(This is a sample cover image for this issue. The actual cover is not yet available at this time.) This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright
Transcript
(This is a sample cover image for this issue. The actual cover is not yet available at this time.)
This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
Possible involvement of polyphenols and polyamines in salttolerance of almond rootstocks
Ahlem Zriga, Taïeb Tounektia, Ahmedou Mohamed Vadela, Hatem Ben Mohameda,Daniel Valerob, María Serranob, Chaker Chtarac, Habib Khemiraa,*a Laboratory of Biotechnology Applied to Crop Improvement, Faculty of Sciences of Gabes, University of Gabes, Cité Erriadh, Zrig, 6072 Gabes, TunisiabUniversity Miguel Hernandez, EPSO. Ctra. Beniel km. 3.2, 03312 Orihuela, Alicante, SpaincDepartment of Chemical Engineering, National Engineering School of Gabes, University of Gabes, Omar Ibnelkhattab St., 6029 Zrig, Gabes, Tunisia
a r t i c l e i n f o
Article history:Received 27 June 2011Accepted 24 August 2011Available online 31 August 2011
Leaf physiological and biochemical adaptive strategies and more particularly the possible involvement ofpolyamines and polyphenols in salt stress tolerance were investigated. Three almond rootstocks (GN15,GF677 and bitter almond) were subjected to 0, 25, 50 and 75 mM NaCl for 30 days. The dry mass ofleaves, stems and roots decreased with increasing salt concentration in the irrigation solution regardlessof genotype. Photosynthetic assimilation rate decreased in the three almond rootstocks, but more so inGF677 and bitter almond. The accumulation of toxic ions was greater in the leaves than in the roots in allgenotypes. GN15 accumulated less Naþ and Cl� than GF677 and bitter almond. GF677 accumulatedpolyphenols, but had less anthocyanin and antioxidant activity in its leaves compared to bitter almond. Itseems that GN15 was more able to tolerate the excess of toxic ions using anthocyanins which areabundant in its red leaves and free polyamines for a more efficient response to stress. However, most ofthe antioxidant activity was found in the leaves and was lower in the roots. Given that the upper part ofthe tree will be of a different cultivar after grafting, this advantage may not be relevant for the tree’ssurvival. GF677 showed a different antioxidant strategy; it maintained a stable carotenoids content andaccumulated polyphenols in its leaves. The three rootstocks used different strategies to deal with theexcess of salt in the growth medium.
� 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Increased salinity affects plant growth and development by iontoxicity, induced nutritional deficiencies, water deficit (osmoticeffect) and lower rate of photosynthetic assimilation [1]. Plantsrespond to salt stress by increasing the concentration of compatibleosmolyte in their tissues (osmotic adjustment) [2]. Moreover, theinhibition of photosynthesis causes an over-reduction of thephotosynthetic electron transport chain and redirects photonenergy into processes that favour the production of reactive oxygenspecies [3], such as superoxide anion radical (�O2
�), hydrogenperoxide (H2O2) and hydroxyl radical (OH�), which are harmful toplant growth due to their detrimental effects on most sensitivebiological macromolecules and membranes. To prevent this
oxidative stress from accessing, plants display a multitude of pho-toprotective processes including leaf positioning and detoxificationof chloroplasts via biochemical compounds [4,5]. Most secondarymetabolites involved in stress tolerance are synthesized fromintermediates of primary carbon metabolism via phenylpropanoid,shikimate, mevalonate or MEP pathways. Among those, chloro-phylls, carotenoids, polyphenols, flavonoids and anthocyanins playan important role in scavenging free radicals [5]. Their synthesis isgenerally stimulated under abiotic stress [6] and they are usedduring the detoxification process. For instance, Naþ accumulationin the leaves affects photosynthetic apparatus by decreasing thelevel of pigments such as chlorophyll and carotenoids, which arethe most important defence line in the chloroplast. Under saltstress conditions, the production of polyphenols is related to theleaf carbon economy. Their accumulation is enhanced when carbonproduction overtakes the metabolic demand for growth [7]. Theantioxidant activity of polyphenols is mainly due to their redoxproperties, which allow them to act as reducing agents, hydrogendonors and singlet oxygen quenchers. Among flavonoids, antho-cyanins are highly water soluble pigments derived from flavonoid
Abbreviations: A, net photosynthetic rate; ATN, anthocyanin; Car, carotenoids;Chl (a þ b), total chlorophylls; PA, polyamine; Put, putrescine; TAA, total antioxi-dant activity; Spd, spermidine.* Corresponding author. Tel.: þ216 75 39 26 00; fax: þ216 75 39 24 21.
precursors via the shikimate pathway [5]. They protect the chlo-roplasts of shade adapted [8] and senescing leaves from photo-oxidative damage during exposure to high solar radiation mainlyby absorbing UVeB. Since anthocyanins are osmotically active,their enhanced expression can increase hardiness throughincreased osmotic control [5]. Kaliamoorthy and Rao [9] reportedup to 40% increase in anthocyanin concentration in maize leaves inresponse to increased salinity. Anthocyanins accumulate also whenleaves are exposed to UVeB, drought low temperature, nutrientdeficiency or to ozone [8].
Another response that helps plants become more tolerant tounfavourable environmental conditions is the accumulation of lowmolecular weight osmolytes such as proline [10] and polyamines(PAs) [8]. Many studies suggest that biosynthesis of PAs may be anintegral part of the plant’s response to stress [8]. A salt-inducedincrease in endogenous polyamines’ content has been reported inseveral plant species [11]. However, ionic and osmotic stressinduced by salinity may influence PA metabolism in different ways,and PAs may have different and specific functions under thesestress conditions [11]. The involvement of these compounds inmetabolic adjustment remains unclear. It has been suggested that,due to their polycationic nature, PAs could be involved in cellularionic balance [12].
Almond tree has been described as being sensitive to salinity[13,14]. Different interspecies Prunus hybrids are used as rootstocksfor almond because of their resistance to pathogens and theirtolerance to adverse soil conditions (pH,water logging, and salinity).Among the most widely used are GF677, GN15 and bitter almond.
The aim of this study was to characterize the response of threealmond rootstocks (GF677, GN15 and bitter almond) to soil salinityand to determine if polyphenols and polyamines are implicated inthis response.
2. Results
2.1. Growth, photosynthetic and ionic characteristics
After 30 days of exposure to NaCl treatment, total plant drymassof all three rootstocks was reduced considerably compared tocontrol plants but with various degrees (Table 1). The addition of75 mM NaCl in the culture medium reduced by 33.5%, 48% and 61%the total plant dry mass of bitter almond, GN15 and GF677,
respectively. In the case of GF677 genotype, 75 mM NaCl in theculture medium affected considerably (60%) both shoots and rootsparts, while in GN15, shoot dry mass (DM) was affected more thanroot DM (51%, 45%). However, in better almond the roots DM wasaffected more than shoot DM (46%, 27%). Still, NaCl treatmentincreased root/shoot ratio in GN15 and GF677 rootstocks, but itdecreased in bitter almond (Table 1).
In control plants, the net photosynthetic assimilation rate (A)was greater in the rootstocks with green leaves (bitter almond andGF677) than in GN15 which has reddish leaves (Table 1). However,A of GN15 leaves was the last affected by salt addition. Forinstance, the addition of 75 mM NaCl in the culture mediumdecreased A by 25%, 30% and 37% for GN15, bitter almond andGF677 respectively.
Our results suggest that the effect of salt addition to the growthmedium on Naþ and Cl� absorption and partitioning depended ongenotype, and salt concentration (Fig. 1). In all three genotypes, theleaves accumulated considerably more Naþ and Cl� than roots. Atthe highest NaCl concentration, GN15’s leaves and roots containedless Naþ levels in their leaves (317 and 188 meq g�1DW respectively)compared to other genotype. However, the pattern of accumulationof Naþ was comparable for the three rootstocks. Naþ was parti-tioned preferentially to the leaves not the roots. The accumulationstarted at 25 mM for GN15 and GF677 and only at 50 mM for bitteralmond, a maximum concentration which depended on genotypewas rapidly reached. This maximum concentration was about fourtimes higher in GF677 and bitter almond compared to GN15. Theaddition of salt to growth medium increased Cl� concentration inthe leaves but not the roots. The accumulation of Cl� in the leaveswas gradual. After one month of treatment with 75 mM NaCl, thehighest increase of Cl� was recorded in the leaves of bitter almond(60%) and GF677 (50%) as compared to GN15 (31%).
In all three rootstocks, adding NaCl salt to the growth mediumsignificantly reduced Kþ concentration and Kþ/Naþ ratio in theleaves (Table 2). The addition of 75 mM NaCl reduced leaf Kþ
concentration by 38%, 34% and 30% in bitter almond and GF677 andGN15 respectively. GN15 leaves maintained the highest Kþ/Naþ
ratio at all salinity levels. Root Kþ content changed little because ofsalinity.
2.2. Leaf pigments
The effect of growth medium salinity on leaf pigments variedbetween cultivars. In bitter almond, Chla, total chlorophyll andcarotenoids’ content were depressed by salinity but Chlb contentand Chla/Chlb and carotenoids/Chl (a þ b) ratio were not affected.
In GF677, chlorophyll’s contents were reduced by salinitywhereas carotenoids were not affected, Chla/Chlb and carotenoids/Chl (a þ b) ratios increased.
In GN15, chlorophylls were not or little affected by growthmedium salinity; Chla/Chlb ratio increased, carotenoids’ contentand carotenoids to chlorophylls ratio decreased (Fig. 2).
The leaves of GN15 which have a reddest appearance had thehighest anthocyanin (ANT) concentration (Fig. 3). These pigmentsdecreased progressively with increasing salt level in GN15 root-stock. However, the concentration of these compounds remainedunchanged in bitter almond and GF677 leaves. The individual ANT,cyanidin-3.5-glucoside and petunidin-3-glcoside showed the samevariation pattern in response to salt stress treatments in bitteralmond. However, petunidin-3-glucoside decreased in GF677mainly with 50 and 75 mM NaCl. Regardless of salt level, GN15 hadthe highest ANT content, especially cyaniding-3.5-glucoside,compared with GF677 and bitter almond. The ANT/Chl (a þ b) ratiodecreased considerably under salt stress conditions in GN15, but itincreased in bitter almond with 75 mM NaCl.
Table 1Salt effect on growth parameters and photosynthetic assimilation rate (A) of threealmond rootstocks. Each point represents themean (�SE) of 4 replicates. Differenceswere considered significant at probability level of P� 0.05 (results of Duncan’s test).Different letters indicate significant differences between treatments for a givenrootstock.
The pattern of variation of total polyphenols was different acrosscultivars, organs and NaCl treatments (Fig. 4). In GF677 rootstock,leaf polyphenols increased only with 75 mM NaCl, while rootspolyphenols decreased progressively with increasing salinity in the
medium. Furthermore, total leaf polyphenols decreased in GN15 at50 and 75 mM NaCl and remained unchanged in bitter almond. Incontrast, we recorded a significant accumulation of total poly-phenols in the GN15 and bitter almond roots with the lowest NaCllevel (25 mM), the concentration of these compounds decreased atthe higher salt levels.
Table 2Effect of salinity on Kþ partitioning and Kþ/Naþ ratio in the roots and leaves of three almond rootstocks. Mean separation within columns by Duncan’s Multiple Range Test(P � 0.05). Different letters indicate significant differences between treatments for a given rootstock.
Fig. 1. Effect of salinity on Naþ and Cl� partitioning in three almond rootstocks. Each point represents the mean (�SE) of four replicates. Mean separation by Duncan’s MultipleRange Test. Different letters indicate significant differences between treatments (P � 0.05).
A. Zrig et al. / Plant Physiology and Biochemistry 49 (2011) 1313e1322 1315
Author's personal copy
2.4. Polyamines
The concentrations of putrescine (Put) and spermidine (Spd) inGN15 leaves increased by more than four folds at the highest NaCllevel (Fig. 5). They were not affected by salinity treatments in theother twocultivars. Sperminewasnotdetected in the leavesor roots.
2.5. Antioxidant activity
Salinity had a significant effect on total antioxidant activity inthe leaves and roots of all three genotypes (Fig. 6). This effect wascultivar dependent. The antioxidant activity of leaf extracts wasreduced by salinity in bitter almond and increased in GF677 andGN15. The activity in the roots was reduced in bitter almond,slightly reduced then increased in GF67 and unchanged in GN15.
2.6. Correlations
The relationships of relative changes in ions, anthocyanins,photosynthetic pigments, polyphenols and polyamines with
photosynthetic assimilation rate (A) were determined for threealmond rootstocks separately (Table 3). Reduction in A was nega-tively correlated with toxic ions contents in the leaves, but posi-tively correlated with the Kþ contents, whichmay suggest a generalionic homeostasis effect on photosynthesis. In GF677, the strongpositive correlation may suppose an evident photo-limitation ofthe photosynthesis by the chlorophylls degradation. Besides, thepositive and the negative correlations of anthocyanins and poly-phenols respectively in GN15 and GF677 may suggest a severalroles of each compound in salt tolerance for the correspondingrootstocks.
3. Discussion
Almond tree is thought to be a salt sensitive glycophyte [14]. Thepresent study supports this contention, since we recordeda considerable reduction in total plant dry mass in all three almondrootstocks. Therefore, irrigating almond trees with moderatelysaline water does not threaten their survival but it affects theirgrowth and yield. However GN15 and GF677 had higher total dry
Fig. 2. Effect of salinity stress on leaf chlorophyll (Chl) and carotenoid (Car) content and their ratios. Each point represents the mean (�SE) of four replicates. Mean separation byDuncan’s Multiple Range Test. Different letters indicate significant differences between treatments (P � 0.05).
A. Zrig et al. / Plant Physiology and Biochemistry 49 (2011) 1313e13221316
Author's personal copy
mass than bitter almond at all salinity levels. Furthermore, thesetow hybrids had higher root dry mass regardless of salt treatmentand higher Root/shoot ratio which may have allowed efficientwater and nutrients’ uptake from the soil. This suggests that GN15and GF677 are less sensitive to soil salinity than bitter almond.
Our results suggest that all three rootstocks accumulated Naþ
preferentially in their leaves, a typical behaviour of an “includer”glycophyte [7]. The inclusion of toxic ions would be advantageous,provided they are sequestered in the cell vacuole, which protectsthe cytoplasm against the toxic effect. Control plants (not treated)of bitter almond and GF677 contained three to four times more Naþ
in their organs than GN15. The latter accumulated more Cl� insteadof Naþ. It appears that GN15 has the capacity to limit Naþ uptake byits roots; this may explain the low Naþ concentration in the leavesunder salt stress conditions. The restriction of Naþ uptake was alsoseen in GF677 and bitter almond roots but to a lesser extent. RootNaþ content did not increase and leaf Naþ content levelled off aftera slight initial increase as NaCl concentration in the growthmedium was increased from 0 to 75 mM. In contrast, Cl� uptakepattern was linear; leaf Cl� concentration increased linearly asexternal NaCl concentration increased. This was less so in the caseof GN15. The increased uptake of Cl� combined with a limitedproduction of new leaves, can lead to its build-up to toxic levels[14]. Our results confirm previous reports that for Prunus most ofNaCl toxicity is due to Cl� [14]. This toxic anion appears that havea determinant role in the sensitivity of almond tree to soil salinity.
Saline conditions can rapidly reduce the capacity of roots toabsorb essential nutrients from the soil [2]. Indeed, Kþ content inmany glycophytes can be reduced substantially under such stress[7]. Our results show that the greatest decrease in Kþ content in thethree almond rootstocks occurred in the leaves, suggestinga reduction in the discrimination in favour of Kþ [2], resulting ina low Kþ/Naþ ratio. Despite the reduction in its leaf Kþ content,
GN15 was the only rootstock which maintained a leaf Kþ/Naþ ratiogreater than one, the minimum level required for normal func-tioning of plants under saline conditions [15]. Thus, a high Kþ/Naþ
ratio under saline conditions could be considered a selectioncriterion for salt tolerance in Prunus. The lack of selectivity for Kþ
may have caused a nutrient unbalance which lead to the decline innet photosynthetic assimilation rate (A). The latter was positivelycorrelated (r ¼ 0.74e0.89, P < 0.05) with Kþ concentration in theleaves. In GF677, the correlation analysis suggests that the declinein A was associated with a reduction in total chlorophyll content(r ¼ þ0.87, P < 0.05), suggesting a photochemical limitation tophotosynthesis. The decrease of total chlorophyll content may bedue to either an increase in chlorophyll degradation or a decrease inde novo synthesis [16]. Our results suggest that the reduction intotal Chl was mainly due to a rapid degradation of Chlb undersalinity stress suggesting that some structural damage has occurredin photosystem II reaction centres [16]. Therefore, salt stressappeared to have caused a reduction in antenna size in chloroplastsof GF677 leaves. Similar results were reported for Cymbopogonnardus (L.) Rendle [17].
Total chlorophyll content of GN15 leaves seems to be lessaffected by the medium salinity compared to GF677 and bitteralmond, whichmay explain the lower decline in A in GN15 leaves. Itappears that latter were able to overcome salt-induced photo-oxydation of their chloroplastmembranes [18]. The lower decline inphotosynthetic pigments’ content is important, as it has a directrelationship with salinity tolerance [16]. The increase in Chla/Chlbratio in GN15 and GF677 mainly with 50 and 75 mM NaCl may bedue to a conversion of Chlb to Chla. The inter-conversion of Chla andChlb plays a significant role in the establishment of required Chla/Chlb ratio during the adaptation of stressful conditions [17]. Thehigher Chla/Chlb ratio indicates also less emphasis on light har-vesting in relation to the rates of PSII photochemistry under stress.
Fig. 3. Salt effect anthocyanins content: ANT, ANT/Chl (a þ b), cyanidin 3.5-glucoside, and Petunidin 3-glucoside. Each point represents the mean (�SE) of 4 replicates. Differentletters indicate significant differences between treatments (Duncan test, P � 0.05).
A. Zrig et al. / Plant Physiology and Biochemistry 49 (2011) 1313e1322 1317
The carotenoids content showed significant differences amongthe three genotypes; this suggests different photoprotectionresponses under salt stress conditions. For instance, salt stress didnot affect leaf carotenoids’ content of GF677 rootstock leaves; thisshould confer some photoprotection for their chloroplasts since ithas been reported that high carotenoids’ content in the leaves is anindication of stress tolerance [19]. The Car/Chl (aþ b) ratio increasedby 40% in GF677 at the highest NaCl level (75 mM), .Such increaseswere reported in several plant species such as sugarcane [19] andC. nardus [17]. Thus, it appears that in GF677, carotenoids play aneffective role against photooxydation by scavenging the reactiveoxygen species which can be generated under salt stress [7].
Chl content of GN15 leaves was not affected by salinity; thissuggests an efficient protection against photooxydation duringsalinity stress despite a significant decline in carotenoids’ content.The protective role of carotenoids here may have been eitherenhanced or replaced by anthocyanins which are abundant in thered leaves of this rootstock. In fact, it has been reported that the
inadequate photochemical and non-photochemical sinks for excessexcitation energy (lower nitrogen contents and xanthophylls cyclepool size) observed in red leaves of species such as Cistus creticusplants are compensated by anthocyanins; this can make up for thedeficiency and alleviate the risk of photodamage [20].
Modulations in the levels of carotenoids and flavonoidsincluding anthocyanins are of great importance in the prevention ofstress induced oxidative damage and the maintenance of osmoticbalance [21]. Apart from the direct, light screening function,anthocyanins may indirectly protect against excess light throughtheir oxy-radical scavenging properties [22]. In the present study,salt stress (mainly Cl� toxicity) decreased progressively the leafanthocyanin content in GN15 rootstock only. The effect of Kþ
decrease on anthocyanins in the leaves of this rootstock is notevident since Kþ deficiency in more commonly associated with theaccumulation of anthocyanins [23]. The Ant/Chl (a þ b) ratiodecreased too suggesting that anthocyanins protected chlorophyllsagainst reactive oxygen species and played a role in
Fig. 4. Effect of growth medium salinity on total polyphenol concentrations in the leaves and roots of three almond rootstocks. Each point represents the mean (�SE) of fourreplicates. Mean separation by Duncan’s Multiple Range Test. Different letters indicate significant differences between treatments (P � 0.05).
A. Zrig et al. / Plant Physiology and Biochemistry 49 (2011) 1313e13221318
Author's personal copy
photoprotection. For instance, cyanidine can serve as a sink forelectrons from H2O2 and lessen the oxidative damage to chloro-phyll pigments [24]. Furthermore, under salt stress conditions,anthocyanins can bind toxic ions, and thus protect chloroplasts andcytoplasmic structures from the adverse effects of salinity, andallow sufficient plant photosynthetic assimilation [22]. In our study,chlorophyll content and Awere least affected by salinity in the red-leafed GN15 rootstock. Furthermore, it was reported that antho-cyanic leaves had a higher antioxidant capacity compared to greenones; anthocyanins contribute to that capacity more than other lowmolecular weight compounds [22].
In higher plants, phenolic compounds were recently recon-sidered to be relevant in oxidative stress tolerance [19]. Theantioxidant activity of phenolics is mainly due to their redoxproperties, which allow them to act as reducing agents, hydrogendonors, and singlet oxygen quenchers [6]. According to our results,the effect of salinity on polyphenols depended on cultivar and theorgan considered. For instance, GF677 rootstock accumulatedhigher amounts of polyphenols in their leaves at 75 mM NaCl,
while their root content decreased. Salt stress induces higherlevels of polyphenolic compounds in different tissues of severalplant species [25,26]. Tattini et al. [27] mentioned a close rela-tionship between oxidative stress tolerance and flavonoids accu-mulation. In contrast, total polyphenols content of GN15 tissuesdecreased when salt concentration in the medium was increased(50 and 75 mM NaCl); this was apparently due to the degradationof anthocyanins.
As for polyamines, we recorded only an accumulation of Put andSpd in the leaves of GN15 plants irrigated with the 75 mM NaClsolution. Similar results were reported in Lupinus luteus [26]. Ahigher Put and Spd concentration in plant tissues can improve theirKþ/Naþ homeostasis [28]. Furthermore, exogenous applications ofPut and Spd reduce salt-induced oxidative damage by activatingenzymatic and non enzymatic antioxidants [8].
Salinity of the culture medium did not affect significantly theantioxidant activity in the leaves and roots of GF677 and GN15rootstocks, while decreased it in the leaves of bitter almond. Itappears, therefore, that the least sensitive rootstocks (GF677 and
Fig. 5. Effect of growth medium salinity on free putriscine and spermidine concentrations in the leaves of three almond rootstocks. Each point represents the mean (�SE) of fourreplicates. Mean separation by Duncan’s Multiple Range Test. Different letters indicate significant differences between treatments (P � 0.05).
A. Zrig et al. / Plant Physiology and Biochemistry 49 (2011) 1313e1322 1319
GN15) had efficient antioxidant mechanisms in their leaves androots. Similar results were reported in cowpea [29]. Generally,carotenoids, low molecular weight antioxidants and mostly poly-phenols including anthocyanins are the most frequent antioxidantcompounds in many plants with red leaves [19].
In conclusion, this study supports the hypothesis that antho-cyanins play an important physiological role in protecting almondrootstocks against salt stress. The response varies between geno-types and organs. For instance, it seems that GN15 was able toovercome salt toxicity using anthocyanins which were abundant inits red leaves and free polyamines. Given that the upper part of thetreewill be of a different cultivar after grafting, this advantage (highantioxidant activity in the leaves) may not be relevant for the tree’ssurvival. GF677 showed a different antioxidant strategy; it main-tained a stable carotenoids’ content and slightly accumulatedpolyphenols in its leaves. Bitter almond was the most sensitiveamong the three rootstocks.
Fig. 6. Effect of growth medium salinity on antioxidant activity in the extracts of leaves and roots of three almond rootstocks. Each point represents the mean (�SE) of fourreplicates. Mean separation by Duncan’s Multiple Range Test. Different letters indicate significant differences between treatments (P � 0.05).
Table 3Correlation coefficients (r) of photosynthetic assimilation rate (A) on Naþ, Cl�, Kþ,total chlorophyll (Chl (a þ b)), carotenoid (Car), anthocyanin (ANT), polyphenols(Poly), putricine (Put) and spermidine (Spd) leaf contents. Asterisks indicatestatistically significant correlations at probability levels of P � 0.05 (*) and P � 0.01(**), while ns denotes not significant correlation.
A. Zrig et al. / Plant Physiology and Biochemistry 49 (2011) 1313e13221320
Author's personal copy
4. Materials and methods
4.1. Plant material and salt treatments
The present study was performed on eight months-old rootedcuttings of three almond rootstocks: bitter almond (Prunusamygdalus) and the two hybrids Prunus rootstocks, GF677(P. amygdalus� Prunus persica) andGarnemGN15 (Garfi�Nemared).The plants were cultivated in perforated-4L-plastic-pots containingdesert dune-sand under controlled conditions (temperature:25 � 2 �C; light intensity (PAR): 500e700 mM m�2 s�1). They wereirrigated every 4dayswith a complete nutrient solution (N,1.8mM;P,0.35 mM; K, 0.64 mM; Ca, 1.0 mM; Mg, 0.35 mM; S, 035 mM; .Fe,0.03 mM; Zn, 0.4 mM, Mn, 5.0 mM; Cu, 0.1 mM and B, 0.02 mM). Aftera period of acclimatization (one month), salt stress was imposed onthese plants by increasing the concentration of NaCl in the solution to25, 50 or 75 mM. To avoid osmotic shock, NaCl concentration wasincreased gradually by 25 mM increment each day until the desiredconcentrationswere reached. Every fourdays, the substrate in thepotwaswashed twicewith tapwater toavoid salt build-upthen500mlofthe nutrient solution, enough to cause some drainage, was applied.The experimental design was a completely randomized block withfour replicates (each pot contained one plant being a replicate). Theseplants were harvested after four weeks of initiating the treatments.Fullyexpanded leaves fromeachplantwereharvested in themorning(between 9 and 11 a.m. local time), weighed and divided into twobatches; one was frozen in liquid nitrogen and then stored at�80 �Cfor biochemical analyses; the other was washed in de-ionized water,dried at 80 �C in a forced-air oven for 48 h and ground into a finepowder to pass through a 30-mesh screen for ion analyses.
4.2. Growth, ion contents and gas exchange
At the end of the experiment, all four plants were individuallyharvested and divided into roots, stems and leaves. After recordingtheir total fresh weight, all plant organs were oven-dried at 80 �Cfor 48 h then weighed again. Plant growth was estimated bydetermining the dry weight of leaves, stems and roots.
For ion analyses, 1 g of dry ground leaves from each plant wasextracted with 20 mL of 0.1 M HNO3. After filtration, Naþ contentswere determined with an atomic absorption spectrometer (Avanta,GBC, Australia), and Cl� contents were determined with a chlorideanalyzer (CorningM926 chloride analyser, Halstead, Essex, UK). Netphotosynthetic rate (A) of fully expanded leaves was determinedwith a portable photosynthesis system (LCp proþ, ADC, UK) undernatural conditions (PAR was 800e1200 mM m�2 s�1 and airtemperature was 20e30 �C).
4.3. Determination of chlorophylls and carotenoids
Leaf chlorophyll and carotenoid contents were determined byusing the method of Arnon [30]. In short, 0.5 g of fresh leaves weregrounded in liquid nitrogen to a fine powder in a pre-cooledmortarand homogenized for 30 s in 5 mL of 95.5% acetone. The pigments’concentrations were estimated from absorbance at 647 nm and664 nm. A solution of 95.5% acetone was used as a blank. Pigmentconcentrations were calculated as follows: Chl a (mg/gFW) ¼ [12.7 � (A664) _ 2.69 � (A647)] � (0.5 � 5), Chl b (mg/gFW) ¼ [22.9 � (A647) _ 4.69 � (A664)] � (0.5 � 5).
Total carotenoids were extracted in duplicates according toMínguez-Mosquera and Hornero-Méndez [31]. One gram of frozenleaf tissue was briefly extracted with acetone and shaken withdiethyl ether and 10% NaCl. Two phases were obtained; the lipo-philic phase was washed with Na2SO4 (2%), saponified with 10%KOH in MeOH, and the pigments were subsequently extracted with
diethyl ether, evaporated and then made up to 25 mL with acetone.Total carotenoids were estimated by reading the absorbance at450 nm in a UNICAM Helios- spectrophotometer (Cambridge, UK),and expressed as mg of b-carotene equivalent per kg fresh weight,taking into account the molar absorption coefficient ( 3
1% cm) of2560. The results are presented as means � SE.
4.4. Anthocyanin content
Leaf anthocyanins were determined according to Serrano et al.[32]. Total anthocyanin was calculated using as a standard cyanidin3-glucoside (molar absorption coefficient of 23.900 L cm�1 mol�1
and molecular weight of 449.2 g mol�1) and the results, expressedas mg of anthocaynin per kg fresh weight, were the mean � SE ofduplicate determinations made on each one of four subsamples.
Anthocyanins were assayed by high performance liquid chro-matography coupled with a diode array detector (HPLC-DAD) [33].One millilitre from the extracts obtained for total anthocyaninquantificationwas filtered through 0.45 mmMillipore filter and theninjected into a HewlettePackard HPLC series 1100 equipped withaC18Supelco column (Supelcogel Ce610H, 30 cm�7.8mm, SupelcoPark, Bellefonte, USA) and detected by absorbance at 510 nm. Thepeaks were eluted by a gradient using the followingmobile phases:95% water þ 5% MeOH (A); 88% water þ 12% MeOH (B); 20%waterþ 80%MeOH (C); andMeOH (D) at a rate of 1mLmin�1. Peakswere identified using authentic standards by comparison of theretention times and peak spectral analysis. The anthocyanin stan-dards were provided by Dr. García-Viguera et al. [33].
4.5. Phenolic compounds contents
Phenolic compounds were extracted according to Tomás-Bar-berán et al. [34] using water: MeOH (2:8) containing 2 mMNaF andquantified using the FolineCiocalteu reagent [35]. The results wereexpressed as mg gallic acid equivalent per kg fresh weight ofduplicate determinations made on each subsample.
4.6. Antioxidant activity
The total antioxidant activity (TAA)wasquantified also induplicatefor each subsample according to Serrano et al. [36], which enables thedetermination of TAA due to hydrophilic compounds (H-TAA) in thesame extraction. Briefly,1 g of fresh leaves or rootswere homogenizedin5mLof50mMphosphatebufferpH7.8and3mLofethylacetate, andthen centrifuged at 15.000 rpm for 15 min at 4 �C. two fractions wereobtained, the lower fraction was used for H-TAA quantification. TAAwas determined using the enzymatic system composed of the chro-mophore 2.2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) dia-mmonium salt (ABTS), the horse radish peroxidase enzyme (HRP) anditsoxidant substrate (hydrogenperoxide), inwhichABTS�þ radicals aregenerated andmonitored at 730 nm. The decrease in absorbance afteradding the extracts was proportional to TAA of the sample. A calibra-tion curve (0e20 nM)was performedwith Trolox (R)-(þ)-6-hydroxy-2.5.7.8-tetramethylcroman-2-carboxylic acid (Sigma, Madrid, Spain)in aqueous media for H-TAA. The results are expressed as themean� SE in mg of Trolox equivalent kg�1 fresh weight.
4.7. Free polyamine contents
For each replicate, 1 g fresh leaves was extracted with 10 mL of5% cold perchloric acid, with 1.6 hexanediamine (100 nmol g�1 oftissue) added as an internal standard. The homogenate was thencentrifuged for 30 min at 20 000 � g. A 2 mL aliquot of thesupernatant was used to determine free polyamines by benzoyla-tion, and derivatives analysed by HPLC according to Serrano et al.
A. Zrig et al. / Plant Physiology and Biochemistry 49 (2011) 1313e1322 1321
Author's personal copy
[37]. The elution system consisted of MeOH/H2O (64:36) solvent,running isocratically with a flow rate of 0.8 mL min�1. The ben-zoylpolyamines were eluted through a reversed-phase column(LiChro Cart 250- 4.5 mm) and detected by absorbance at 254 nm. Arelative calibration procedure was used to determine the poly-amines in the samples, using 1.6 hexanediamine as the internalstandard and standard curves covered the range 1e320 nM. Thecalibration curves were y ¼ 10.66x þ 170.00, r2 ¼ 0.94 for Put,y¼ 10.19x� 39.96, r2¼ 0.96 for Spd, and y¼ 11.52x� 4.32, r2¼ 0.90for Spermine.
4.8. Statistical analyses
Variance of the data was analyzed with GLM procedure of SASsoftware [38] for a Randomized Complete Block design with fourreplicates. Both main effects (salinity level and rootstock) andinteractions were considered in the analyses. Where applicable,means were separated by Duncan’s Multiple Range Test(P � 0.05).
Acknowledgments
The authors are grateful to Groupe Chimique Tunisien (GCTGabes) for their financial support and the Technical Services of theUniversity Miguel Hernandez, EPSO Spain which helped with partof the biochemical analyses.
References
[1] T.J. Flowers, Improving crop salt tolerance, J. Exp. Bot. 55 (2004) 307e319.[2] I. Türkana, T. Demiral, Recent developments in understanding salinity toler-
potato tubers: a self amplifying response to stress, Plant Physiol. 13 (2003)1440e1449.
[4] R. Munns, Comparative physiology of salt and water stress, Plant Cell Environ.25 (2002) 239e250.
[5] L. Chalker-Scott, Environmental significance of anthocyanins in plant stressresponses, Photochem. Photobiol. 70 (1999) 1e9.
[6] A.K. Parida, A.B. Das, Salt tolerance and salinity effects on plants: a review,Ecotoxicol. Environ. Safe 60 (2006) 324e349.
[7] S. Barthod, Z. Cerovic, D. Epron, Can dula chlorophyll fluorescence excitationbe used to assess the variation in the content of UV radiation? J. Exp. Bot. 58(2007) 1753e1760.
[8] R. Alcázar, T. Altabella, F. Marco, C. Bortolotti, M. Reymond, C. Koncz,P. Carrasco, A. Tiburcio, Polyamines: molecules with regulatory functions inplant abiotic stress tolerance, Planta 231 (2010) 1237e1249.
[9] S. Kaliamoorthy, A.S. Rao, Effect of salinity on anthocyanins accumulation inroots of maize, Ind. J. Plant Physiol. 37 (1994) 169e170.
[10] B. Zhu, J. Su, M. Chang, D.P.S. Verma, Y.L. Fan, R. Wu, Overexpression of D1-pyrroline-5-carboxylate synthetase gene and analysis of tolerance to water-and salt-stress in transgenic rice, Plant Sci. 139 (1998) 41e48.
[11] J.H. Liu, H. Inoue, T. Moriguch, Salt stress-mediated changes in free polyaminetiters and expression of genes responsible for polyamine biosynthesis of applein vitro shoots, Environ. Exp. Bot. 62 (2008) 28e35.
[12] M. Janicka-Russak, K. Kaba, E. Modzinska, G. Kobus, The role of polyamines inthe regulation of the plasma membrane and the tonoplast proton pumpsunder salt stress, J. Plant Physiol. 167 (2010) 261e269.
[13] A. Ranjbartoreh, G. Samson, P. Van Damme, Chlorophyll fluorescenceperformance of sweet almond (Prunus dulcis Miller) in response to salinitystress induced by NaCl, Photosynthetica 44 (2006) 513e522.
[14] S. Najafian, M. Rahemi, V. Tavallali, Effect of salinity on tolerance of two bitteralmond rootstocks, American-Eurasion, J. Agric. Environ. Sci. 3 (2008) 264e268.
[15] R.G. Wyn Jones, Salt tolerance. in: C.B. Johnson (Ed.), PhysiologicalProcesses Limiting Plant Productivity. Butterworth, London, 1981, pp.271e291.
[16] M. Ashraf, G. Sarwar, Salt tolerance potential in some members of Brassica-ceae. Physiological studies on water relations and mineral contents. in:R. Ahmed, K.A. Malik (Eds.), Prospects for Saline Agriculture. Kluwer AcademicPublishers, Netherland, 2002, pp. 237e245.
[17] A.V. Mane, B.A. Karadge, J.S. Samant, Salinity induced changes in photosyn-thetic pigments and polyphenols of Cymbopogon nardus (L.) Rendle, J. Chem.Pharm. Res. 2 (2010) 38e347.
[18] T. Tounekti, A.M. Vadel, M. Onate, H. Khemira, S.M. Bosch, Salt-inducedoxidative stress in rosemary plants: damage or protection? Environ. Exp. Bot.71 (2011) 298e305.
[19] A. Wahid, A. Ghazanfar, Possible involvement of some secondary metab-olites in salt tolerance of sugarcane, J. Plant Physiol. 163 (2006) 723e730.
[20] V.P. Kytridis, Y. Manetas, Mesophyll versus epidermal anthocyanins aspotential in vivo antioxidants: evidence linking the putative antioxidantrole to the proximity of oxy-radical source, J. Exp. Bot. 57 (2006)2203e2210.
[21] Y. Manetas, Why some leaves are anthocyanic and why most anthocyanicleaves are red? Flora 201 (2006) 163e177.
[22] K.S. Gould, J. McKelvie, K.R. Markham, Do anthocyanins function as antioxi-dants in leaves; imaging of H2O2 in red and green leaves after mechanicalinjury, Plant Cell Environ. 25 (2002) 1261e1269.
[23] S. Neill, K. Gould, Optical properties of leaves in relation to anthocyaninconcentration and distribution, Can. J. Bot. 77 (1999) 1777e1782.
[24] I. Hernández, L. Alegre, F. Van Breusegem, S. Munné-Bosch, How relevantare flavonoids as antioxidants in plants? Trends Plant Sci. 14 (2009) 125e132.
[25] A. Parida, A.B. Das, P. Das, NaCl stress causes changes in photosyntheticpigments, proteins and other metabolic components in the leaves of a truemangrove, Bruguiera parviflora, in hydroponic cultures, J. Plant Biol. 45 (2002)28e36.
[26] J. Legocka, A. Kluk, Effect of salt and osmotic stress on changes in polyaminecontent and arginine decarboxylase activity in Lupinus luteus seedlings,J. Plant Physiol. 162 (2005) 662e668.
[27] M. Tattini, D. Remorini, P. Pinelli, G. Agati, E. Saracini, M.L. Traversi, R. Massai,Morpho-anatomical, physiological and biochemical adjustments in responseto root zone salinity stress and high solar radiation in two Mediterraneanevergreen shrubs, Myrtus communis and Pistacia lentiscus, New Phytol. 170(2006) 779e794.
[28] F. Zhao, C.P. Song, J. He, H. Zhu, Polyamines improve Kþ/Naþ homeostasis inBarley seedlings by Regulating root ion Channel Activities, Plant Physiol. 145(2007) 1061e1072.
[29] F.R. Cavalcanti, J. Lima, S. Ferreira-Silva, R. Viégas, J. Silveira, Roots and leavesdisplay contrasting oxidative response during salt stress and recovery incowpea, J. Plant Physiol. 164 (2007) 591e600.
[30] D.I. Arnon, Copper enzyme in isolated chloroplasts. Polyphenoloxidase in Betavulgaris, Plant Physiol. 24 (1949) 1e15.
[31] M.I. Mınguez-Mosquera, D. Hornero-Mendez, Separation and quantificationof the carotenoid pigments in red peppers (Capsicum annuum L.), paprika,and oleoresin by reversed phase HPLC, J. Agric. Food Chem. 41 (1993)1616e1620.
[32] M. Serrano, F. Guillén, D. Martínez-Romero, S. Castillo, D. Valero, Chemicalconstituents and antioxidant activity of sweet cherry at different ripeningstages, J. Agric. Food Chem. 53 (2005) 2741e2745.
[33] C. García-Viguera, P. Zafrilla, F. Romero, P. Abellá, F. Artés, F.A. Tomás-Bar-berán, Color stability of straw berry jam as affected by cultivar and storagetemperature, J. Food Sci. 64 (1999) 243e247.
[34] F.A. Tomás-Barberán, M.I. Gil, P. Cremin, A.L. Waterhouse, B. Hess-Pierce,A.A. Kader, HPLDeDADeESIMS analysis of phenolic compounds in nectarines,peaches, and plums, J. Agric. Food Chem. 49 (2001) 4748e4760.
[35] V.L. Singleton, R. Orthofer, R.M. Lamuela-Raventos, Analysis of total phenolsand other oxidation substrates and antioxidants by means of FolineCiocalteureagent, Meth. Enzymol. 299 (1999) 152e178.
[36] M. Serrano, H.M. Díaz-Mula, P.J. Zapata, S. Castillo, F. Guillén, D. Martínez-Romero, J.M. Valverde, D. Valero, Maturity stage at harvest determines thefruit quality and antioxidant potential after storage of sweet cherry cultivars,J. Agric. Food Chem. 57 (2009) 32e46.
[37] M. Serrano, D. Martínez-Romero, F. Guillén, D. Valero, Effects of exogenousputrescine on improving shelf life of four plum cultivars, Postharvest Biol.Technol. 30 (2003) 259e271.
[38] Sas, Sas Institute User’s Guide: Statistics, Version 550 6 551. SAS Institute,Cary, NC. USA, 1996.
A. Zrig et al. / Plant Physiology and Biochemistry 49 (2011) 1313e13221322
