Plant Physiology and Biochemistry 45 (2007) 39e46www.elsevier.com/locate/plaphy

Research article

Modulation of polyamine balance in Lotus glaber by salinityand arbuscular mycorrhiza

Analıa I. Sannazzaro a, Mariela Echeverrıa a, Edgardo O. Alberto a,Oscar A. Ruiz a, Ana B. Menendez a,b,*

a Instituto Tecnologico de Chascomus (IIB-INTECH), CONICET, 1428 Buenos Aires, Argentinab University of Buenos Aires, Biodiversity and Experimental Biology, DBBE, Piso 4, PAB II, CIUDAD UNIV, 1428 Buenos Aires, Argentina

Received 22 March 2006

Available online 28 December 2006

Abstract

In this work we investigated the involvement of Glomus intraradices in the regulation of plant growth, polyamines and proline levels of twoLotus glaber genotypes differing in salt tolerance, after longterm exposure to saline stress.

The experiment consisted of a randomized block design with three factors: (1) mycorrhizal treatments (with or without AM fungus); (2) twosalinity levels of 0 and 200 mM NaCl; and (3) L. glaber genotype. Experiments were performed using stem cuttings derived from L. glaberindividuals representing a natural population from saline lowlands.

One of the most relevant results was the higher content of total free polyamines in mycorrhized plants compared to non-AM ones. Sincepolyamines have been proposed as candidates for the regulation of root development under saline situations, it is possible that AM plants (whichcontained higher polyamine levels and showed improved root growth) were better shaped to cope with salt stress.

Colonization by G. intraradices also increased (Spd þ Spm)/Put ratio in L. glaber roots. Interestingly, such increment in salt stressed AMplants of the sensitive genotype, was even higher than that produced by salinization or AM symbiosis separately. On the other hand, salinity butnot mycorrhizal colonization influenced proline levels in both L. glaber genotypes since high proline accumulation was observed in both geno-types under salt stress conditions.

Our results suggest that modulation of polyamine pools can be one of the mechanisms used by AM fungi to improve L. glaber adaptation tosaline soils. Proline accumulation in response to salt stress is a good indicator of stress perception and our results suggest that it could be used assuch among L. glaber genotypes differing in salt stress tolerance.� 2006 Elsevier Masson SAS. All rights reserved.

Keywords: Lotus; Salinity; Arbuscular mycorrhiza; Polyamine; Proline

1. Introduction

Lotus glaber Mill. (narrow-leaf trefoil; syn L. tenuis Waldstet Kit. Ex Wild.) is a glycophytic, perennial legume of Euro-pean origin that occurs frequently in saline habitats. Previousobservations indicate the occurrence of genotypes highly

* Corresponding author. University of Buenos Aires, Biodiversity and Ex-

perimental Biology, DBBE, Piso 4, PAB II, CIUDAD UNIV, 1428 Buenos

Aires, Argentina. Tel.: þ54 11 4542 7396.

E-mail address: anamen@bg.fcen.uba.ar (A.B. Menendez).

0981-9428/$ - see front matter � 2006 Elsevier Masson SAS. All rights reserved

doi:10.1016/j.plaphy.2006.12.008

tolerant to salinity in different populations of L. glaber [34],which is the best-adapted forage species used for cattle pro-duction in the Salado River basin (9 � 106 Ha). Mendozaand Pagani [33] showed this species has a high dependenceof arbuscular mycorrhizal (AM) colonization in P-deficientsoils. More recently, highly diverse AM fungal colonizationpatterns in roots of L. glaber were found in saline fields [43].

Several researchers have shown AM fungi can protect theplant against adverse effects of salinity. For instance, salttolerance of banana plants was enhanced in the presence ofGlomus isolates [52]. In cotton, improvement of plant growth,

.

40 A.I. Sannazzaro et al. / Plant Physiology and Biochemistry 45 (2007) 39e46

biomass accumulation and P-nutrition varied depending onboth the AM fungal isolate and severity of salt stress.

Free polyamines are small organic cations that are absolutelyrequired for eukaryotic cell growth. The three main polyaminesfound in plants are putrescine (Put), spermidine (Spd) and sper-mine (Spm). These compounds are thought to play an importantrole in plant responses to a wide array of environmental stressessuch as low and high temperatures [6,21,40,49], salinity[3,25,44], high osmolarity [4], hypoxia [36], and oxidativestress [26,28]. For instance, exogenously added Spd and Spmprotected rice plants from saline stress [8] whereas transgenicplants overexpressing enzymes intervening in Spd and Spm bio-synthesis were more tolerant to saline and hyperosmotic stress[23,41,51]. In plants and bacteria, the diamine Put can be syn-thesized directly from ornithine via ornithine decarboxylaseor indirectly following decarboxylation of arginine by argininedecarboxylase. In turn, Spd and Spm are synthesized from Putby successive additions of aminopropyl groups.

Information regarding polyamines in mycorrhizal fungi orin plant-fungal symbiotic interactions is limited to few re-cords. Similar pathways for Put synthesis to those describedin plants and bacteria have been found in ectomycorhizae[16] and in an AM fungus [42]. Free polyamines have beensuggested to play an important role in the initial stages ofthe infection of pea roots by Glomus intraradices (Schenckand Smith) [14]. In addition, mycorrhization may change thepolyamine balance of plants. Kytoviita and Sarjala [27]showed that ectomycorrhizal symbiosis increased free Putlevels in mycorrhizal roots of Scots pine seedlings.

On another hand, proline is one of the most common com-patible osmolytes in plants [20]. It is believed to increase plantadaptation to drought and salinity [13,19] and it may also berelated with survival of non-halophytes upon salt stress

[9,46,47]. This molecule serves as an osmosolute and as a pro-tectant for enzymes and cellular structures [24].

Proline accumulation in mycorrhizal plants subjected todrought has been reported [2,18] and variable effects ofmycorrhizal colonization on proline levels of plants undersalt stress have been observed. Proline accumulation wasgreater in mycorrhized moong plants at 12.5 mM and25 mM NaCl at 40 and 62 days after sowing [22], whereasnon-AM faba bean plants accumulated much more prolinethan AM plants at various salinity ranges [39].

Lotus glaber is known to accumulate high levels of prolinein response to salinity [29], but so far there is no informationregarding the influence of AM fungal colonization on suchaccumulation.

The present work was aimed at evaluating the influence ofthe AM fungus G. intraradices on the polyamine and prolinebalances of two L. glaber genotypes differing in their toleranceto saline stress.

2. Materials and methods

2.1. Experimental design

The experiment consisted of a randomized block designwith three factors: (1) mycorrhizal treatments (with or withoutAM fungus); (2) two salinity levels of 0 and 200 mM NaCl;and (3) L. glaber genotype. One salt shock of 200 mM NaClshowed to induced high titers of spermine and proline underlong term salinization [29].

A total of 20 plants distributed in 5 pots (4 plants per pot)per treatment were used. Plants were harvested and processedat two different sampling times: just before salt addition (ini-tial time, Ti) and 4 weeks after salt application (final time, Tf).

Fig. 1. Shoot and root growth (%) estimated as (DWf � DWi)/DWi � 100, where DWf and DWi represent dry weights at final and initial times, respectively. Two

months old plants were watered with nutrient solution containing 0 or 200 mM NaCl during 4 weeks. M�: non-mycorrhizal, Mþ: mycorrhizal, S�: non-salinized

plants, Sþ: salinized plants. Data are average of 5 replicates. Means (�SD) with the same letter are not significantly different (P < 0.05).

41A.I. Sannazzaro et al. / Plant Physiology and Biochemistry 45 (2007) 39e46

The experiment was repeated once. Only data from the firstexperiment are presented.

2.2. Biological material

G. intraradices (BAFC 3108) was multiplied in one litrepot cultures with soil-perlite (1:3 V/V) and Sorghum halepense(L.) Pers. (¼ Andropogon halepensis Brot.) as host duringfour months. The inoculum consisted of thoroughly mixed rhi-zosphere samples containing spores, hyphae and root frag-ments with no less than 70% of their root length infected.

Experiments were performed using stem cuttings derivedfrom L. glaber individuals representing a natural populationfrom saline lowlands. Stem cuttings were used in order to limitgrowth rate variability arising from the intrinsic high geno-typic heterogeneity of L. glaber seed populations. Differencesin sensitivity to saline stress among 80 L. glaber individualswere previously determined in our laboratory, according tosurvival time after continuous exposure to 300 mM NaCl. Amore tolerant genotype (TG, 27 day survival) and a moresensitive one (SG, 16 day survival) were chosen for this studyand were vegetatively propagated [35]. Fifteen day old rootedcuttings were transferred to pots containing 250 ml of washed,sterilized sand-vermiculite (1:1 V/V) and inoculated with 5 gof G. intraradices inoculum. Control plants received an equalamount of autoclaved (120 �C, 20 min) inoculum.

2.3. Plant growth conditions

Plants were grown in a greenhouse, with a 16/8 h photope-riod at 25 �C/21 �C (day/night) and 55/75 � 5% RH. Lightintensity (80 mmol m�2 s�1) was provided by daylight and Gro-lux� fluorescent lamps (F 40 W). Plants were weekly suppliedwith nutrient solution containing 1.5 mM CaCl2, 0.25 mMMgSO4, 0.02 mM KH2PO4 and micronutrients equivalent to1⁄4 of Hoagland solution [15]. After two months (Ti) half ofthe plants were salt stressed with nutrient solution containing200 mM NaCl. From this time on and in order to prevent oscil-lations in electrical conductivity (EC), saline solutions wererenewed every two days. Previous work performed in our labo-ratory has shown that this procedure avoids soil EC oscillationsduring the experiment. Plants were further cultured during 4additional weeks (Tf). Mycorrhizal root length colonizationof plants at initial time ranged 70e80%.

2.4. Measurements and analytical determinations

Plants were harvested and divided into shoots and roots. Thepresence of G. intraradices was determined visually by clearingand staining the roots with Trypan blue in lactophenol [38]. Per-centage of mycorrhizal root colonization (% MC) was performedaccording to McConigle et al. [32]. Dry weights of shoots androots were determined. Shoot and root growth (%) were esti-mated according to the formula: (DWf � DWi)/DWi � 100,where DWf and DWi represent dry weights at initial and finaltimes, respectively. Free polyamines were estimated by analy-sing dansyl-derivatives by reversed phase HPLC as described

previously [31]. Proline was estimated spectrophotometricallyby ninhydrin reaction under conditions described elsewhere [29].

2.5. Statistical analysis

Data were subjected to analysis of variance and meanscompared by T-Test for independent samples.

3. Results

No symptoms of injury were visually observed in leaves ofsalt-treated plants during the course of the experiment.

Growth responses to salt and mycorrhizal treatments weredifferent in both the TG and SG genotypes (Fig. 1). Salinityhad no clear significant effect on growth of TG plants,whereas mycorrhization improved shoot and root growthof this genotype. In contrast, salinity reduced growth of

Fig. 2. Total free polyamine contents (mmol/g DW) of tolerant and sensitive

Lotus glaber plants at initial and final times. Two months old plants were

watered with nutrient solution containing 0 or 200 mM NaCl during 4 weeks.

M�: non-mycorrhizal, Mþ: mycorrhizal, S�: non-salinized plants, Sþ:

salinized plants. Data are average of 5 replicates. Means (�SD) with the

same letter within shoots or roots are not significantly different (P < 0.05).

42 A.I. Sannazzaro et al. / Plant Physiology and Biochemistry 45 (2007) 39e46

0

0,05

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Tolerant

ab a

b b

a

aa

a a bb

M- M+

Put (

µmol

/g D

W)

Spd

(µm

ol/g

DW

)Sp

m (µ

mol

/g D

W)

Put (

µmol

/g D

W)

Spd

(µm

ol/g

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)Sp

m (µ

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W)

0

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Sensitive

ab a

b b

a

aa

a a bb

M- M+

(b)(a)

Fig. 3. aeb. Polyamine contents (mmol/g DW) of Lotus glaber plants at initial time. a) Tolerant; and b) sensitive. M�: non-mycorrhizal, Mþ: mycorrhizal plants.

Data are average of 5 replicates. Means (�SD) with the same letter within shoots or roots are not significantly different (P < 0.05). Shoot L, root .

non-mycorrhized SG plants, whereas mycorrhization counter-acted that effect by enhancing root growth. In the controltreatment, SG plants presented higher growth percentagesthan the TG genotype. Besides, shoot growth responses tomycorrhizal colonization were different in SG and TG plantsunder no stress condition.

Total free polyamine pools were increased by G. intraradi-ces colonization in roots and shoots of both L. glaber geno-types at the initial time Ti (Fig. 2). Particularly, incrementsof free Put in shoots of TG plants, and of free Spd and Spmin both TG and SG plants were observed (Fig. 3).

Salinity induced a reduction of free polyamine pools in TGroots, whereas mycorrhization led to polyamine increases inshoots and roots of non-salinized plants and in roots of stressedplants (Fig. 2). However, variations of individual polyamines inresponse to salinity and mycorrhization differed depending onthe plant genotype and organ (root or shoot) considered

(Fig. 4). Thus, in TG plants salinity led to decreases of shootPut, Spd and root Spd in both AM and non-AM plants, and ofroot Put in non-AM plants, whereas shoot Spm increased androot Spm levels decreased in salinized non-AM plants (Fig. 4a).Under non-saline conditions, mycorrhization increased levelsof the three polyamines in shoots and of Spd in roots of TGplants, decreased root Put contents and did not affect rootSpm. Under salt stress, mycorrhized TG plants showed higherlevels of Spd in shoots and roots and higher levels of rootSpm as compared to non-AM plants.

Regarding the SG genotype (Fig. 4b), salinization of non-mycorrhizal plants caused decreases in shoot Put, Spd, androot Put levels, increased shoot Spm content and had no effecton Spd and Spm root contents. In contrast, non-salinized AMSG plants increased their shoot Put, Spd and Spm and root Spdcontents, whereas root Put and Spm were not affected by theinteraction with the fungus under salt stress. Contrasting

43A.I. Sannazzaro et al. / Plant Physiology and Biochemistry 45 (2007) 39e46

Fig. 4. aeb. Shoot and root polyamine contents (mmol/g DW) of tolerant (a); and sensitive (b) Lotus glaber plants at final time. Two months old plants were watered

with nutrient solution containing 200 mM NaCl during 4 weeks. M�: non-mycorrhizal, Mþ: mycorrhizal, S�: non-salinized plants, Sþ: salinized plants. Data are

average of 5 replicates. Means (�SD) with the same letter within shoots or roots are not significantly different (P < 0.05).

with the TG genotype, mycorrhizal SG plants under salinityshowed higher root Spm, lower shoot and root Put and lowershoot Spd levels than the corresponding non-AM plants.

Mycorrhization increased (Spd þ Spm)/Put ratios in TGplants and shoot (Spd þ Spm)/Put ratio in SG at initial time.Mycorrhizal colonization also increased root (Spd þ Spm)/Put ratio in plants of both genotypes at final time regardlessthe presence or absence of salt (Fig. 5). In turn, salinity in-creased shoot (Spd þ Spm)/Put ratios regardless the genotypeand the root (Spd þ Spm)/Put ratio in SG plants.

Salinity significantly increased root and shoot proline levelsof both plant genotypes (Table 1). However, proline accumu-lation in response to salt stress in roots and shoots of SG plantswas considerably higher than in the more tolerant ones. My-corrhization had no effect on proline levels. Although a slightincrement of proline level was observed in AM-roots of TGplants under no stress condition, this was not significant.

4. Discussion

In this work we investigated the involvement of G. intrara-dices in the regulation of plant growth, polyamines and prolinelevels of two L. glaber genotypes differing in salt tolerance,after long-term exposure to saline stress.

One of the most relevant results was the higher content oftotal free polyamines in mycorrhized plants compared tonon-AM ones. Since polyamines have been proposed as candi-dates for the regulation of root development under saline situ-ations [11], it is possible that AM plants (which containedhigher polyamine levels and showed improved root growth)were better shaped to cope with salt stress.

Several reports have supported a protective role of Spd andSpm against salinity [6,8,30,53]. In line with this, the higher(Spd þ Spm)/Put ratio of salinized plants observed in bothTG and SG genotypes would be reflecting the intrinsic salttolerance trait of L. glaber.

Colonization by G. intraradices also increased(Spd þ Spm)/Put ratio in L. glaber roots. Interestingly, suchincrements in salt stressed AM plants of the SG genotypewere even higher than those produced by salinization or AMsymbiosis separately, suggesting an additive effect of both fac-tors on the root (Spd þ Spm)/Put ratio.

Considering these results and the high AM dependence ofL. glaber [33,43], we propose that modulation of polyaminepools can be one of the mechanisms used by AM fungi toimprove L. glaber adaptation to saline soils. However, verifica-tion of this hypothesis would require more in depth studies touncover relationships among mycorrhization, root topology,conjugated polyamines, hormonal balances, (gibberellin, eth-ylene) and aminoacid metabolisms.

On the other hand, the increments of free Spm concentra-tions in shoots of non-AM plants under salt stress observedin this study, could be explained by at least two differentmechanisms: 1- changes in the conjugated versus free poly-amines ratios; and 2- de novo Spm synthesis from its meta-bolic precursors, Put and Spd. The fact that free Spmaccumulation was concomitant with decreases in Put andSpd levels suggests that the later mechanism could be operat-ing in L. glaber plants under salt stress.

Similar Spm increments were previously described by Goi-coechea and collaborators [18] in leaves of non-stressed alfalfaplants mycorrhized with G. fasciculatum. It is intriguing the

44 A.I. Sannazzaro et al. / Plant Physiology and Biochemistry 45 (2007) 39e46

role that such increase of shoot Spm level could play in theinteraction of plants with their fungal partners. On otherhand, it seems reasonable the occurrence of hormonal signalsin this process. A candidate hormone to regulate free Spmpools in the shoots would be abscisic acid (ABA). Amongthe phytohormones tested, ABA is the only one found atconsiderably higher levels in AM-colonized plants than innon-AM plants [5,12].

Unfortunately, we cannot further compare our results withother reports since information regarding polyamine metabo-lism in AM associations is scarce and the few reports onthis subject refer to different hosts or stress conditions[17,18,37].

Our results showed that salinity but not mycorrhizal coloni-zation influenced proline levels in both L. glaber genotypes. Ithas been suggested that the increment in the (Spd þ Spm)/Putratio under salt stress conditions could be explained by thecontribution of putrescine catabolism to proline accumulation

Fig. 5. Shoot and root (Spd þ Spm)/Put ratios of tolerant and sensitive geno-

types of L. glaber at initial and final times. Two months old plants were

watered with nutrient solution containing 0 or 200 mM NaCl during 4 weeks.

M�: non-mycorrhizal, Mþ: mycorrhizal, S�: non-salinized plants, Sþ:

salinized plants. Data are average of 5 replicates. Means (�SD) with the

same letter within shoots or roots are not significantly different (P < 0.05).

via diamine oxidase [3]. In the present work, higher(Spd þ Spm)/Put ratios did not always correlate with prolineaccumulation (the main compatible osmolite in L. glaber),therefore some other mechanism different from putrescine ca-tabolism must be operating.

Taking into account that L. glaber is well adapted to salinesoils, we should assume that the two studied genotypes are, toa certain extent, tolerant to salinity. However, the more sensi-tive genotype accumulated higher proline levels than the moretolerant one. This result is in agreement with several reportscomparing genotypes in other plant species [10,29,48,50]and supports the view that proline accumulation in responseto salt stress is a good indicator of stress perception among ge-notypes differing in tolerance degree. Accordingly, mycorrhi-zal and non-mycorrhizal plants grown under saline conditionswould be equally stressed, since their proline levels did notdiffer.

Under 0 mM NaCl, mycorrhizal colonization increasedshoot growth of TG, whereas the opposite effect was observedin SG plants. Plant genotype determines how the interactionwith AM fungi influences the final shoot P concentration[45], therefore it is possible that the different responses to my-corrhizal colonization of TG and SG plants under no stresscould be reflecting differences in carbon flow from the plantto the fungus against Pi. This possibility should be addressedin future work.

Finally, the fact that growth improvements of L. glaberplants by G. intraradices were more visible under 200 mMNaCl supports previous findings that AM plants perform betterthan non-AM plants under salt stress [1,7] and shows thecapacity of the AM fungus to realise its symbiotic activityespecially under detrimental conditions.

Acknowledgements

This work was supported by grants from the AgenciaNacional de Promocion Cientıfica y Tecnologica/FONCyT, No14194, Lotus Adaptation and Sustainability in South America(LOTASSA) and Universia-Banco Rıo (Argentina). Authorsare grateful to the Comision de Investigaciones Cientıficas y

Table 1

Proline (mmol/g DW) content at final time

Tolerant Shoot Root

M � S� 1.9 � 0.1 c 1.3 � 0.1 d

M � Sþ 160.6 � 2.9 b 89.7 � 2.7 b

M þ S� 1.7 � 0.1 c 2.6 � 0.0 c

M þ Sþ 153.0 � 2.1 b 101.9 � 2.1 b

Sensitive Shoot Root

M � S� 1.9 � 0.0 c 2.2 � 0.04 c

M � Sþ 723.1 � 148.8 a 274.2 � 40.7 a

M þ S� 1.9 � 0.0 c 2.7 � 0.3 c

M þ Sþ 712.2 � 18.6 a 267.3 � 50.9 a

M�: non-mycorrhized, Mþ: mycorrhized, S�: non-salinized plants, Sþ:

salinized plants. Means with the same letter within each set of data are not

significantly different (P < 0.05).

45A.I. Sannazzaro et al. / Plant Physiology and Biochemistry 45 (2007) 39e46

Tecnicas (CONICET) and the Agencia Nacional de PromocionCientıfica y Tecnologica for granting the fellowships of A.Sannazzaro and M. Echeverrıa, respectively.

References

[1] G.N. Al-Karaki, Growth of mycorrhizal tomato and mineral acquisition

under salt stress, Mycorrhiza 10 (2000) 51e54.

[2] R. Azcon, M. Gomez, R.M. Tobar, Physiological and nutritional re-

sponses by Lactuca sativa to nitrogen sources and mycorrhizal fungi

under drought, Biology and Fertility of Soils 22 (1996) 156e161.

[3] A. Aziz, J. Martin-Tanguy, F. Larher, Stress-induced changes polyamine

and tyramine levels can regulate proline accumulation in tomato leaf

discs treated with sodium chloride, Physiologia Plantarum 104 (1998)

195e202.

[4] R.T. Besford, C.M. Richardson, J.L. Campos, A.F. Tiburcio, Effect of

polyamines on stabilization of molecular complexes in thylakoid mem-

branes of osmotically stressed oat leaves, Planta 189 (1993) 201e206.

[5] H. Bothe, A. Klingner, M. Kaldorf, O. Schmitz, H. Esch,

B. Hundeshagen, H. Kernebeck, Biochemical approaches to the study

of plant-fungal interactions in arbuscular mycorrhiza, Experientia 50

(1994) 919e925.

[6] A. Bouchereau, A. Aziz, F. Larher, J. Martin-Tanguy, Polyamines and environ-

mental challenges: recent development, Plant Science 140 (1999) 103e125.

[7] I.C. Cantrell, R.G. Linderman, Preinoculation of lettuce and onion with

VA mycorrhizal fungi reduces deleterious effects of soil salinity, Plant

Soil 233 (2001) 269e281.

[8] M.K. Chattopadhyay, B.S. Tiwari, G. Chattopadhyay, A. Bose,

D.N. Sengupta, B. Ghosh, Protective role of exogenous polyamines on

salinity-stressed rice (Oryza sativa) plants, Physiologia Plantarum 116

(2002) 192e199.

[9] T.M. Chu, D. Aspinall, L.G. Paleg, Stress metabolism VII Salinity and

proline accumulation in barley, Australian Journal of Plant Physiology

3 (1976) 219e228.

[10] T.D. Colmer, E. Epstein, J. Dvorak, Differential solute regulation in leaf

blades of various ages in salt-sensitive wheat and a salt-tolerant wheat -

� Lophopyrum elongatum (Host) A. Love amphiploid, Plant Physiology

108 (1995) 1715e1724.

[11] I. Couee, I. Hummel, C. Sulmon, G. Gouesbet, A. El Amrani, Involve-

ment of polyamines in root development. Plant Cell, Tissue and Organ

Culture 76 (2004) 1e10.

[12] G. Danneberg, C. Latus, W. Zimmer, B. Hundeshagen, H. Schneider-Po-

etsch, H. Bothe, Influence of vesicular-arbuscular mycorrhiza on phyto-

hormone balances in maize (Zea mays L), Journal of Plant Physiology

141 (1992) 33e39.

[13] A.J. Delauney, D.P.S. Verma, Proline biosynthesis and osmoregulation in

plants, Plant Journal 4 (1993) 215e223.

[14] N. El Ghachtouli, M. Paynot, D. Morandi, J. Martintanguy,

S. Gianinazzi, The effect of polyamines on endomycorrhizal infection

of wild-type Pisum sativum, cv Frisson (Nod Myc) and 2 mutants

(Nod- Myc and Nod- Myc-), Mycorrhiza 5 (1995) 189e192.

[15] F. Fodor, E. Cseh, A. Varga, G. Zaray, Lead uptake, distribution and remo-

bilization in cucumber, Journal of Plant Nutrition 21 (1998) 1363e1373.

[16] S. Fornale, T. Sarjala, N. Bagni, Endogenous polyamine content and

metabolism in the ectomycorrhizal fungus Paxillus involutus, New Phy-

tologist 143 (1999) 581e587.

[17] N. Goicoechea, M.C. Antolin, M. Sanchez Diaz, Gas exchange is related

to the hormone balance in mycorrhizal or nitrogen-fixing alfalfa

subjected to drought, Physiologia Plantarum 100 (1997) 989e997.

[18] N. Goicoechea, G. Szalai, M.C. Antolin, M. Sanchez-Diaz, E. Paldi, In-

fluence of arbuscular Mycorrhizae and Rhizobium on free polyamines

and proline levels in water-stressed alfalfa, Journal of Plant Physiology

153 (1998) 706e711.

[19] H. Greenway, R. Munns, Mechanisms of salt tolerance in non-halo-

phytes, Annual Review. Plant Physiology 31 (1980) 149e190.

[20] P.M. Hasegawa, R.A. Bressan, J.K. Zhu, H.J. Bohnert, Plant cellular and

molecular response to high salinity, Annual Review of Plant Physiology

and Plant Molecular Biology 51 (2000) 463e499.

[21] L. He, K. Nada, Y. Kasukabe, S. Tachibana, Enhanced susceptibility of

photosynthesis to low-temperature photoinhibition due to interruption

of chill-induced increase of S-adenosylmethionine decarboxylase activity

in leaves of spinach (Spinacia oleracea L), Plant Cell Physiology 43

(2002) 196e206.

[22] V. Jindal, A. Atwala, B.S. Sekhon, R. Singh, Effect of vesicular-arbuscu-

lar mycorrhizae on metabolism of moong plants under NaCl salinity,

Plant Physiology and Biochemistry 31 (1993) 475e481.

[23] Y. Kasukabe, L. He, K. Nada, S. Misawa, I. Ihara, S. Tachibana, Overex-

pression of spermidine synthase enhances tolerance to multiple

environmental stresses and up-regulates the expression of various

stress-regulated genes in transgenic Arabidopsis thaliana, Plant and

Cell Physiology 45 (2004) 712e722.

[24] B.P.K. Kishor, Z. Hong, G. Miao, C.A. Hu, D.P.S. Verma, Over expres-

sion of D1-pyrroline-5-carboxylate synthetase increases proline produc-

tion and confers osmotolerance in transgenic plants, Plant Physiology

108 (1995) 1387e1394.

[25] R. Krishnamurthy, K.A. Bhagwat, Polyamines as modulators of salt

tolerance in rice cultivars, Plant Physiology 91 (1989) 500e504.

[26] J. Kurepa, J. Smalle, M.V. Montagu, D. Inze, Polyamines and paraquat

toxicity in Arabidopsis thaliana, Plant Cell Physiology 39 (1998)

987e992.

[27] M.M. Kytoviita, T. Sarjala, Effects of defoliation and symbiosis on poly-

amine levels in pine and birch, Mycorrhiza 7 (1997) 107e111.

[28] C. Langebartels, K. Kerner, S. Leonardi, M. Schraudner, M. Trost,

W. Heller, H. Sandermann Jr., Biochemical plant responses to ozone. I

Differential induction of polyamine and ethylene biosynthesis in tobacco,

Plant Physiology 95 (1991) 882e889.

[29] S. Maiale, D.H. Sanchez, A. Guirado, A. Vidal, O.A. Ruiz, Spermine accu-

mulation under salt stress, Journal of Plant Physiology 161 (2004) 35e42.

[30] M.M.F. Mansour, M.M. Al-Mutawa, Stabilization of plasma membrane

by polyamines against salt stress, Cytobios 100 (1988) 7e17.

[31] M. Marce, D.S. Brown, T. Capell, X. Figueras, A.F. Tiburcio, Rapid

high-performance liquid chromatographic method for the quantitation

of polyamines as their dansyl derivatives: application to plants and

animals tissues, Journal of Chromatography B 666 (1995) 329e335.

[32] T.P. McConigle, M.H. Miller, D.G. Evans, G.L. Fairchild, J.A. Swan, A

new method which gives an objective measure of colonization of roots by

vesicular-arbuscular mycorrhizal fungi, New Phytologist 115 (1990)

495e501.

[33] R.E. Mendoza, E.A. Pagani, Influence of phosphorus nutrition on mycor-

rhizal growth response and morphology of mycorrhizae in Lotus tenuis,

Journal of Plant Nutrition 20 (1997) 625e639.

[34] L. Montes, Lotus tenuis, Revista Argentina de Produccion Animal 8

(1988) 367e376.

[35] M.M. Mujica, C.P. Rumi, A technique of vegetative propagation by stem

cuttings was fitted to Lotus tenuis, Lotus Newsletter 29 (1998).

[36] K. Nada, E. Iwatani, T. Doi, S. Tachibana, Effect of putrescine

pre-treatment to roots on growth and lactate metabolism in the roots of

tomato (Lycopersicon esculentum Mill.) under root-zone hypoxia,

Journal of the Japanese Society for Horticultural Science 73 (2004)

337e339.

[37] I. Paradi, Z. Bratek, F. Lang, Influence of arbuscular mycorrhiza and

phosphorus supply on polyamine content, growth and photosynthesis

of Plantago lanceolata, Biologia Plantarum 46 (2003) 563e569.

[38] J.M. Philips, D.S. Hayman, Improved procedures for clearing roots and

staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid

assessment of infection, Transactions of the British Mycological Society

55 (1970) 158e161.

[39] G.H. Rabie, A.M. Almadini, Role of bioinoculants in development of

salt-tolerance of Vicia faba plants under salinity stress, African Journal

of Biotechnology 4 (2005) 210e220.

[40] M. Roy, B. Ghosh, Polyamines, both common and uncommon, under

heat stress in rice (Oryza sativa) callus, Physiologia Plantarum 98

(1996) 196e200.

46 A.I. Sannazzaro et al. / Plant Physiology and Biochemistry 45 (2007) 39e46

[41] M. Roy, R. Wu, Overexpression of S-adenosylmethionine decarboxylase

gene in rice increases polyamine level and enhances sodium chloride-

stress tolerance, Plant Science 163 (2002) 978e992.

[42] A. Sannazzaro, C. �Alvarez, A. Menendez, F. Pieckenstain, E. Alberto,

O. Ruiz, Ornithine and arginine decarboxilase activities and effect of

some polyamine biosynthesis inhibitors on Gigaspora rosea germinating

spores, FEMS Microbiology Letters 230 (2004) 115e121.

[43] A. Sannazzaro, O. Ruiz, E. Alberto, A. Menendez, Presence of differ-

ent arbuscular mycorrhizal infection patterns in roots of Lotus glaber

plants growing in the Salado River basin, Mycorrhiza 14 (2004)

139e142.

[44] L. Simon-Sarkadi, G. Kocsy, Z. Sebestyen, Effect of salt stress on free

amino acid and polyamine content in cereals, Acta Biologica Szegedien-

sis 46 (2002) 73e75.

[45] S.E. Smith, D.J. Read, Mycorrhizal Symbiosis, Academic Press, San

Diego, 1997.

[46] R. Storey, R.G. Wyn Jones, Betaine and choline levels in plants and

their relationship to NaCl stress, Plant Science Letters 4 (1975)

161e166.

[47] R. Storey, R.G. Wyn Jones, Responses of Atriplex spongiosaa and

Suaeda monoica to salinity, Plant Physiology 63 (1979) 156e162.

[48] M. Tal, A. Katz, H. Heiken, K. Dehan, Salt tolerance in the wild relatives

of the cultivated tomato: proline accumulation in Lycopersicone sculen-

turn Mill., L. peruvianumm Mill. and Solanum pennellii cor. treated

with NaCl and polyethylene glycole, New Phytologist 82 (1979)

349e355.

[49] A.F. Tiburcio, M.A. Masdeu, F.M. Dumortier, A.W. Galston, Polyamine

metabolism and osmotic stress I. Relation to protoplast viability, Plant

Physiology 82 (1986) 369e374.

[50] H. Vaidyanathan, P. Sivakumar, R. Chakrabarty, G. Thomas, Scavenging

of reactive oxygen species in NaCl-stressed rice (Oryza sativa L)-differ-

ential response in salt-tolerant and sensitive varieties 173H, Plant Science

165 (2003) 1411e1418.

[51] B. Waie, M.V. Rajam, Effect of increased polyamines biosynthesis on

stress responses in transgenic tobacco by introduction of human S-adeno-

sylmethionine gene, Plant Science 164 (2003) 727e734.

[52] A.M. Yano-Melo, O.J. Saggin Jr., L. Costa Maia, Tolerance of mycorrh-

ized banana (Musa sp cv. Pacovan) plantlets to saline stress. Agriculture,

Ecosystems and Environment 95 (2003) 343e348.

[53] P.J. Zapata, M. Serrano, M.T. Pretel, A. Amoros, M.A. Botella, Poly-

amines and ethylene changes during germination of different plant spe-

cies under salinity, Plant Science 167 (2004) 781e788.