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: [email protected] (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
0,1
0,15
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0,45
0
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1,2
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0
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0,25
0,3
0,35
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
DW
)Sp
m (µ
mol
/g D
W)
0
0,1
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0
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0,5
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.
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