Physiological and metabolomic analysis of a knockoutmutant suggests a critical role of MtP5CS3 gene in osmoticstress 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

� 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 � Proline �D1-Pyrroline-5-carboxylate synthetase 3 gene �Knockout mutant � Metabolomic analysis �Drought � Salinity � Osmotic stress tolerance �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 ofthis article (doi:10.1007/s10681-013-0957-4) contains supple-mentary material, which is available to authorized users.

M. L. Nguyen � G.-B. Kim � Y.-W. Nam (&)

Department of Life Science, Sogang University,

Seoul 121-742, Korea

e-mail: ywnam@sogang.ac.kr

S.-H. Hyun � S.-Y. Lee � H.-K. Choi

College of Pharmacy, Chung-Ang University,

Seoul 156-756, Korea

C.-Y. Lee � 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

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 (Szekely

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 (Abraham 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; Abraham 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 (Szek-

ely et al. 2008). In leaf mesophyll cells, AtP5CS1 was

imported into chloroplasts under osmostic stress,

while AtP5CS2 remained in the cytoplasm (Szekely

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 nuclear magnetic

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

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 MgSO4�7H2O, 0.7 mM KH2PO4,

0.8 mM Na2HPO4�2H2O, 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-2s-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

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-32P]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) were ground in liquid 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 (Garcıa-Rıos 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

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 Szekely 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 Szekely et al. (2008).

Extraction and derivatization of metabolites

Four-week-old wild type and Mtp5cs3 plants grown 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/mL methoxylamine

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

(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

data were imported into the SIMCA-P software (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

metabolites that highly contributed to the 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 root length (Fig. 1e, f; Supplementary Table

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

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 p3Control 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

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 p3Control 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 PEGp5cs3WT p5cs3WT

p3WT5 cm

(G)

(B) (D)(C)

C S3 S6 D C S3 S6 D0

20

40

60

∗∗∗

∗∗∗

∗

∗∗∗

∗∗∗∗ns ns

Root Shoot

Pro

line

(µm

ole

s/g

fw

)

C PEG0

2

4

WTp5cs3

∗∗∗

ns

Greenhouse Hydroponic

0

10

20

30

40

50 WTp5cs3

∗∗∗

Control 150 g/L PEGRoot Shoot Root Shoot

H2O

2 (n

g/g

fw

)

(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

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

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 to MtP5CS3 but placed distantly, was introduced

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

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

5WT-GUSp5cs3-GUSp5cs3-P5CS3

control 100 g/L 150 g/L

∗∗∗

∗

∗∗∗∗∗∗

∗∗ns

PEG

Pro

line

(μm

ole

s/g

fw

)

Control 100 g/l PEG 150 g/L PEG0

10

20

30

40

50

WT-GUS-Rp3-GUS-R

p3-P5CS3-R WT-GUS-Sh

p3-GUS-Shp3-P5CS3-Sh

∗∗

Ele

ctro

lyte

leak

age

(%)

0.00

0.05

0.10

0.15

0.20Control100 g/L PEG

A17-GUS A17-P5CS3Root Shoot

∗∗∗

Root Shoot

Fre

sh w

eig

ht

(g)

Control NaCl PEG0

5

10

15

20 A17-GUSA17-P5CS3

∗∗∗

Pro

line

(µm

ole

s/g

fw

)

Control NaCl0.0

0.5

1.0

1.5A17-GUSA17-P5CS3

∗∗∗

To

tal C

hl.

ab (

µg

/mg

fw

)

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

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

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 (Abraham 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

(Szekely 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 eight sample

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

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 (Szekely et al. 2008), the germination rate of the

Mtp5cs3 mutant seeds estimated in this study

0

1

23

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

Fructose NS

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

(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 (Szekely 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 (Szekely 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. Significant differences in P5CS activity were only

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

activity correlated with the elevated expression levels of

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

MtP5CS3 activity led to increased expression of MtOAT

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

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

(Szekely 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

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 roots were 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

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.

truncatula under saline condition (Kim and Nam 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 energy source (Kohl et al. 1988), the Mtp5cs3 mutant

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.

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