
e and i, asterisks denote statistical significance between
genotypes as determined by two-way analysis of variance
(ANOVA) test followed by Bonferroni posttest (*P 0.05,
**P 0.01, and ***P 0.001)
108 Euphytica (2013) 193:101–120
123

9. type, although the measured values fell within the
range of errors (Fig. 2h). Mtp5cs3 also accumulated
H2O2 by 1.6 times more than the wild type (Fig. 2i).
When H2O2 was colorimetrically detected, Mtp5cs3
roots showed stronger reddish-brown color than the
wild type (Fig. 2j). Apparently, all these changes were
associated with cotyledon opening responses—
roughly a half of the Mtp5cs3 cotyledons remained
unopened under PEG treatment while most wild type
cotyledons were fully open (not shown).
The expression profiles of the three MtP5CS genes
were monitored in the Mtp5cs3 mutant using real-time
PCR and RNA gel blot analysis (Fig. 3). To discrim-
inate each transcript, gene-specific probes were used
(Supplementary Fig. S1a). Although the probe for
MtP5CS3a was expected to hybridize with both
MtP5CS3 and MtP5CS3a transcripts, their signals
were distinguishable due to different transcript lengths
of the two isoforms (Fig. 3c, f). Under high salinity,
the transcript levels of MtP5CS1 and MtP5CS2
changed little in the wild type or Mtp5cs3, whereas
that of MtP5CS3 increased 125-fold in shoots and
23-fold in roots of the wild type (Fig. 3a, b). While no
MtP5CS3 transcript was detected in the Mtp5cs3
mutant, that of MtP5CS3a increased approximately
6-fold in Mtp5cs3, although its absolute quantity was
significantly lower than that of MtP5CS3 (Fig. 3c).
Under drought condition, the transcript level of
Table 1 Survival rates of the wild type and Mtp5cs3 mutants
grown in a greenhouse, in vitro, or in a hydroponic container
under various stress conditions
Treatment Survival rate (%)
Wild type Mtp5cs3
150 mM NaCl for 16 days
(greenhouse)
95.8 62.5
Recovery after 7-day drought
treatment (greenhouse)
93.8 68.8
150 g/L PEG on mFM agar for
1 month (in vitro)
84.4 46.9
200 g/L PEG in a hydroponic
chamber for 13 days
(hydroponic)
62.2 32.4
Drought
(F)
(C)
NaCl
(A) (B)
(D) (E)
WT
WT
Mtp5cs3
Mtp5cs3
Fig. 3 Expression of the three MtP5CS genes in the wild type
and Mtp5cs mutant under salt and drought stresses. Plants were
treated with 150 mM NaCl for 8 days or drought for 4–7 days,
and the transcript levels of MtP5CS1, MtP5CS2, MtP5CS3, and
MtP5CS3a were estimated. Results from real-time PCR and
RNA gel blot analyses of MtP5CS transcript levels in salt-
treated (a–c) or drought-treated (d–f) plants are shown.
MtCorA1 was used as a drought-responsive marker. Thin bars
indicate standard errors. In c and f, total RNAs, 20 lg per lane,
were agarose gel-separated, transferred to nylon filter, and
hybridized with MtP5CS3- (top panel) or MtP5CS3a-specific
(middle panel) probe. Note that the MtP5CS3a probe hybridizes
with either MtP5CS3 or MtP5CS3a mRNA. Ethidium bromide
(EtBr)-stained gels (bottom panel) show equally loaded RNA.
‘‘–’’ untreated (control), ‘‘?’’ treated
Euphytica (2013) 193:101–120 109
123

10. MtP5CS3 increased 27-fold in the wild type, although
as in salt treatment, those of MtP5CS1 and MtP5CS2
changed little (2-fold). The MtP5CS3a transcript
accumulated 26-fold in Mtp5cs3, but not in the wild
type (Fig. 3d–f). These results indicate that MtP5CS3,
among others, was most strongly expressed and its
encoded protein was most likely responsible for
proline accumulation under drought or high salt stress
condition. To verify this, P5CS enzyme activity was
measured from the wild type and Mtp5cs3 under
drought or salinity. In both shoots and roots, P5CS
activities of Mtp5cs3 were slightly lower than those of
the wild type under most conditions. The magnitudes
were statistically significant, however, only in the
measurements made with 1-day salinity-treated roots,
8-day salinity-treated shoots, and 4-day drought-
treated shoots (Supplementary Fig. S2).
At low temperature (4 °C), Mtp5cs3 showed
reduced growth, especially of root (Supplementary
Fig. S3a). However, proline contents of Mtp5cs3
remained at similar levels to those of the wild type
(Supplementary Fig. S3b). When cold-acclimated
(4 °C for 7 days) leaves were subjected to freezing
treatment, no significant difference was observed in
electrolyte leakage between the wild type and the
Mtp5cs3 mutant, although the unacclimated wild type
showed higher levels of electrolyte leakage than the
unacclimated Mtp5cs3 at -2 to -6 °C (Supplemen-
tary Fig. S3c). When cold-acclimated plants were
successively transferred to a freezing temperature,
4 °C, and back to room temperature, only a few wild
type plants recovered (3/20), whereas none of Mtp5cs3
did (0/20) (Supplementary Fig. S3d).
Enhancement of osmotic stress tolerance
by ectopic expression of MtP5CS3
To examine whether the defective phenotypes of
Mtp5cs3 could be restored by the wild-type allele
(MtP5CS3), a construct for expressing MtP5CS3 (35S–
P5CS3), in which GFP was not fused to the full-length
cDNA toMtP5CS3butplaceddistantly,wasintroduced
into the mutant by A. rhizogenes-mediated transforma-
tion. The resulting composite transgenic plant in which
the wild type MtP5CS3 cDNA was ectopically
expressed (p3_35S–P5CS3) grew significantly more
robustly than the mutant reporter control (p3_35S–
GUS) or the wild type reporter control (WT_35S–GUS)
under PEG treatment (Fig. 4a). Its proline contents
were more than twice and 128 % those of the mutant
reporter control under 100 and 150 g/L of PEG,
respectively (Fig. 4b). However, electrolyte leakage
from roots and shoots was not significantly different
between the composite transgenic plants except that
electrolyte leakage from the roots of p3_35S–P5CS3
showed significant difference from that from the roots
of the mutant reporter control (p3_35S–GUS) when
placed under 150 g/L PEG (P 0.01) (Fig. 4c). The
effects on electrolyte leakage in shoots were not
significant (P = 0.06) possibly due to large variations
in shoot growth (Fig. 4c). These results indicate that
ectopic expression of MtP5CS3 in the mutant back-
ground complemented P5CS activity, restoring the
mutant’s tolerance to PEG treatment, at least in roots.
MtP5CS3 was also overexpressed in the wild-type
M. truncatula A17 and the effects on tolerance to
osmotic stresses were examined. The transgenic over-
expresser (A17_35S-P5CS3) grew more robustly than
the wild type reporter control (WT_35S–GUS) under
salinity (Fig. 4d). Under 100 g/L PEG, the FWs of
roots and shoots of the overexpresser were significantly
higher than those of the wild type reporter control. A
particularly large reduction was observed in shoot
growth of the wild type reporter control compared with
the overexpresser (Fig. 4e). The proline content of the
overexpresser increased by 16-fold under PEG treat-
ment while only by 10-fold in the wild type reporter
control (Fig. 4f). The overexpresser showed signifi-
cantly less premature senescence and higher chloro-
phyll ab contents than the wild type reporter control
(Fig. 4g). The roots and shoots of the overexpresser
also suffered from 20 % (NaCl) and 50 % (PEG) less
electrolyte leakage than the wild type reporter control,
although the measured values fell within the range of
errors (Fig. 4h). Finally, the transcript level of
MtP5CS3 was substantially higher in the roots of the
overexpresser than in the wild type reporter control
under PEG (Fig. 4i). These results indicate that
overexpression of MtP5CS3 effectively enhanced the
tolerance of M. truncatula to osmotic stresses.
Stress-induced metabolic changes in the wild type
and the mutant
To evaluate possible effects of the Mtp5cs3 mutation
on the accumulation of global metabolites under
osmotic stress, metabolomic analysis was carried
out. The wild type and Mtp5cs3 mutant placed under
110 Euphytica (2013) 193:101–120
123

11. drought condition were collected in three replicates
and each subjected to the GC/MS-based analysis. The
resulting nonbiased metabolite profiles had 58 soluble
metabolites including amino acids, nitrogenous com-
pounds, organic acids, fatty acids, sugars, and polyols,
approximately 65 % of which were detected in
significant quantities (Supplementary Table S3). Prin-
cipal component analysis (PCA) indicated that the first
component (PC1) clearly distinguished root versus
shoot by covering 33.4 % of the total variance, while
another first component discriminated drought
treatment versus control by covering 23.1 % of the
total variance (Fig. 5). Although further discrimina-
tion by the second component (PC2) was statistically
significant only with the root (Supplementary Fig.
S4a), not with the shoot sample (Supplementary Fig.
S4b), the analysis of variance (ANOVA) test was
carried out with the data from either root or shoot
metabolome (Supplementary Table S3).
To estimate possible differences in the relative
change in each metabolite level between the wild type
and the Mtp5cs3 mutant, Z-scores of the relative
(A)
Control +150 g/l PEG GFP
(C)(B)
(H)
(D)
GFPControl +100 mM NaCl
(E) (F) (G)
(I)
0
1
2
3
4
5
WT-GUS
p5cs3-GUS
p5cs3-P5CS3
control 100 g/L 150 g/L
∗∗∗
∗
∗∗∗
∗∗∗
∗∗
ns
PEG
Proline(μmoles/gfw)
Control 100 g/l PEG 150 g/L PEG
0
10
20
30
40
50
WT-GUS-R
p3-GUS-R
p3-P5CS3-R WT-GUS-Sh
p3-GUS-Sh
p3-P5CS3-Sh
∗∗
Electrolyteleakage(%)
0.00
0.05
0.10
0.15
0.20
Control
100 g/L PEG
A17-GUS A17-P5CS3
Root Shoot
∗∗∗
Root Shoot
Freshweight(g)
Control NaCl PEG
0
5
10
15
20 A17-GUS
A17-P5CS3
∗∗∗
Proline(µmoles/gfw)
Control NaCl
0.0
0.5
1.0
1.5
A17-GUS
A17-P5CS3
∗∗∗
TotalChl.ab(µg/mgfw)
Fig. 4 Effects of ectopic expression of the wild type gene on
plant responses to salinity and low water potential stresses. a–
c Ectopic expression of MtP5CS3 in the Mtp5cs3 mutant. Binary
plasmids carrying GUS or the wild-type MtP5CS3 gene under
CaMV 35S promoter were introduced into the wild type or
Mtp5cs3 via A. rhizogenes-mediated root transformation. a The
resulting WT_GUS, p3_GUS, and p3_P5CS3 transgenic plants,
selected by GFP expression (right panel) in 4 weeks, were
treated with 100 or 150 g/L of PEG for 5 days. b Free proline
contents of the treated plants. c Electrolyte leakage of the treated
plants (R, root; Sh, shoot). d–i Effects of overexpressing
MtP5CS3 in M. truncatula A17. d Composite transgenic plants
generated by hairy root transformation of 35S-GUS or 35S-
MtP5CS3–carrying binary plasmids were selected by GFP
signals in 4 weeks (right panel) and treated with 100 mM of
NaCl for 10 days or 100–150 g/L of PEG for 4 days (not
shown). e Quantification of fresh weights of the plants in
d. f Free proline contents of the treated plants. g Total
chlorophyll contents of the treated plants. h Electrolyte leakage
of the treated plants in d (R, root; Sh, shoot). i RT-PCR analysis
of MtP5CS2 and MtP5CS3 transcript levels in the PEG-treated
plants. Asterisks denote statistical significance between geno-
types as determined by two-way ANOVA test followed by
Bonferroni posttest (*P 0.05, **P 0.01, and ***P
0.001)
Euphytica (2013) 193:101–120 111
123

12. intensities of each metabolite were calculated and used
to construct a heat map of 56 quantified metabolites
(Fig. 6). The heat map showed that, in roots, both the
wild type and Mtp5cs3 showed substantial changes in
metabolite accumulation under drought. The trends of
changes in the accumulation pattern were generally
common for the wild type and the mutant for each
metabolite group. Most amino acids accumulated
highly under drought condition. Eight and ten of them
accumulated more abundantly in the stressed wild type
and Mtp5cs3 roots, respectively, with the extents of
fold changes slightly higher in the Mtp5cs3 mutant
than in the wild type. Other nitrogenous compounds,
such as putrescine, urea, and uric acids, accumulated
less abundantly under drought in either genotype. In
contrast, carbohydrates and polyols accumulated
abundantly in the two genotypes under drought
condition, while the accumulation patterns of organic
and fatty acids were not consistent. The shoot
metabolome also exhibited drought-induced changes
largely similar to those of the root metabolome, but the
differential accumulation pattern of each metabolite
between the wild type and the mutant was not
consistent with that of the root metabolome. To
provide an alternative perspective of the metabolomic
data, logarithmic ratios of the measured intensities for
each metabolite between drought-treated and
untreated samples were calculated, and the resulting
values were hierarchically clustered in a diagram for
roots and shoots (Supplementary Fig. S5). Together,
these results indicate that the global metabolite
composition of roots changed markedly in response
to drought and the patterns were common for both
genotypes but often more pronounced in the mutant
than in the wild type.
To show the possible relationship among the
differentially accumulated metabolites, a diagram
was constructed in which individual metabolites were
connected with the central metabolic pathway
(Fig. 7). The extent of contribution of each metabolite
to the whole metabolome was variable. Significantly
decreased accumulation of proline was observed in the
Mtp5cs3 mutant under drought condition, which
presumably reflected the lowered biosynthetic activity
due to the absence of the MtP5CS3 enzyme. Consis-
tently, aspartate and glutamate, the precursors of
asparagine, glutamine, and proline, accumulated more
abundantly in the Mtp5cs3 mutant than in the wild type
under drought condition. Similar patterns of change
were observed in alanine, lysine, methionine, threo-
nine, isoleucine, and valine, all of which are synthe-
sized from pyruvate, aspartate, or both, although
leucine was not detected at a significant level.
Phenylalanine and tryptophan, the products of
Fig. 5 Principal component analysis (PCA) showing the
patterns of 58 metabolites that accumulated in M. truncatula
seedlings after drought treatment. Four-week-old wild type and
Mtp5cs3 mutant plants were treated with or without drought for
5 days and roots and shoots that were separately harvested were
subjected to GC/MS. The results from PCA of all eight samples
including the wild type and mutant roots and shoots with or
without drought treatment are shown. W wild type, p mutant,
R root, Sh shoot
112 Euphytica (2013) 193:101–120
123

13. aromatic amino acid biosynthetic pathway, accumu-
lated 1.5-fold under drought in both genotypes.
Among carbohydrates, glucose and galactose accu-
mulated 1.5-fold in both genotypes under drought
condition. However, fructose, one of the precursors of
sucrose (detected as glucopyranoside herein), showed
little change under drought in either genotype. Nota-
bly, the highest accumulation was observed with
carbohydrate-derived polyols, including inositol,
glycerol, mannitol, and ribitol, the major osmoprotec-
tants described previously (Hare et al. 1988).
The metabolites that showed relatively large quan-
titative changes under drought condition also showed
comparatively high values of relative GC/MS inten-
sities. Particularly, the sum of the relative intensities of
asparagine, isoleucine, proline, and valine amounted
to approximately 90 % of the total amino acid
intensities. Likewise, the sum of the relative intensities
of galactose, glucose, glucopyranoside, inositol, and
glycerol amounted to approximately 80 % of the total
intensities derived from sugars and polyols in the
stressed roots (Supplementary Table S3). Remarkably,
proline occupied approximately 70 % of the total
relative intensities of all amino acids in roots under
drought condition.
Discussion
The Mtp5cs3 mutant shows much compromised
tolerance to osmotic stress
Plants may possess duplicated genes of a biosynthetic
pathway that are often regulated differentially. The
functions of the isozymes are not always identical to
ensure avoidance of possible catastrophic effects
resulting from gene mutation. In Arabidopsis, it has
been known that AtP5CS1 is strongly induced by
dehydration, high salinity, and ABA, whereas
AtP5CS2 is expressed in dividing cells and during
incompatible pathogenic interaction (A´ braha´m et al.
2003). A knockout mutant of AtP5CS1 is defective in
salinity-induced proline synthesis, hypersensitive to
salt stress, and accumulates more ROS, while a
knockout mutant of AtP5CS2 is embryo abortive
(Sze´kely et al. 2008). Thus, proline synthesized in
different plant parts or subcellular compartments may
actually be involved in diverse cellular functions such
Fig. 6 Heat map of 56 quantified metabolites that accumulated in
shoots and roots of the wild type and Mtp5cs3 mutant plants
exposed to drought treatment. Z-scores derived from GC/MS
intensities measured for each metabolite are shown in eightsample
groups based on plant part, treatment, and genotype. Metabolites
are grouped in seven representative categories: (1) amino acids,(2)
other nitrogen compounds, (3) inorganic acids, (4) organic acids,
(5) fatty acids, (6) sugars, and (7) alcohols. In each group,
metabolites are placed hierarchically from higher (top) to lower
(bottom) GC/MS intensities. The Z-scores were calculated using
the equation, {x – mean (x)}/sd (x), wherein x and sd denote
measured intensity and standard deviation, respectively. The map
was generated in R language using the heatmap method (http://
www.r-project.org/). - normal condition, D drought condition
Euphytica (2013) 193:101–120 113
123

14. as osmotic, structural, and redox adjustments, which
collectively contribute to osmotic stress tolerance
(Szabados and Savoure´ 2010). Recently, a third P5CS
gene (MtP5CS3) predominantly expressed under abi-
otic stresses was first discovered in M. truncatula
(Kim and Nam 2013), and its loss-of-function mutant
(Mtp5cs3) has been analyzed in this study.
Despite nearly normal phenotypes under optimal
growth conditions (Supplementary Fig. S1c), the
Mtp5cs3 mutant exhibited considerably lower sizes
and weights of fruits and seeds than the wild type
(Supplementary Fig. S1e). It has been reported that
proline tends to accumulate highly in flowers and flower
buds of M. truncatula (Armengaud et al. 2004), in
immature seeds of Vicia faba (Venekamp and Koot
1984), and in mature seeds of Arabidopsis (Schmidt
et al. 2007). Consistently, flowering, embryogenesis,
and seed sets have been shown to be more sensitive to
proline deficiency (Mattioli et al. 2009). Thus, repro-
ductive organs appear to demand a high level of proline
and be sensitive to a moderate level of proline
deficiency from which other organs may not suffer
seriously. Although it has been shown that proline plays
an important role in providing dessication tolerance in
seeds (Sze´kely et al. 2008), the germination rate of the
Mtp5cs3 mutant seeds estimated in this study
0
1
2
3
4
5
Asparagine
0.0
0.5
1.0
1.5
Aspartate
Isoleucine
0.0
0.5
1.0
1.5
2.0
Valine
0
1
2
3
Lysine
0.00
0.05
0.10
0.15
0.20
0.25
Serine
0.0
0.5
1.0
1.5
2.0
Glutamine
0.00
0.05
0.10
0.15
Threonine
0.0
0.2
0.4
0.6
0.8
0.00
0.02
0.04
0.06
0.08
0.10
Alanine
Tryptophan
0.0
0.1
0.2
0.3
0.4
0.5
Mannose
0.0
0.1
0.2
0.3
0.4
Galactose
0
1
2
3
4
5
Ribose
0.00
0.05
0.10
0.15
Glycerol
0.0
0.1
0.2
0.3
Ribitol
0.00
0.02
0.04
0.06
Mannitol
0.00
0.02
0.04
0.06
0.08
Inositol
0.0
0.2
0.4
0.6
0.8
1.0
Pyruvate
PEP
3PGA
Glucose-6-P
LeucineND
Glycine NS
Arabinose NS
FructoseNS
Glucose
0
1
2
3
Sucrose
Urea
0.0
0.1
0.2
0.3
Glutamate
0.0
0.2
0.4
0.6
Proline
0
10
20
30
40
Methionine
0.00
WT-Control p3-Control p3-DroughtWT-Drought
0.05
0.10
0.15
TCA cycle
0.00
0.05
0.10
0.15
Phenylalanine
Fig. 7 Metabolic
relationship among
compounds that showed
changes in the relative
quantities in drought-treated
M. truncatula roots. A
subset of the most
discriminatory organic
compounds that contributed
to differential drought stress
responses between the wild
type and Mtp5cs3 mutant
roots is shown. The
catabolic pathway of
glucose is shown at center
(bold line). Abbreviated
biosynthetic pathways to the
individual metabolites are
shown as straight (single
reaction) or broken
(multiple reactions) arrows.
WT the wild type, p3
Mtp5cs3. Thin lines indicate
standard errors (n = 3). NS
not significantly different
between WT and Mtp5cs3
114 Euphytica (2013) 193:101–120
123

15. (95–98 %) was not significantly different from that of
the wild type under normal condition.
Under drought and salinity conditions, the Mtp5cs3
mutant showed significantly compromised tolerance
compared to the wild type (Figs. 1, 2). Mtp5cs3 could
not accumulate proline adequately, showed much
lower biomass, more senescing young leaves, and a
lower survival rate than the wild type (Table 1). Under
PEG treatment, the Mtp5cs3 mutant accumulated
proline only two times that of the untreated control,
while the wild type up to seven times (Fig. 2e). The
Mtp5cs3 mutant also showed more unopened cotyle-
dons than the wild type, and generated more H2O2
(Fig. 2i, j), a main component of ROS (Matysik et al.
2002). Similarly, the compromised proline accumula-
tion in the Atp5cs1 mutant resulted in reduced
activities of key antioxidant enzymes involved in the
glutathione-ascorbate cycle and increased ROS accu-
mulation, which consequently led to more severe
oxidative damage (Sze´kely et al. 2008). Nevertheless,
externally supplied proline during salt treatment
increased lateral root number and total lateral root
length (Fig. 1a–f). Consistent with our study, other
researchers reported that externally supplied proline
helped the Atp5cs1 mutant to overcome its salt
hypersensitivity in Arabidopsis (Sze´kely et al. 2008)
and proline supplied to osmotically stressed rice calli
increased their growth in vitro (Kishor 1989). These
results indicate that proline deficiency caused by the
absence of the MtP5CS3 enzyme exerted a fairly large
negative effect on the plant’s proline accumulating
capacity and tolerance to abiotic stresses such as
drought, salinity, and PEG treatment.
The results that MtP5CS3 was most strongly
expressed among the three MtP5CS genes suggested
that its encoded protein was likely responsible for
proline accumulation under drought or salinity condi-
tion, P5CS enzyme activity, as determined from the
wild type and Mtp5cs3 under drought or salinity,
however, did not convincingly support these conclu-
sions.SignificantdifferencesinP5CSactivitywereonly
observed with 1-day salinity-treated roots, 8-day salin-
ity-treated shoots, and 4-day drought-treated shoots
(Supplementary Fig. S2). To closely monitor patterns of
change in enzyme activity along the time course after
treatment, it might have been helpful to carry out assay
with samples harvested at narrower time points. Alter-
natively, use of a more sensitive analytical method that
allows measuring P5CS activity more specifically in
plant extracts, such as one described by Parre et al.
(2010), would have been helpful for evaluating the
enzyme activity more accurately. Although our data fell
short of providing convincing evidence that the enzyme
activitycorrelatedwiththe elevated expressionlevelsof
MtP5CS3, the extents of decrease in proline content
were smaller than what would have resulted from the
lack of MtP5CS3 gene expression in the Mtp5cs3
mutant (Fig. 2e). These observations suggest that
activities other than P5CS be involved in maintaining
the proline level. For instance, MtOAT, a gene encoding
OAT, an enzyme catalyzing synthesis of proline from
ornithine, showed approximately 5-fold induction
under 5-day salinity treatment (Kim and Nam 2013).
Although its expression patterns have not been exam-
ined in the Mtp5cs3 mutant, it is possible that the lack of
MtP5CS3activityledtoincreasedexpressionofMtOAT
to compensate for the proline deficiency as a regulatory
response to the challenging stresses.
Unlike the expression of AtP5CS1, which was
induced by dehydration, high salt, and ABA treatment
but not by low temperature (Yoshiba et al. 1995), the
expression of MtP5CS3 was induced at 4 °C in 24 h
(Kim and Nam 2013). These results were supported in
part by our observation herein that the wild type and
Mtp5cs3 mutant plants grew differently at low tem-
perature. The growth of the mutant roots was affected
more severely by cold treatment (4 °C) (Supplemen-
tary Fig. S3a). Nonetheless, no significant change was
observed in proline content between the two geno-
types (Supplementary Fig. S3b). These results indicate
that at low temperature plants grew in a manner
apparently uncorrelated with proline content and the
observed decrease in plant growth was an indirect
consequence of Mtp5cs3 mutation. Concerning elec-
trolyte leakage at freezing temperature, the unaccli-
mated wild type plants showed slightly higher levels
of electrolyte leakage than the unacclimated Mtp5cs3
mutant. However, these results were only confined in a
narrow range of temperature (-2 to -4 °C), and there
was virtually no difference between the two genotypes
over the colder temperature range and when the two
genotypes were acclimated (Supplementary Fig. S3c).
These findings may have resulted from our prelimin-
ary treatment carried out without a programmed
acclimation stage as proposed previously (Thapa
et al. 2008). Together, our results suggest that
MtP5CS3 gene is unlikely associated with cold
tolerance.
Euphytica (2013) 193:101–120 115
123

16. The differential abiotic stress responses were also
associated with differential regulation of the three
MtP5CS genes. Under drought and salinity conditions,
the expression of MtP5CS3 was highly induced, while
those of MtP5CS1 and MtP5CS2 remained nearly
constant (Fig. 3). However, the transcript level of
MtP5CS3a, a splice variant apparently transcribed
from a separate promoter (Kim and Nam 2013),
remained constant with or without stresses in the wild
type but showed considerable induction under drought
and salinity in the Mtp5cs3 mutant (Fig. 2f; 3). This
observation, apparently contradictory to the previous
observation that MtP5CS3a was expressed rather
constitutively (Kim and Nam 2013), could possibly
be due to different time points selected for sampling
after treatment. Nonetheless, the increased expression
of MtP5CS3a was barely sufficient to compensate for
the absence of the canonical MtP5CS3 transcript in the
mutant. Alternatively spliced genes are an important
factor in plant responses to environmental stimuli. For
example, an alternatively spliced variant of AtP5CS1,
which lacked one of the canonical exons in the mature
transcript, constituted a half of the total AtP5CS1
transcript population and was related to the natural
variation in proline and climate adaptation (Kesari
et al. 2012). Thus, it is necessary to carefully examine
the expression patterns and roles of possible MtP5CS
splice variant(s) to fully understand the regulation of
these proline biosynthetic genes.
Mtp5cs3 mutation augments drought-induced
metabolic changes in roots
Recently, the use of a mutant for metabolomic analysis
has been particularly useful for efforts to dissect the
complex regulatory network (Saito and Matsuda
2010). For example, a knockout mutant of a gene
responsible for ABA-mediated drought response
showed that the accumulation patterns of amino acids
and sugars changed dramatically via different meta-
bolic networks modulated coordinately in response to
drought stress (Urano et al. 2009). Here, we examined
the effects of the mutation of MtP5CS3 gene on the
overall metabolism. The wild type and Mtp5cs3 roots
accumulated dozens of low-molecular-weight metab-
olites under drought condition, many of which had
been considered as compatible osmolytes. The result-
ing changes in the metabolite pools mostly depended
on the treatment (drought or none) as well as on the
genotype as shown by PCA analysis (Fig. 5, Supple-
mentary Fig. S4). With a few exceptions, however,
metabolic changes that occurred in the Mtp5cs3
mutant were similar to those of the wild type. The
most notable changes were observed in amino acids,
sugars, and polyols, and the extents of the changes
were generally larger in the Mtp5cs3 mutant than in
the wild type.
An increased accumulation of amino acids and the
enzymes catalyzing their breakdown has been associ-
ated with osmotically stressed plants (Rizhsky et al.
2002; Less and Galili 2008; Lugan et al. 2010).
However, glutamate and glutamine, the two amino
acids directly participating in nitrogen assimilation
pathways, decreased in the wild type but increased in
the Mtp5cs3 mutant under drought condition. Similar
observations were made for asparagine and glutamine,
which accumulated in L. japonicus under saline
condition (Sanchez et al. 2008b). This finding was
attributed to the reduced capacity of ammonia assim-
ilation through the conserved glutamine synthetase
(GS)-glutamate synthase (GOGAT) pathway, because
the GS/GOGAT activity reportedly decreased under
salinity and drought in ABA-dependent or indepen-
dent manner (Debouba et al. 2007; Planchet et al.
2011). The general increase in the accumulation of
amino acids could also have resulted from increased
degradation of proteins. Taking into account that ROS
generated during osmotic stress could cause oxidation
of proteins such as those involved in major cellular
activities, the higher accumulation of H2O2 in the
Mtp5cs3 mutant than in the wild type under low water
potential stress (Fig. 2) could have caused concomi-
tantly high accumulation of amino acids in the mutant
(Sze´kely et al. 2008). Such detrimental effect of
protein breakdown might have affected the photosyn-
thetic ability and growth of the mutant more severely
than the wild type, as shown by active degradation of
rubisco in drought response of alfalfa leaves (Aranju-
elo et al. 2011). However, recent expression profiling
of all Arabidopsis proteases suggested that general
protein degradation did not contribute significantly to
amino acid accumulation in response to drought and
other abiotic stresses (Less and Galili 2008). Rather, it
is differential regulation of protein breakdown
machinery such as specific proteases and E3 ubiquitin
ligases that would more likely have caused increased
degradation of certain proteins leading to elevated
amino acid pools (Degenkolbe et al. 2009).
116 Euphytica (2013) 193:101–120
123

17. Alternatively, decreased synthesis of certain proteins
from altered metabolism of proline and its precursors
(Szabados and Savoure´ 2010) or increased synthesis of
branched-chain amino acids such as isoleucine (Joshi
and Jander 2009) may have contributed to an increase
in amino acids that have been hypothesized to act as
osmolytes. Meanwhile, a decreased accumulation of
nitrogenous compounds including growth regulators
such as indole may reflect the mutant’s lowered
demand for growth compared to the wild type under
drought condition (Lugan et al. 2010). Nonetheless,
the precise roles of changes in the amounts of amino
acids and nitrogenous compounds during osmotic
stress remain to be elucidated.
It has been suggested that proline can significantly
contribute to restoring osmotic homeostasis (Hare and
Cress 1997). Herein, proline accumulated most abun-
dantly among all amino acids in both genotypes under
drought condition. The content of this stress-induced
amino acid increased more highly in the wild type than
in the mutant. Since glutamate is a substrate for proline
biosynthesis, an increased demand for proline accu-
mulation as a response to osmotic stress would exploit
the pools of glutamate and glutamine. Thus, changes
in glutamate and glutamine levels would reflect the
intrinsic flux changes that occur to support the
elevated biosynthesis of proline. The absence of
MtP5CS3 activity in the mutant would have lowered
such requirement for glutamine and glutamate, and
consequently, led to elevated levels of the two amino
acids (Fig. 7).
The wild type and the Mtp5cs3 mutant also accu-
mulated significantly high levels of sugars and polyols
under drought condition. Particularly, substantial
increases in the level of galactose, glucose, glycerol,
and mannitol were observed in drought-stressed
mutant roots (Fig. 6). These results indicate that
carbohydrate metabolites accumulated to compensate
for the decreased level of proline as an osmoprotectant
in drought-stressed root cells (Munns and Tester 2008).
However, the level of glucopyranoside, a significant
proportion of which would represent the disaccharide
sucrose, showed an exceptionally opposite pattern of
accumulation (Supplementary Table S3 and Supple-
mentary Fig. S5). This appears to be related to the
lower photosynthetic ability of the mutant, as shown by
its retarded growth (Fig. 2) and reduced chlorophyll
content (Fig. 4), and as a result, the lower capacity to
transport sucrose synthesized in leaves to the stressed
roots. Consistent with this hypothesis, generally larger
extents of metabolite change were observed in roots
than in shoots, indicating that rootswere more sensitive
than shoots to the relatively mild and transient drought
condition applied in this study.
In general, the observed metabolic changes were
similar to what has been reported from other legume
plants. In a systems comparison of two alfalfa cultivars
which differed in drought tolerance, metabolites such
as raffinose, galactinol, inositol, and glucopyranoside
accumulated more highly in the drought-tolerant
cultivar (Kang et al. 2011). In contrast, the accumu-
lation of most organic acids decreased. Just as in this
study, proline accumulated most highly among the
analyzed metabolites regardless of the cultivar, indi-
cating its conserved role in drought tolerance (Kang
et al. 2011). In another comparative study with six
Lotus species under drought condition, the observed
metabolic changes were similarly conserved among
species (Sanchez et al. 2012). Particularly, metabolites
such as proline, fructose, glucose, and maltose accu-
mulated more than 5-fold, while the accumulation of
most amino acids decreased regardless of species.
Taken together, drought-induced accumulation of
sugars and carbohydrate polyols as well as proline
was broadly conserved among legume cultivars and
species. Since all of the changes associated with the
observed metabolite accumulation were quantitative,
qualitative characterization of certain metabolites as
indicators of drought tolerance was not possible
(Sanchez et al. 2012).
We showed that Mtp5cs3, a knockout mutant of an
amino acid biosynthetic gene, resulted in a fairly broad
range of metabolite changes at least in roots. These
results reinforce the importance of proline and its
biosynthetic enzymes in the regulation of cellular
homeostasis against osmotic stress. In particular,
MtP5CS3 is only one of the three isozymes that can
be synthesized from the MtP5CS gene family, and our
results corroborate the previous finding that this third
copy of MtP5CS gene, the first example of a third
P5CS gene in plants (Kim and Nam 2013), plays a
crucial role in osmotic stress regulation in legumes.
Thus, M. truncatula responds to drought stress by
altering metabolism of amino acids, sugars, and
polyols, which ultimately results in changes in global
metabolic profiles.
Euphytica (2013) 193:101–120 117
123

18. The MtP5CS3 gene plays a critical role
in regulating osmotic tolerance of M. truncatula
Numerous studies showed that overexpression of
homologous or heterologous P5CS genes enhanced
tolerance of the transgenic plants to osmotic stress in
various species. A gene from moth bean (Vigna
aconitifolia) increased proline accumulation, leading
to the tolerance to salt and water stress in grasses (Su
and Wu 2004; Vendruscolo et al. 2007). Transgenic
plants expressing a mutated V. aconitifolia P5CS gene
(P5CSF129A), whose feedback inhibition site was
disrupted by a replacement of a phenylalanine residue
with alanine, resulted in higher proline accumulation
and increased osmotolerance than those overexpress-
ing the wild type gene (Hong et al. 2000). In M.
truncatula, an overexpressed V. aconitifolia P5CS
gene enhanced proline accumulation and osmotoler-
ance during nitrogen fixation (Verdoy et al. 2006). In
this study, we also showed that the mutant’s suscep-
tibility to water stress was significantly restored by
ectopic expression of the wild type MtP5CS3 gene
(Fig. 4). However, accumulation of a higher level of
proline under low water potential stress than under
salinity stress (Fig. 4b, f) may be due to the ion uptake
and its accumulation that likely compromised the need
of proline accumulation under salinity stress (Sharma
and Verslues 2010).
Although overexpression of P5CS genes has rou-
tinely been associated with increased osmotic stress
tolerance, the proline level resulting from such ectopic
expression often did not reach a critical level high
enough to provide adequate osmotic tolerance unless
the apparatus for feedback inhibition was removed
(Hong et al. 2000). Moreover, overabundance of
proline is also toxic to cellular metabolism (Nanjo
et al. 2003). Thus, rather than simply overexpressing
proline biosynthetic genes to enhance stability of
plants, manipulating the ability to increase the rate of
proline synthesis was suggested to be crucial for
engineering stress-tolerant plants (Szabados and Sa-
voure´ 2010). The three P5CS genes of M. truncatula
can potentially be useful for meeting the demand for a
higher level of proline as a protecting molecule under
osmotic stress.
We previously showed that the Mtp5cs3 mutation
exerted negative effects on nitrogen fixing ability of M.
truncatulaunder saline condition (Kim andNam 2013).
In an aeroponic container, the Mtp5cs3 mutant formed
significantly fewer nodules than the wild type upon
inoculation with Sinorhizobium meliloti (Supplemen-
tary Fig. S6a). Although nodules that formed on the
roots of the two genotypes were similar in size and
shape (Supplementary Fig. S6b), they exhibited mark-
edly different nitrogen fixing abilities when the plants
were placed under mild NaCl treatment. Acetylene
assay revealed that nitrogenase activity of the nodules
on the Mtp5cs3 mutant decreased to 12 % while the
wild type retained 58 % of the activity under salinity
(Supplementary Fig. S6c). Similarly, remarkable accu-
mulation of proline in alfalfa nodules under drought
also suggested the importance of proline in nitrogen
fixation (Aranjuelo et al. 2011). Because nodule
symbiosomes also require proline at elevated levels as
an energysource (Kohl et al. 1988), the Mtp5cs3mutant
analyzed herein may provide an interesting opportunity
to investigate the roles of proline in stress tolerance
during symbiosis. The relative importance of this
legume-specific MtP5CS3 gene will be helpful for
efforts to improve legume crops by targeted manipu-
lation of proline biosynthetic genes.
References
A´ braha´m E, Rigo´ G, Sze´kely G, Nagy R, Koncz C, Szabados L
(2003) Light-dependent induction of proline biosynthesis
by abscisic acid and salt stress is inhibited by brassinos-
teroid in Arabidopsis. Plant Mol Biol 51:363–372
Alexieva V, Sergiev I, Mapelli S, Karanov E (2001) The effect of
drought and ultraviolet radiation on growth and stress
markersinpea and wheat. Plant, Cell Environ 24:1337–1344
Aranjuelo I, Molero G, Erice G, Avice JC, Nogue´s S (2011)
Plant physiology and proteomics reveals the leaf response
to drought in alfalfa (Medicago sativa L.). J Exp Bot
62:111–123
Armengaud P, Thiery L, Buhot N, Grenier de March G, Savoure´
A (2004) Transcriptional regulation of proline biosynthesis
in Medicago truncatula reveals developmental and envi-
ronmental specific features. Physiol Plant 120:442–450
Barker DG, Pfaff T, Moreau D, Groves E, Ruffel S, Lepetit M,
Whitehand S, Maillet F, Nair RM, Journet EP (2006)
Growing M. truncatula: choice of substrates and growth
conditions. In: Mathesius U (ed) The Medicago truncatula
Handbook. The Samuel Roberts Noble Foundation,
Ardmore
Boisson-Dernier A, Chabaud M, Garcia F, Be´card G, Rosenberg
C, Barker DG (2001) Hairy roots of Medicago truncatula
as tools for studying nitrogen-fixing and endomycorrhizal
symbioses. Mol Plant-Microbe Interact 14:639–700
Bradford MM (1976) Rapid and sensitive method for the quan-
titation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem 72:248–254
118 Euphytica (2013) 193:101–120
123

19. Chomczynski P, Sacchi N (1987) Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloro-
form extraction. Anal Biochem 162:156–159
Cook D, Fowler S, Fiehn O, Thomashow MF (2004) A promi-
nent role for the CBF cold response pathway in configuring
the low-temperature metabolome of Arabidopsis. Proc Natl
Acad Sci USA 101:15243–15248
Debouba M, Maaˆroufi-Dghimi MH, Suzuki A, Ghorbel MH,
Gouia H (2007) Changes in growth and activity of enzymes
involved in nitrate reduction and ammonium assimilation
in tomato seedlings in response to NaCl stress. Ann Bot
99:1143–1151
Degenkolbe T, Do PT, Zuther E, Repsilber D, Walther D,
Hincha DK, Kohl KI (2009) Expression profiling of rice
cultivars differing in their tolerance to long-term drought
stress. Plant Mol Biol 69:133–153
Delauney A, Verma DPS (1990) A soybean gene encoding delta
1-pyrroline-5-carboxylate reductase was isolated by func-
tional complementation in Escherichia coli and is found to
be osmoregulated. Mol Gen Genet 221:299–305
Deuschle K, Funck D, Forlani G, Stransky H, Biehl A, Leister D,
van der Graaff E, Kunze R, Frommer WB (2004) The role
of D1
-pyrroline-5-carboxylate dehydrogenase in proline
degradation. Plant Cell 16:3413–3425
Fujita T, Maggio A, Garcı´a-Rı´os M, Bressan RA, Csonka LN
(1998) Comparative analysis of the regulation of expres-
sion and structures of two evolutionarily divergent genes
for D1
-pyrroline-5-carboxylate synthetase from tomato.
Plant Physiol 118:661–674
Funck D, Stadelhofer B, Koch W (2008) Ornithine-d-amino-
transferase is essential for arginine catabolism but not for
proline biosynthesis. BMC Plant Biol 8:40
Gagneul D, Aı¨nouche A, Duhaze´ C, Lugan R, Larher FR, Bou-
chereau A (2007) A reassessment of the function of the so-
called compatible solutes in the halophytic plumbaginaceae
Limonium latifolium. Plant Physiol 144:1598–1611
Garcı´a-Rı´os M, Fujita T, LaRosa PC, Locy RD, Clithero JM,
Bressan RA, Csonka LN (1997) Cloning of a polycistronic
cDNA from tomato encoding c-glutamyl kinase and c-
glutamyl phosphate reductase. Proc Natl Acad Sci USA
94:8249–8254
Ginzberg I, Stein H, Kapulnik Y, Szabados L, Strizhov N, Schell
J, Koncz C, Zilberstein A (1998) Isolation and character-
ization of two different cDNAs of D1
-pyrroline-5-carbox-
ylate synthase in alfalfa, transcriptionally induced upon salt
stress. Plant Mol Biol 38:755–764
Handa S, Handa A, Hasegawa PM, Bressan RA (1986) Proline
accumulation and the adaptation of cultured plant cells to
water stress. Plant Physiol 80:938–945
Hare PD, Cress WA (1997) Metabolic implications of stress-
induced proline accumulation in plants. Plant Growth Re-
gul 21:79–102
Hare PD, Cress WA, Van Staden J (1988) Dissecting the roles of
osmolyte accumulation during stress. Plant, Cell Environ
21:535–553
Hellmann H, Funck D, Rentsch D, Frommer WB (2000) Hyper-
sensitivity of an Arabidopsis sugar signaling mutant toward
exogenous proline application. Plant Physiol 123:779–789
Hiscox JD, Israelstam GF (1979) A method for the extraction of
chlorophyll from leaf tissue without maceration. Can J Bot
51:1332–1334
Hong Z, Lakkineni K, Zhang Z, Verma DP (2000) Removal of
feedback inhibition of D1
-pyrroline-5-carboxylate synthe-
tase results in increased proline accumulation and protection
of plants from osmotic stress. Plant Physiol 122:1129–1136
Hu CAA, Delauney AJ, Verma DPS (1992) A bifunctional
enzyme (D1
-pyrroline-5-carboxylate synthetase) catalyses
the first two steps in proline biosynthesis in plants. Proc
Natl Acad Sci USA 89:9354–9358
Joshi V, Jander G (2009) Arabidopsis methionine gamma-lyase
is regulated according to isoleucine biosynthesis needs but
plays a subordinate role to threonine deaminase. Plant
Physiol 151:367–378
Kang Y, Han Y, Torres-Jerez I, Wang M, Tang Y, Monteros M,
Udvardi M (2011) System responses to long-term drought
and re-watering of two contrasting alfalfa varieties. Plant J
68:871–889
Karimi M, Inze´ D, Depicker A (2002) GATEWAY vectors for
Agrobacterium-mediated plant transformation. Trends
Plant Sci 7:193–195
Kesari R, Lasky JR, Villamor JG, Marais DLD, Chen YJC, Liu
TW, Lin W, Juenger TE, Verslues PE (2012) Intron-med-
iated alternative splicing of Arabidopsis P5CS1 and its
association with natural variation in proline and climate
adaptation. Proc Natl Acad Sci USA 109:9197–9202
Kim GB, Nam YW (2013) A novel D1
-pyrroline-5-carboxylate
synthetase gene of Medicago truncatula plays a predomi-
nant role in stress-induced proline accumulation during
symbiotic nitrogen fixation. J Plant Physiol 170:291–302
Kishor KPB (1989) Salt stress in cultured rice cells: effects of
proline and abscisic acid. Plant, Cell Environ 12:629–633
Kiyosue T, Yoshiba Y, Yamaguchi-Shinozaki K, Shinozaki K
(1996) A nuclear gene encoding mitochondrial proline
dehydrogenase, an enzyme involved in proline metabo-
lism, is upregulated by proline but downregulated by
dehydration in Arabidopsis. Plant Cell 8:1323–1335
Kohl DH, Schubert KR, Carter MB, Hagedorn CH, Shearer G
(1988) Proline metabolism in N2-fixing root nodules:
energy transfer and regulation of purine synthesis. Proc
Natl Acad Sci USA 85:2036–2040
Last RL, Jones AD, Shachar-Hill Y (2007) Towards the plant
metabolome and beyond. Nature Rev Mol Cell Biol
8:167–174
Less H, Galili G (2008) Principal transcriptional programs
regulating plant amino acid metabolism in response to
abiotic stresses. Plant Physiol 147:316–330
Lugan R, Niogret MF, Leport L, Gue´gan JP, Larher FR, Savoure´
A, Kopka J, Bouchereau A (2010) A metabolome and
water homeostasis analysis of Thellungiella salsuginea
suggests that dehydration tolerance is a key response to
osmotic stress in this halophyte. Plant J 64:215–229
Maggio A, Miyazaki S, Veronese P, Fujita T, Ibeas JI, Damsz B,
Narasimhan ML, Hasegawa PM, Joly RJ, Bressan RA
(2002) Does proline accumulation play an active role in
stress-induced growth reduction? Plant J 31:699–712
Mattioli R, Falasca G, Sabatini S, Altamura M, Costantino P,
Trovato M (2009) The proline biosynthesis genes P5CS1 and
P5CS2 play overlapping roles in Arabidopsis flower transi-
tion but not in embryo development. Physiol Plant137:72–85
Matysik J, Alia Bhalu B, Mohanty P (2002) Molecular mecha-
nisms of quenching of reactive oxygen species by proline
under stress in plants. Curr Sci 82:525–532
Euphytica (2013) 193:101–120 119
123

20. Merchan F, de Lorenzo L, Rizzo SG, Niebel A, Manyani H,
Frugier F, Sousa C, Crespi M (2007) Analysis of regulatory
pathways involved in the reacquisition of root growth after
salt stress in Medicago truncatula. Plant J 51:1–17
Michel BE, Kaufmann MR (1973) The osmotic potential of
polyethelene glycol 6000. Plant Physiol 51:914–916
Munns R, Tester M (2008) Mechanisms of salinity tolerance.
Annu Rev Plant Biol 59:651–681
Nanjo T, Fujita M, Seki M, Kato T, Tabata S, Shinozaki K
(2003) Toxicity of free proline revealed in an Arabidopsis
T-DNA-tagged mutant deficient in proline dehydrogenase.
Plant Cell Physiol 44:541–548
Parre E, de Virville J, Cochet F, Leprince AS, Richard L, Le-
febvre-De Vos D, Ghars MA, Bordenave M, Zachowski A,
Savoure´ A (2010) A new method for accurately measuring
Delta(1)-pyrroline-5-carboxylate synthetase activity.
Methods Mol Biol 639:333–340
Planchet E, Rannou O, Ricoult C, Mercey BS, Grondard MA,
Limami AM (2011) Nitrogen metabolism responses to
water deficit act through both abscisic acid (ABA)-
dependent and independent path ways in Medicago trun-
catula during post-germination. J Exp Bot 62:605–615
Quandt HJ, Pu¨hler A, Broer I (1993) Transgenic root nodules of
Vicia hirsuta: a fast and efficient system for the study of
gene expression in indeterminate-type nodules. Mol Plant
Microbe Interact 6:699–706
Rizhsky L, Liang H, Mittler R (2002) The combined effect of
drought stress and heat shock on gene expression in
tobacco. Plant Physiol 130:1143–1151
Saito K, Matsuda F (2010) Metabolomics for functional
genomics, systems biology, and biotechnology. Annu Rev
Plant Biol 61:463–489
Sanchez DH, Lippold F, Redestig H, Hannah MA, Erban A,
Kra¨mer U, Kopka J, Udvardi MK (2008a) Integrative
functional genomics of salt acclimatization in the model
legume Lotus japonicus. Plant J 53:973–987
Sanchez DH, Siahpoosh MR, Roessner U, Udvardi M, Kopka J
(2008b)Plantmetabolomicsrevealsconservedanddivergent
metabolic responses to salinity. Physiol Plant 132:209–219
Sanchez DH, Pieckenstain FL, Szymanski J, Erban A, Bromke
M, Hannah MA, Kraemer U, Kopka J, Udvardi MK (2011)
Comparative functional genomics of salt stress in related
model and cultivated plants identifies and overcomes
limitations to translational genomics. PLoS ONE 6:e17094
Sanchez DH, Schwabe F, Erbani A, Udvardi MK, Kopka J
(2012) Comparative metabolomics of drought acclimation
in model and forage legumes. Plant, Cell Environ
35:136–149
Schmidt R, Stransky H, Koch W (2007) The amino acid per-
mease AAP8 is important for early seed development in
Arabidopsis thaliana. Planta 226:805–813
Sharma S, Verslues PE (2010) Mechanisms independent of
ABA or proline feedback have a predominant role in
transcriptional regulation of proline metabolism during
low water potential and stress recovery. Plant, Cell Environ
33:1838–1851
Sharma S, Villamor JG, Verslues PE (2011) Essential role of
tissue-specific proline synthesis and catabolism in growth
and redox balance at low water potential. Plant Physiol
157:292–304
Strizhov N, A´ braha´m E, O¨ kre´sz L, Blickling S, Zilberstein A,
Schell J, Koncs C, Szabados L (1997) Differential
expression of two P5CS genes controlling proline accu-
mulation during salt-stress requires ABA and is regulated
by ABA1, ABI1 and AXR2 in Arabidopsis. Plant J
12:557–569
Su J, Wu R (2004) Stress-inducible synthesis of proline in
transgenic rice confers faster growth under stress condi-
tions than that with constitutive synthesis. Plant Sci
166:941–948
Szabados L, Savoure´ A (2010) Proline: a multifunctional amino
acid. Trends Plant Sci 15:89–97
Sze´kely G, A´ braha´m E, Cse´plo¨ A, Rigo´ G, Zsigmond L, Csisza´r
J, Ayaydin F, Nicolai Strizhov N, Ja´sik J, Schmelzer E,
Koncz C, Szabados L (2008) Duplicated P5CS genes of
Arabidopsis play distinct roles in stress regulation and
developmental control of proline biosynthesis. Plant J
53:11–28
Thapa B, Arora R, Knapp AD, Brummer EC (2008) Applying
freezing test to quantify cold acclimation in Medicago
truncatula Gaertn. J Am Soc Hortic Sci 133:684–691
Urano K, Maruyama K, Ogata Y, Morishita Y, Takeda M,
Sakurai N, Suzuki H, Saito K, Shibata D, Kobayashi M,
Yamaguchi-Shinozaki K, Shinozaki K (2009) Character-
ization of the ABA-regulated global responses to dehy-
dration in Arabidopsis by metabolomics. Plant J
57:1065–1078
Vendruscolo ACG, Schuster I, Pileggi M, Scapim CA, Molinari
HBC, Marur CJ, Vieira LGC (2007) Stress-induced syn-
thesis of proline confers tolerance to water deficit in
transgenic wheat. J Plant Physiol 164:1367–1376
Venekamp JH, Koot JTM (1984) The distribution of free amino
acids, especially of proline, in the organs of field bean
plants, Vicia faba L., during development in the field.
J Plant Physiol 116:343–349
Verbruggen N, Hermans C (2008) Proline accumulation in
plants: a review. Amino Acids 35:753–759
Verdoy D, de la Pen´a TC, Redondo FJ, Lucas MM, Pueyo JJ
(2006) Transgenic Medicago truncatula plants that accu-
mulate proline display nitrogen-fixing activity with
enhanced tolerance to osmotic stress. Plant, Cell Environ
29:1913–1923
Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu JK
(2006) Methods and concepts in quantifying resistance to
drought, salt and freezing, abiotic stresses that affect plant
water status. Plant J 45:523–539
Yoshiba Y, Kiyosue T, Katagiri T, Ueda H, Mizoguchi T,
Yamaguchi-Shinozaki K, Wada K, Harada Y, Shinozaki K
(1995) Correlation between the induction of a gene for D1
-
pyrroline-5-carboxylate synthetase and the accumulation
of proline in Arabidopsis thaliana under osmotic stress.
Plant J 7:751–760
Zhang CS, Lu Q, Verma DPS (1995) Removal of feedback
inhibition of D1
-pyrroline-5-carboxylate synthetase, a
bifunctional enzyme catalyzing the first two steps of pro-
line biosynthesis in plants. J Biol Chem 270:20491–20496
120 Euphytica (2013) 193:101–120
123