www.elsevier.com/locate/plantsci

Plant Science 168 (2005) 541–546

Free spermidine and spermine content in Lotus glaber

under long-term salt stress

Diego H. Sanchez, Juan C. Cuevas, Maria A. Chiesa, Oscar A. Ruiz*

Unidad de Biotecnologıa 1, IIB-IINTECH/UNSAM-CONICET, Camino circunvalacion laguna, km6 CC164,

(B7130IWA) Chascomus, Provincia de Buenos Aires, Argentina

Received 24 June 2004; received in revised form 21 July 2004; accepted 23 September 2004

Available online 18 October 2004

Abstract

Polyamine metabolism has long been involved in plant stress responses, although physiological roles of putrescine, spermidine and

spermine are still under debate. Regarding long-term salinization, it have been suggested that high titers of spermidine and/or spermine, but

not putrescine, are correlated with the response of plants to salinity. In this work, we use the moderately salt tolerant glycophyte Lotus glaber

as a model to test the hypothesis that free spermidine and spermine are biochemical indicators of salt stress response. For such purpose, we

evaluated polyamine content in three different long-term salt stress approaches: germinating and growing seedlings under salinity, salinization

of growing plants and imposition of salt stress to clone stem cuttings obtained from plants recovered from saline lowlands. Proline was also

tested to evaluate if polyamines levels correlate with this compatible solute response. Results from these experiments showed a similar trend

concerning higher polyamines content i.e., a salt induced a decrease of free spermidine and an increase of free spermine, in line with the idea

that polyamine are biochemical indicators of salt stress. However, polyamine levels not always paralleled the accumulation of proline. These

observations are discussed with regard of the putative roles reported for polyamines in plant abiotic stresses.

# 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Lotus; Polyamines; Salinity; Salt stress; Polyamines; Spermidine; Spermine

1. Introduction

Salt stress is becoming an ever-increasing threat for food

production, being irrigation a major problem of agricultural

fields due to gradual salinization [1,2]. Salt stress impose

two constraints: a hyperosmotic effect due to lower soil

water potential and a hyperionic effect due to direct toxicity

of ions over metabolism and nutrition of plants [3].

According to the two-phase growth model, growth reduction

during salinity arises by the progressive accumulation of

toxic ions in the aerial part of a plant [2,4]. As a

consequence, the hyperionic cue is responsible for varietals

differences in salt tolerance, which are evident after long-

term salinization, namely days or weeks [2,4].

* Corresponding author. Tel.: + 54 2241 424045; fax: +54 2241 424048.

E-mail address: ruiz@intech.gov.ar (O.A. Ruiz).

0168-9452/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved

doi:10.1016/j.plantsci.2004.09.025

Common polyamines (putrescine, spermidine and sper-

mine) are ubiquitous aliphatic polycations that have been

long recognized as modulators of plant growth and

development [5], and are also implicated in plant responses

to environmental cues [6]. Stress induced expression and

activity of arginine decarboxylase is the most commonly

accepted feature of polyamine metabolism, a phenomenon

that is responsible for the typical accumulation of putrescine

observed under stress. In the particular case of salt stress,

induction of arginine decarboxylase and accumulation of

putrescine seems to be a consequence of the osmotic

elicitation due to salt shock [6–8]. Under long-term

salinization, high titers of spermidine and/or spermine,

but not putrescine, are correlated with the response of plant

to salinity [9–11]. However, these reports are contradictory

with regard to the role of spermidine and spermine as

biochemical markers of salt stress tolerance. Our aim was to

get insight into this matter in a moderately salt tolerant

.

D.H. Sanchez et al. / Plant Science 168 (2005) 541–546542

glycophyte: Lotus glaber. This self-incompatible plant is an

economically important forage legume occurring in Argen-

tine that has been established in the Salado River Basin

[12,13], the most important area devoted to beef cattle

production in the country. It has been shown that L. glaber is

useful in the reclamation of soils in the alkaline–saline

lowlands of the Argentinean pampas, due to its high

tolerance to different abiotic stress factors [12,14].

In this work, we used L. glaber as a model to test the

hypothesis that free spermidine and spermine are biochem-

ical indicators of salt stress response. Since compatible

solutes are expected to be accumulated under salinity, in

some experiments we used proline as marker to test if

polyamine content parallels the known accumulation of this

osmoticum.

2. Materials and methods

2.1. Plant material, growth conditions and treatments

Three different salt stress experiments were performed

using L. glaber Mill. (Narrow-leaf trefoil; syn L. tenuis

Waldst et Kit. ex Wild.). For experiments named ‘‘Seeds

germinating and growing under salt stress’’ and ‘‘1-month-

old plants under salt stress’’ (see below), seeds of L. glaber

were scarified with emery paper and surface sterilized for

2 h with gaseous Cl2. At least 50 seeds were directly sown in

pots filled with sand:perlite (1:1), irrigated with half-

strength Hoagland’s nutrient solution and kept at 4 8C for

48 h. Plants were grown in a growth room with a 16/8 h

photoperiod at 24/20 � 2 8C and 45/75 � 5% RH (day/

night) and a photon flux density of 100 mmol m�2 s�1

provided by day-light and grolux1 fluorescent lamps. The

experiment where seeds were germinated and growth under

salt stress consisted on germinating and growing plants for

15 days (two true leaves) at different salt treatments (0, 25,

50 and 75 mM NaCl, added to nutrient solution). This

experiment was repeated twice and a representative

experiment is shown. The experiment called ‘‘1-month-

old plants under salt stress’’ consisted on germinating and

growing L. glaber for 1 month and then salt stress was

imposed, as one salt shock of 200 mM NaCl that lasted 2

weeks. For biochemical determinations, plant material from

non-salinized controls and salt stress treatment were

sampled every 5 days. This experiment was repeated four

times and a representative experiment is shown. The last

experiment (‘‘Stem cuttings under salt stress’’) was

performed using stem cuttings derived from at least 20 L.

glaber plants recovered from saline lowlands. These plants

are named ‘‘genotypes’’ or ‘‘lines’’ throughout this report.

Each genotype was vegetatively propagated by stem cuttings

according to Mujica and Rumi [15] in pots filled with sand

sub-irrigated with half-strength Hoagland’s nutrient solu-

tion; rooting was allowed for 1 month. Once rooted, total

plant height was measured each 7 days for 3 weeks (see

below). According to Munns [2], a high intrinsic growth rate

might reduce the rate at which toxic ions are accumulated in

the shoot. Therefore, four different genotypes were selected

for further evaluation according to their height growth rate.

For salt stress experiments, at least 15 individual stem

cuttings obtained from the same genotype were subjected to

different treatments (0, 75 and 150 mM NaCl, imposed as

one salt shock) for 3 weeks. Plant material for biochemical

determinations was sampled at the end of the experiment,

which was carried out twice under field conditions during

the spring.

2.2. Growth analysis

For the ‘‘Stem cuttings under salt stress’’ experiment,

plant growth was estimated from total plant height. Height at

time n (Hn) from each cutting was normalized to its own

initial height (Hi) at the beginning of the experiment,

according to the formula:

Shoot growth ¼ Hn � Hi

Hi

In this regard, shoot growth shown in Fig. 3 reflects the

increment in height of the shoot cutting at time n compared

with its height at the beginning of the experiment. The

coefficient of variation of shoot growth in replicated stem

cuttings derived from the same genotype never exceeded

20%. Relative growth rate (RGR) was determined according

to the formula:

RGR ¼ ½LnðHt2Þ � LnðHt1

Þ�t2 � t1

where Ht2 and Ht1 represents the cutting height at times t2and t1, respectively. The coefficient of variation of RGR in

replicated cuttings derived from the same genotype never

exceeded 25%.

2.3. Analytical determinations

Water content, expressed as a percentage (WC%), was

determined as:

WC% ¼ 100 � ðFW � DWÞFW

where FW and DW means fresh weight and dry weight,

respectively. Na+ and K+ were extracted with 100 mM HCl

and estimated by standard flame photometry [16]. Free

polyamines were estimated as described previously, analyz-

ing their dansyl-derivatives by reversed phase HPLC [11].

Free putrescine content of L. glaber is not shown since it was

below the sensitivity of our detection system when was

measured in vegetal materials obtained from plants under

salt stress. Proline was estimated spectrophotometrically by

ninhydrin reaction under conditions described elsewhere

[17].

D.H. Sanchez et al. / Plant Science 168 (2005) 541–546 543

3. Results

3.1. Seeds germinating and growing under salt stress

When L. glaber seeds were germinated and grown for 15

days in different salt treatments, an evident inhibition of

growth was observed as salt concentration increased in the

nutrient solution. Noteworthy, no difference in germination

efficiency (radicle emergency) was observed between 0, 25

and 50 mM NaCl. In these cases, all germinating seeds

rendered growing seedlings, indicating that no selection to

salinity takes place during germination and growth in salt

added pots. In other words, the plant material sampled does

represent the genotype heterogeneity in the seed population.

However at 75 mM NaCl, although all seeds do germinate, at

least 25% of seedlings did not further grow. As salt

concentration increased in the nutrient solution, seedling

Na+ content gradually increased while K+ decreased (Fig. 1A).

Accordingly, the compatible solute proline content

increased gradually but its accumulation was highly induced

at 75 mM NaCl (Fig. 1B). In addition, WC% did not differ

among 0, 25 and 50 mM NaCl treatments (they were

Fig. 1. Sodium, potassium, proline, spermidine and spermine content of L.

glaber seedlings germinated and grown for 15 days at 0, 25, 50 and 75 mM

NaCl treatments. Na: sodium, K: potassium, DW: dry weight, Spd: sper-

midine, Spm: spermine. Bars represent media + S.E.M. of triplicates. (*)

Means statistically different among salt stress treatment and controls at

p < 0.05, according to Student’s t-test. (A) Sodium and potassium content.

(B) Proline content. (C) Free spermidine and spermine content.

93.5 � 0.1, 93.8 � 0.1 and 93.8 � 0.3, respectively), but

was statistically diminished to 91.9 � 0.3 at 75 mM NaCl.

These data suggest that there is no threat to L. glaber seedling

survival up to 50 mM NaCl. Yet, 75 mM NaCl treatment

imposed a threat to germination and growth, and those

seedlings that survived were under high ionic and osmotic

stresses. In this context, free spermidine content remained

unaltered among 0, 25 and 50 mM NaCl treatments, while free

spermine content increased steeply (Fig. 1C). At 75 mM NaCl,

free spermidine statistically diminished, while free spermine

increased, although not beyond spermine content of the

50 mM NaCl treatment (Fig. 1C).

3.2. 1-month-old plants under salt stress

As expected, 1-month-old L. glaber plants increased Na+

and decreased K+ content when subjected to salt stress (data

not shown), and the compatible solute proline was also

accumulated (Fig. 2A). In addition, free spermidine content

decreased, while free spermine content increased as salt

stress progressed compared to non-salinized controls (Fig. 2

B and C).

Fig. 2. Proline, spermidine and spermine content of 1-month-old L. glaber

plants subjected to 0 and 200 mM NaCl treatments for 2 weeks. d: Days,

Spd: spermidine, Spm: spermine. Bars represent media + S.E.M. of tripli-

cates. (*) Means statistically different among salt stress treatment and

controls at p < 0.05, according to Student’s t-test. (A) Proline content.

(B) Free spermidine content. (C) Free spermine content.

D.H. Sanchez et al. / Plant Science 168 (2005) 541–546544

Fig. 3. (A and B) Shoot growth and RGR of 0, 75 and 150 mM NaCl treated stem cuttings of lines 3, 13, 15 and 20. Data represent the media of five independent

cuttings. RGR: relative growth rate, Spd: spermidine, Spm: spermine. Error bars were removed to aid comprehension. For more details, see Section 2. (C) Free

spermidine and spermine content of control and salt treated cuttings at the end of the experiment (week 3). Bars represent media + S.E.M. of triplicates. (*)

Means statistically different among salt stress treatments and controls at p < 0.05, according to Student’s t-test.

3.3. Stem cuttings under salt stress

Four different genotypes recovered from the field were

chosen for further physiological and biochemical evaluation.

As shown in Fig. 3A, two of them presented a high intrinsic

growth (lines 15 and 20), while the other two were low

growing lines (lines 3 and 13). However, no great differences

in shoot growth and RGR were observed among high and

low intrinsic growing lines when subjected to salt stress (Fig.

3A and B). In this regard, line 3 does not decreased RGR in

week 1 when salinized and also appeared to resume growth

rate in week 3 when subjected to 75 mM NaCl. Line 15

presented an increased growth at week 1 when subjected to

150 mM NaCl, suggesting a halophytic-like response (Fig.

3A and B). As a general rule, all genotypes presented a

decrease in free spermidine and an increase in free spermine

contents under long-term salt treatment, with no apparent

relationship of absolute polyamine content among high and

low intrinsic growing lines under control or salt stress

treatments (Fig. 3C). Particularly, line 15 showed a lower

free spermidine content under control and salinized

conditions compared with the rest of the genotypes. In

the case of free spermine, line 20 showed higher contents,

while lines 3 and 15 presented lower increments as a

consequence of salt stress.

4. Discussion

Since L. glaber is an out-breeder species [12,14], genetic

differences are expected to be the basis of the differential

growth rate and response to salinity among different plants.

In addition, ions may well be compartmentalized in the

stems of legumes as a strategy to avoid salt build up in the

leaves [18]. These characteristics might be linked, given that

a high intrinsic growth rate may reduce the rate at which

toxic ions are accumulated in the shoot [2,4]. However, a

conclusion arises from the results of the plants recovered

from the field: at least for L. glaber, differences in salt

induced growth reduction among genotypes seemed not to

be dependent of their intrinsic height growth rate (Fig 3A

and B). Note that L. glaber appeared to be a moderate salt

tolerant glycophyte and also must be considered that the

genotypes utilised in this study were all collected from

alkaline–saline constrained lowlands. In addition, we

observed that seeds of a population commonly sowed in

farm-technological applications in our Institute, were able to

germinate and grow even at 50 mM NaCl without any

evident salt selection, and plants of the same population still

grow when exposed to 200 mM NaCl. Also, height growth

of stem cuttings in the last experiment was decreased only

25–50% when exposed to 150 mM NaCl.

D.H. Sanchez et al. / Plant Science 168 (2005) 541–546 545

Polyamine accumulation under stress has been well

documented in several plant species. However, their role in

stress responses is still elusive and even contradictory. For

example, although the fact that putrescine accumulation

under several stress conditions might argue in favor of the

idea that this aliphatic diamine act as a compatible solute, an

excessive content over the normal level is toxic to plants

[19]. Also, spermidine and spermine are known anti-

senescent compounds [20] and yet, they may inhibit growth

and induced oxidative stress [21–23]. It has been previously

suggested that polyamines may be used as biochemical

indicators of salt stress tolerance [6,9]. Concerning free

higher polyamine levels in L. glaber under salinity, similar

results were obtained using different experimental

approaches, i.e. a salt induced decrease of free spermidine

and an increase of free spermine (Figs. 1–3). Taking into

account the role of polyamines in growth and development

[24], one may suggest a relationship among the salt induced

reduction of growth rate and spermidine content. However,

results from the first and the third experiments argue against

this idea. Firstly, seeds germinating and growing even under

50 mM NaCl presented lower growth but no decrease in their

free spermidine level (Fig. 1C), and secondly, no correlation

in the free spermidine content was observed among high and

low growing genotypes in control or salinized conditions

(Fig. 3). On the other hand, spermine accumulation as a

consequence of salinization is in accordance with previous

results in other plant species [9–11,25]. It has been shown

that A. thaliana spermine synthase gene AtSPMS

(At5g53120) is induced by stress hormone ABA and salt

shock [26]. Spermine seems to play physiological roles on

membrane functions and in reducing radical generation

under oxidative stress [25,27,28], while it has been

suggested as a signaling molecule in HR cell death and

mitochondrial dysfunction [29,30]. However, it has been

reported that spermine is not essential for plant survival [31]

and also that its accumulation under salt stress do not

represent a salt tolerance trait [11]. Taken these results

together, we hypothesized that spermine is a biological

molecule involved in plant response to salinity that might be

related to stress signaling. Whether AtSPMS gene is

involved in such response waits for further elucidation.

When 1-month-old plants where salinized, the changes in

polyamine content induced by salinity did parallel those of

proline (Fig. 2). However, this was not the case in growing

seedlings under salt stress. Although proline was accumu-

lated steeply, while increasing salt concentration in the

nutrient solution, seedlings germinated and grown in 50 mM

NaCl did not diminished their spermidine content. Also,

those at 75 mM NaCl increased spermine content but

without a further increase compared to those at 50 mM NaCl

(Fig. 1C). Therefore, free spermidine and spermine changes

under salt stress not necessarily correlates with ion and/or

water disbalance, suggesting a complex regulation of salt

stress-induced changes in polyamine metabolism that might

reasonably be related to polyamines physiological roles.

In conclusion, our results and those previously reported

argue in favor of the idea that free spermidine and spermine

are biochemical indicators of the response of plants to long-

term salinization, although the former seemed to be

diminished or increased depending on plant species and

growth conditions [9–11,25]. Spermine accumulation

appears as a general feature of plant response to salinity,

although it may not be considered as a salt tolerance trait.

Its physiological role under salinity deserves further

research.

Acknowledgments

We are grateful to Vanesa Ixtania and Mercedes M.

Mujica for their outstanding support on technical features

concerning L. glaber culture. This work was supported by

grants from the Consejo Nacional de Investigaciones

Cientıficas y Tecnicas (CONICET, Argentina), The Third

World Academy of Sciences (TWAS) and the Agencia

Espanola de Cooperacion Internacional (AECI). DHS is a

fellow from CONICET, while OAR is a member of the

research committee from CONICET.

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