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Environmental and Experimental Botany 97 (2014) 1– 10
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Environmental and Experimental Botany
jou rn al h om epa ge: www.elsev ier .com/ locate /envexpbot
roline and reactive oxygen/nitrogen species metabolism is involvedn the tolerant response of the invasive plant species Ailanthusltissima to drought and salinity
anagiota Filippoua, Pavlos Bouchagierb, Effie Skottib, Vasileios Fotopoulosa,∗
Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, PO BOX 50329, 3603 Limassol, CyprusTechnological Educational Institution of Ionian Islands, Department of Technology of Organic Agriculture and Food Science, 281 00 Argostoli, Kefallonia,reece
r t i c l e i n f o
rticle history:eceived 15 July 2013eceived in revised form 4 September 2013ccepted 26 September 2013
eywords:biotic stresseactive oxygen specieseactive nitrogen species
a b s t r a c t
Ailanthus altissima (Miller) Swingle (family Simaroubaceae), commonly known as the ‘Tree of Heaven’,grows aggressively in harsh environments where it invades abandoned fields or cracked city sidewalks.The present study deals with the adaptation of defence mechanisms of A. altissima seedlings subjectedto two of the most important abiotic stress factors worldwide, drought and salinity. Salinity-stressed A.altissima seedlings were obtained by watering the plants with two different NaCl concentration solutions(150 and 300 mM) for 48 h, while drought-stressed plants were obtained after withholding watering for14 d. Physiological parameters, reactive oxygen/nitrogen species and malondialdehyde content measure-ments in stressed plants indicated the abiotic stress factor-specific regulation of its defence response.
rolineitrate reductaseilanthus altissima
Moreover, the content of the osmoprotective molecule proline was also affected by both stresses inparallel to the oxidative/nitrosative markers. Nitrate reductase enzymatic activity and protein contentinvolved in nitric oxide biosynthesis, �1-pyrroline-5-carboxylate synthetase enzymatic activity involvedin proline biosynthesis, as well as the activity of H2O2-generating and scavenging enzymes (superoxidedismutase and catalase, respectively), provided further biochemical support for the specific abiotic stress
his in
tolerance mechanism of t. Introduction
Ailanthus altissima (Miller) Swingle (family Simaroubaceae) is aioecious plant species which produces a large number of sama-as assisted by wind and water dispersion, grows quickly and has aigh ability to resprout and form dense clonal stands (Kowarik andaumel, 2007). This species grows aggressively in harsh environ-ents where it invades abandoned fields or cracked city sidewalks
Kowarik and Saumel, 2007). The highly invasive behaviour of. altissima, or “Tree of Heaven”, can be attributed to its physi-logical characteristics and the phytotoxic compounds found ints roots and leaves (Tsao et al., 2002), although the presencef phytotoxins in Ailanthus plants was not proven to result inhe occurrence of allelopathy (Motard et al., 2011). It was alsoeported that extracts or semi-purified fractions of A. altissimaere strong plant growth inhibitors (Tsao et al., 2002), with
ilanthone having been identified as the principal plant growthnhibitor in A. altissima against Brassica juncea, Eragrostis tef andemna minor (Lin et al., 1995). The combined effects on the early
∗ Corresponding author. Tel.: +357 25002418; fax: +357 25002632.E-mail address: [email protected] (V. Fotopoulos).
098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.envexpbot.2013.09.010
vasive plant species.© 2013 Elsevier B.V. All rights reserved.
stages of this invasive species, the interaction between geneticand environmental factors, and whether or not they are related tohabitat type, is still poorly understood (Constan-Nava and Bonet,2012).
Previous phytochemical studies have demonstrated the pres-ence of phenolic, flavonoid and total alkaloid contents and theircorrelation with the antioxidant activity of A. altissima crudeextracts (Luis et al., 2012). Interestingly, the secondary metabolitesphenylpropanoids, isolated from the genus Ailanthus, are mainlyused for protection against biotic or abiotic stresses, such as micro-bial infection, wounding, UV radiation, exposure to pollutants andherbivores and have antibacterial and radical scavenging activities(Hwang et al., 2012).
Nowadays, plants experience different types of abiotic stresses,such as drought and salinity, partly as a result of severe climaticchanges (Cramer et al., 2011). Salinity, one of the most importantabiotic stresses limiting crop production, affects many morpholog-ical, physiological and biochemical processes, including water andnutrient uptake (Munns, 2002; Zhao et al., 2007). Drought has been
considered as one of the serious environmental stresses on plantgrowth and development. At the primary level, water deficit altersthe water relation or water balance (Mahdieh et al., 2008), whereas,at the cellular level, it affects the integrity of membranes and
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roteins, which in turn leads to metabolic dysfunction (Fariduddint al., 2009).
The objective of the present study was to identify the relativeffect of key environmental stress factors (drought and salinity)nd investigate how they interact with the emergence and estab-ishment of this invasive tree. These comprehensive approachesre especially needed for invasive species, as they may be essen-ial for predicting alien plant invasion and developing managementechniques to prevent such invasions in natural ecosystems,ncluding protected areas (Mack et al., 2000). Therefore, theresent study deals with the tolerance mechanism of A. altissimalants, subjected to the two most important abiotic stress factorsorldwide.
Both drought and salinity stresses are linked by the fact thathey decrease the availability of water to plant cells (Verslues etl., 2006), thus leading to a destructive plant damage dependingn the resistance type of each plant or tissue. It is well knownhat in plants subjected to environmental stress, the increasedroduction of reactive oxygen species (ROS) (Tanou et al., 2009)nd/or reactive nitrogen species (RNS) (Filippou et al., 2011)esults in cell membrane damage caused by lipid peroxidationnd affects respiratory activity in mitochondria, causing pigmentreak down, leakage of cellular contents and eventually cell deathKarabal et al., 2003). Fortunately, plants have developed variousrotective mechanisms to eliminate or reduce ROS, which are effec-ive at different levels of stress-induced deterioration (Beak andkinner, 2003). They have thus developed a complex defence sys-em to reduce damage by regulating antioxidant enzymes suchs superoxide dismutase (SOD) and catalase (CAT) and produc-ng non-enzymatic antioxidant protective molecules (Woo et al.,007).
Plants accumulate protective compatible solutes, such as pro-ine, in response to drought and salinity in order to facilitate
ater uptake (Ashraf and Foolad, 2007). In addition to osmoticdjustments, proline is important for protecting cells againstOS accumulation under stress conditions. The main pathway forroline biosynthesis during osmotic stress in plants uses gluta-ate which is converted to proline by two successive reductions
atalyzed by �1-pyrroline-5-carboxylate synthetase (P5CS) and1-pyrroline-5-carboxylate reductase (P5CR). Proline accumula-
ion under stress might occur due to an increase in P5CS, theate-limiting enzyme in proline biosynthesis (Spoljarevic et al.,011) and a decrease of proline dehydrogenase (PDH) activityZhang et al., 1995).
It is also known that excessive salinity and drought stressesffect several plant metabolic processes, such as nitrogen assimila-ion. It has been reported for instance that the effect of NaCl on plantitrogen assimilation is strictly related to stress-induced modi-cations of the enzymes involved in NO3
− assimilation pathwayDebouba et al., 2006). Namely, the activity of the key enzyme ofhe nitrate assimilation pathway in higher plants, nitrate reductaseNR; EC 1.6.6.1.), has been shown to be modified by both salinityReda et al., 2011) and drought (Fresneau et al., 2007). Generally, NRnzymatic activity in plant tissues is subjected to complex regula-ion in response to different environmental stimuli. Modulation ofR activity by a number of external factors is very rapid and usu-lly occurs due to post-translational modifications of NR proteinKaiser et al., 2002).
Overall, the present study attempts to elucidate the modusperandi of the tolerance mechanisms of A. altissima seedlingsubjected to different concentrations of NaCl as well as droughttress by estimating physiological parameters (such as chloro-
hyll fluorescence and stomatal conductance), cellular integritynd ROS/RNS interplay, as well as by examining intracellular regu-ation of osmolytes (proline content and P5CS enzymatic activity)nd nitrogen assimilation (NO content and NR activity).xperimental Botany 97 (2014) 1– 10
2. Materials and methods
2.1. Plant material and stress conditions
Forty day-old A. altissima seedlings were used in this study.Seeds (obtained from wild populations growing in the Island ofKefallonia, Greece) were sown in sterile perlite:sand (1:3) potsand placed at 4 ◦C for 4 d for stratification. Plants were grown ina growth chamber at 22/16 ◦C day/night temperatures, at 60–70%RH, with a photosynthetic photon flux density of 100 �mol m−2 s−1
and a 16/8 h photoperiod. Plants were watered twice weekly withdeionized water, while a commercial nutrient solution (Plant-Prod20-20-20 Fertilizer, Lambrou Agro, Cyprus) was applied every twoweeks. Drought stress was imposed by withholding watering for14 days, while salinity stress was imposed by watering plants withtwo different concentrations of NaCl (150 and 300 mM) for 48 h.Control samples were treated with water in both cases.
Leaf samples were harvested after 48 h (salinity) and 14 d(drought), flash-frozen in liquid nitrogen and stored at −80 ◦C untiluse. The analyses were carried out using a minimum of three inde-pendent biological replicates (consisting of pooled samples from aminimum of three independent plants per replicate) in each exper-iment.
2.2. Physiological measurements
Stomatal conductance was measured using a �T-Porometer AP4(Delta-T Devices-Cambridge, U.K.) according to the manufacturer’sinstructions. Chlorophyll fluorescence parameters of leaves repre-senting the maximum photochemical efficiency of photosystem II(PSII) (Fv/Fm) were measured with an OptiSci OS-30p ChlorophyllFluorometer (Opti-Sciences, U.S.A.). Leaves were incubated in thedark for 30 min prior to measurements.
2.3. Lipid peroxidation assay
Lipid peroxidation was determined from measurement ofmalondialdehyde (MDA) content resulting from the thiobarbituricacid (TBA) reaction (Filippou et al., 2011) using an extinction coef-ficient of 155 mM−1 cm−1.
2.4. Hydrogen peroxide and nitric oxide quantification
Hydrogen peroxide was quantified using the KI method, asdescribed by Velikova et al. (2000). Nitrite-derived NO content wasmeasured using the Griess reagent in homogenates prepared inan ice-cold Na-acetate buffer (pH 3.6) as described by Zhou et al.(2005).
2.5. Proline content
Free proline levels were determined using the ninhydrin reac-tion according to the method of Bates et al. (1973). Prolineconcentration was determined from a proline standard curve.
2.6. P5CS enzyme activity assay
Plant cell extraction and P5CS activity measurements wereprocessed according to Wang et al. (2011). Leaves were homog-enized in an extraction buffer (100 mM Tris–Cl, pH 7.5, 10 mM�-mercaptoethanol, 10 mM MgCl2, 1 mM PMSF) in pre-chilledeppendorf tubes on ice. Extracts were centrifuged at 4 ◦C for 20 min
at 10,000 × g. Supernatants were further clarified by centrifuga-tion at 10,000 × g for 20 min at 4 ◦C. p5CS enzymatic assay wascarried out in 100 mM Tris–Cl (pH 7.2), 25 mM MgCl2, 75 mM Na-glutamate, 5 mM ATP, 0.4 mM NADPH, and the appropriate crude
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P. Filippou et al. / Environmental
rotein extract. The reaction velocity was measured as the ratef consumption of NADPH, monitored as the decrease in absorp-ion at 340 nm as a function of time. Total protein content wasetermined according to Bradford method (Bradford, 1976). P5CSpecific enzyme activity was expressed as units/mg protein.
.7. Nitrate reductase (NR) assay
The assay was performed essentially as described (Liu et al.,011), with some modifications. The buffer used for preparation ofrude extracts contained 100 mM potassium phosphate (pH 7.5),
mM (CH3COO)2Mg, 10% (v/v) glycerol, 10% (w/v) polyvinylpyrol-idone, 0.1% (v/v) Triton X-100, 1 mM EDTA, 1 mM DTT, 1 mM PMSF,
mM benzamidine (prepared fresh) and 1 mM 6-aminocaproiccid. Leaf tissue was extracted in the appropriate buffer using aortar and pestle and the mixture was thoroughly homogenized.
ell extract was centrifuged at 14,000 × g for 15 min and the clearupernatant was used immediately for measurement of enzymectivity (Wray and Filner, 1970). Total protein content was deter-ined according to Bradford method (Bradford, 1976). NR activityas expressed as specific enzymatic activity (units/mg protein).
.8. Antioxidant enzyme activities
For SOD and CAT extraction, leaf samples (100 mg) were homog-nized in ice-cold extraction buffer (0.1 M phosphate buffer pH.5, 0.5 mM EDTA, 1 mM PMSF) using mortar and pestle. Eachomogenate was centrifuged at 16,000 × g at 4 ◦C for 20 min andotal supernatant was used for enzymatic activity assay. Totaluperoxide dismutase (SOD) activity was determined by measur-ng its ability to inhibit the photochemical reduction of nitro blue
etrazolium chloride (NBT) as described by Giannopolitis and Ries1977), with minor modifications. The reaction mixture contained0 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 �M NBT,.1 mM EDTA, 2 �M riboflavin and 50 �L of enzyme extract in a finalig. 1. Phenotypic response of A. altissima seedlings after 48 h application of varying NaCl2 d of withholding watering (B).
xperimental Botany 97 (2014) 1– 10 3
assay volume of 1.5 mL. The photoreduction of NBT was assayedspectrophotometrically at 560 nm and it was inversely propor-tional to SOD activity (Jiang and Zhang, 2002). The reaction mixturewith no enzyme developed maximum colour due to maximumreduction of NBT and was taken as control. The blank solutionhad the same complete reaction mixture but it was kept in thedark. One unit of SOD activity (U) was defined as the amount ofenzyme required to cause 50% inhibition of the NBT photoreduc-tion rate. The results were expressed as specific activity units/mgprotein. Catalase (CAT) activity was measured according to Aebi(1984) with minor modifications. The reaction mixture consistedof 50 mM potassium phosphate buffer (pH = 7), 10 mM H2O2 and0.15 ml enzyme extract to a final volume of 1.5 ml. The rate of H2O2disappearance was monitored at 240 nm during 3 min. Protein con-tent of samples was determined by the Bradford method (Bradford,1976).
2.9. Western blot analysis of NR protein
Leaves were homogenized and extracted with 50 mM HEPES-KOH pH 7.5, 1 mM EDTA, 1 mM DTT and a cocktail of proteaseinhibitors (1 mM PMSF, 1 mM 6-aminocaproic acid, 1 mM ben-zamidine). The extract was centrifuged at 16,000 × g for 15 minat 4 ◦C and the protein concentration in the supernatant wasdetermined according to Bradford (Bradford, 1976). Proteins wereseparated on a 7.5% SDS-PAGE in Mini PROTEAN III equipment(Bio-Rad) and blotted on to nitrocellulose membrane using awet blotter at 100 V for 1.5 h. Membranes were blocked withblocking solution (5%, w/v) non-fat milk in PBS 1× for 2 h atroom temperature and incubated overnight with the primaryanti-NR antibody dissolved in blocking solution (1/500 dilution).
Nitrate reductase was detected using a primary rabbit antibodyraised against NR (kindly provided by Prof. Steven Huber, Depart-ment of Plant Biology, University of Illinois, USA), a secondaryalkaline-phosphatase-labelled anti-rabbit IgG (1/2000 dilution)concentrations (150 and 300 mM) (A) and of drought-stressed plants after 14 d and
4 P. Filippou et al. / Environmental and Experimental Botany 97 (2014) 1– 10
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ig. 2. Physiological effects of salinity and drought stress on A. altissima seedlings.
ystem II (PSII) (Fv/Fm) and (B) leaf stomatal conductance was measured in leaves olants (B2) respectively.
nd BCIP/NBT solution for the visualization of the protein bandsRosales et al., 2011).
. Results
.1. Phenotypic characterization of salinity and drought-stressed
. altissima plants
As shown in Fig. 1, macroscopic observation 48 h after NaClpplication revealed no phenotypic differences between 150 mMaCl-treated and control plants, whereas small chlorotic lesionsere observed in plants treated with 300 mM NaCl (Fig. 1A). More-
ver, 14 d drought-stressed plants showed increased damage levelsndicated by wilted, chlorotic leaves compared with control plants.oss of turgor and leaf necrosis was observed after 22 d droughttress imposition in A. altissima seedlings (Fig. 1B).
.2. Effect of abiotic stresses on physiological parameters
Physiological processes were monitored by means of chloro-hyll fluorescence and stomatal conductance measurements in
eaves of drought and salinity-stressed A. altissima plants. Photo-
hemical efficiency of PSII (Fv/Fm) was not significantly affectedfter 48 h salt stress application (150 and 300 mM NaCl) (Fig. 2A1),hereas it was significantly lower after withholding watering for4 d compared with control samples (Fig. 2A2).
lorophyll fluorescence representing maximum photochemical efficiency of photo-ity (A1) and drought-stressed plants (A2) and in salinity (B1) and drought-stressed
In a similar fashion, no significant alterations in stomatalconductance were observed under moderate (150 mM NaCl) orsevere salt stress (300 mM NaCl) (Fig. 2B1). Contrarily, 14 ddrought-stressed plants exhibited significant reduction of stomatalconductance values compared with control plants (Fig. 2B2).
3.3. Cellular damage levels and ROS content measurements
An examination of the ROS content (H2O2 content) was per-formed, indicating significant increases under severe salinitystress (300 mM NaCl) (Fig. 3A1) and 14 d drought-stressed plants(Fig. 3A2).
Similarly, significant membrane damage (shown as higher MDAcontent) was observed upon severe salinity stress (Fig. 3B1) anddrought stress conditions (Fig. 3B2) in comparison with con-trol plants. Notably, lower salt concentration (150 mM NaCl)also resulted in significant (albeit smaller) membrane damage(Fig. 3B1).
3.4. RNS content and NR enzymatic activity
Nitric oxide content was measured in leaves of drought and
salinity-stressed A. altissima seedlings. Similar trends to those forH2O2 content were observed in stressed A. altissima seedlings com-pared with control plants (Fig. 4). More specifically, salt stressconditions at the higher NaCl concentration (300 mM) resulted in
P. Filippou et al. / Environmental and Experimental Botany 97 (2014) 1– 10 5
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ig. 3. Effect of salinity and drought stress on hydrogen peroxide content (A) and celtissima plants (n = 3).
ignificantly increased NO content compared with control (Fig. 4A),s did drought stress conditions (Fig. 4B).
In order to provide biochemical support on the observed NOontent increase in stressed plants, we examined the regulationf the NO-generating enzyme nitrate reductase (NR). Therefore,R enzymatic activity and protein content was estimated inrought and salinity-stressed plants. NR activity demonstratedtress factor-dependent differential regulation (Fig. 5A and B).evere salt stress (300 mM NaCl) resulted in significant inductionf NR activity compared with control plants (Fig. 5A). By contrast,rought-stressed plants demonstrated a significant suppression ofR activity compared with control plants (Fig. 5B). This suppres-
ion of NR activity was related to a notable decrease in NR proteinontent (Fig. 5D), as indicated by Western blot analysis of equal pro-ein amounts of control and drought-stressed A. altissima extracts.
oreover, NR protein content slightly increased in low (150 mM)nd high (300 mM) salt-stressed plants compared with controlFig. 5C).
.5. Response of antioxidant enzymes to salinity and droughttress
To better understand how A. altissima seedlings deal with
xidative stress under salinity and drought stress conditions, thenzymatic activity of catalase (CAT) and superoxide dismutaseSOD) was measured. Antioxidant enzyme activity profiles wereimilar under both stress conditions. More specifically, withholdingdamage indicated by MDA content (B) in leaves of salinity and drought-stressed A.
watering for 14 d resulted in significant induction in SOD andCAT activity in comparison with control plants (Fig. 6A2 and B2).Seedlings subjected to moderate and severe salt stress (150 and300 mM NaCl respectively) also demonstrated higher enzymaticactivity for SOD and CAT compared with control samples, reachingsignificantly maximal levels at 150 mM NaCl (Fig. 6A1 and B1).
3.6. Effect of drought and salinity in proline content and P5CSenzymatic activity
Free proline content was analyzed in leaves of control andstressed A. altissima seedlings. Under salinity stress conditions,proline content increased in a concentration-dependent manner,reaching maximal (significantly higher) levels at 300 mM NaCl(Fig. 7A1). In regard with drought-stressed A. altissima plants, pro-line content increased significantly in comparison with controlplants (Fig. 7A2), reaching higher values compared with salt-stressed plants (Fig. 7A1).
In addition to proline content, enzymatic activity of P5CS, thekey regulatory and rate-limiting enzyme in proline biosyntheticpathway, was further investigated. Under both stress conditions,P5CS activity increased in parallel with the observed increasedproline levels (Fig. 7), although significant induction was observed
only in 14 d drought-stressed plants (Fig. 7B2). Moreover, maximalproline levels in drought-stressed plants correlated with high-est P5CS activity levels recorded between all examined samples(Fig. 7).
6 P. Filippou et al. / Environmental and E
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4
twoceche(mtaaifsis
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. altissima seedlings. NO content was measured using the Griess reagent in salinity-tressed plants after lower (150 mM) and higher (300 mM) NaCl application (A) andn 14 d drought-stressed plants (B) (n = 3).
. Discussion
Drought and salinity stresses are two of the most impor-ant environmental factors limiting plant growth and productivityorldwide (Krasensky and Jonak, 2012). The tolerance to salinity
r water stress could be related to different genetically determinedapacity of plants to cope with oxidative stress events (Golldackt al., 2011). Therefore, the identification of physiological and bio-hemical components of the antioxidative defense system, whichave a potential to confer drought or salinity tolerance, could bessential for the characterization of stress-tolerant plant speciesVaseva et al., 2012) like A. altissima (Trifilo et al., 2004). Although
any studies have been carried out characterizing plant responseso drought/salinity stresses in plants, limited information is avail-ble on the effect of water deficiency in invasive plants such as A.ltissima (Trifilo et al., 2004) at the level of nitro-oxidative stressmposed. Results presented in this study clearly indicate the dif-erential response of the tolerance mechanism of 40 d A. altissimaeedlings after imposition of salinity and drought, whilst compar-ng the observed effect between A. altissima and other, model plantpecies.
Initially, phenotypic plant observations revealed that droughtas a significant damaging stress factor, since 14 d drought-
tressed A. altissima seedlings showed increased damage levels
ltimately resulting in complete plant necrosis after 22 d droughttress imposition. Drought stress imposition and subsequentacroscopic observation revealed retarded damage levels in A.ltissima compared with similar age Medicago truncatula and
xperimental Botany 97 (2014) 1– 10
Arabidopsis thaliana plants, grown under similar stress conditions.More specifically, macroscopic tissue damage was observed after14 d in A. altissima, compared with 7–9 d depending on the modelplant species (see Kalamaki et al., 2009; Filippou et al., 2011).
This result was further supported by the subsequent decrease inchlorophyll fluorescence (Fv/Fm) and stomatal conductance valuesin drought-stressed compared with control A. altissima seedlings,suggesting that water deficit condition can disrupt photosynthe-sis and photorespiration, altering the normal homeostasis of cells(Faize et al., 2011). Interestingly, the level of decrease in bothphysiological parameters was notably lower compared with thatobserved in other similar age plants (i.e. M. truncatula plantsdemonstrated a more dramatic decrease in both physiologicalparameters after 9 and 11 d drought imposition) (Filippou et al.,2011).
Salinity stress in A. altissima seedlings did not cause signifi-cant tissue damage even at the higher NaCl application (300 mM),in agreement with the non-significant change in physiologicalparameter values examined under salt stress conditions (150 and300 mM). Previous studies demonstrated that maintenance of pho-tosynthetic capacity at high salt concentrations appeared to beassociated with salt tolerance (Zhao et al., 2007). Further reports(e.g. Mehta et al., 2010) demonstrated that high salinity decreasedthe Fv/Fm ratio, mainly due to inhibition of electron transport at theacceptor side of the PSII reaction centre. Our findings therefore sup-port the notion of increased salt tolerance capacity in A. altissimaseedlings. Moreover, results by El-Hendawy et al. (2005) showedgreater reduction in stomatal conductance of salt sensitive ratherthan salt tolerant wheat cultivars caused by saline conditions, fur-ther strengthening this notion.
In order to further evaluate the defence mechanism of A.altissima seedlings under these two major stress conditions, weexamined components of the nitro-oxidative response. Waterstress and salinity are inevitably associated with increased oxida-tive stress due to enhanced accumulation of ROS, particularly O2
•−
and H2O2 (Bian and Jiang, 2009). In the present study, increasedH2O2 and MDA content in drought and salinity-stressed plantsreflected the degree of oxidative stress. More specifically, a clearlink is provided between the observed increases in ROS content indrought-stressed A. altissima plants compared with controls andthe analogous increased cellular damage levels, similar to severesalinity-stressed plants (300 mM NaCl). When comparing MDAcontent with that of other stressed, similar age plant species (i.e.M. truncatula MDA content after drought stress imposition was14-fold higher compared with A. altissima drought-stressed plantsthat showed 4-fold MDA increase; see Filippou et al., 2011), anotable amelioration of cellular damage was observed, further sup-ported by a similar drop in ROS content. This result is in accordancewith previous studies indicating that drought acclimation reduceslipid peroxidation in wheat seedlings (Selote and Khanna-Chopra,2010). Furthermore, present findings indicate a general trend of aconcentration-dependent increase in the levels of H2O2 and MDAcontent following moderate and severe salt stress application, sug-gesting that the accumulation of H2O2 is mainly due to the osmoticstress induced by the external NaCl (Hernandez et al., 2010).
Recent findings highlight the increased sensitivity of M. trun-catula, strawberry and citrus among other plants to oxidative andnitrosative damage, mainly manifested through major inductionof ROS (H2O2) and RNS (NO) production, under stress conditionssuch as drought and salinity (Filippou et al., 2011; Tanou et al.,2012; Christou et al., 2013). For this reason, NO content was alsomeasured as a biomarker of nitrosative stress in A. altissima plants.
Drought-stressed plants showed a similar, significant increase inNO content as that of severe salinity-stressed plants compared withcontrol samples, indicative of nitrosative damage. In a similar fash-ion to the smaller increases in MDA and ROS content in stressed
P. Filippou et al. / Environmental and Experimental Botany 97 (2014) 1– 10 7
Fig. 5. NR activity in A. altissima leaf segments treated with increasing concentrations of NaCl (A) and under drought stress (B) after 48 h and 14 d stress exposure respectively.W -stressh contrS
AleRsp
geiinsraafiitdldc
dbiThl
estern blot analysis was performed indicating NR protein expression in salinityomogenates of control (C1, C2) and drought-stressed plants (D1, D2), as well asDS-PAGE and NR content evaluated using a polyclonal anti-NR antibody.
. altissima plants compared with other stressed plants of simi-ar age (i.e. M. truncatula 9 d drought-stressed plants; see Filippout al., 2011), smaller comparative induction was also observed inNS content (i.e. NO content increase was 4-fold higher in drought-tressed M. truncatula, whereas it was 2-fold higher in A. altissimalants under drought stress).
Interestingly NR, which is the key biosynthetic enzyme for NOeneration (Yamasaki and Sakihama, 2000), appears to be differ-ntially regulated under the two distinct abiotic stress factors. Thencrease in NR activity is analogous to the increase in NO contentn salt-stressed plants in a NaCl concentration-dependent man-er. Moreover, results by Reda et al. (2011), suggested that thealt-induced increase in NR activity resulted from the osmoticather than the ionic component of salt stress. Contrarily, A.ltissima drought-stressed plants demonstrated suppressed NRctivity compared with control plants, in accordance with previousndings (Fresneau et al., 2007; Rosales et al., 2011). The reduction
n NR activity was further verified by a decrease in protein con-ent, thus indicating that the regulatory mechanism of NR underrought conditions might be at the transcriptional or translational
evel. Interestingly, the reduction in NR activity under drought con-itions correlates with low photosynthetic activity due to stomatallosure, similar to findings by Fresneau et al. (2007).
Previous findings suggested that the degree of toleranceepends on the balance between formation of ROS and its removaly the antioxidative scavenging systems, thus representing an
mportant stress-tolerance trait (Selote and Khanna-Chopra, 2010).he tolerance strategies of A. altissima seedlings are likely to includeighly inducible antioxidative defence composed of low molecu-
ar weight antioxidants (e.g., proline), as well as ROS-scavenging
ed (C) and drought-stressed (D) A. altissima leaf seedlings. Equal amounts of leafol (CS1, CS2) and salt-stressed plants (150 and 300 mM) were subjected to 7.5%
enzymes (Radic et al., 2013). However, little is known about theantioxidant capacity of A. altissima crude extracts and the poten-tial correlation with plant-derived phenylpropanoids mainly usedfor protection against biotic or abiotic stresses (Hwang et al., 2012;Luis et al., 2012). A main aspect in the present study was thereforeto investigate the A. altissima antioxidative system in order to ame-liorate damage by regulating antioxidant enzymes such as SOD andCAT (Woo et al., 2007). Activities of both CAT and SOD showed dra-matic increases in plants under moderate salt stress (150 mM NaCl)compared with control, similar to findings by Barakat (2011). Highsalinity (300 mM NaCl) and drought-stressed plants also showedsignificant (albeit lower) increase in both anti-oxidative enzymeactivities.
In A. altissima plants under moderate salt stress conditions(150 mM NaCl), the efficient up-regulation of both SOD and CATresulted in limited ROS accumulation, thus providing a higher pro-tection during stress. Our results suggest that SOD activity canbe induced by ROS under moderate salinity stress and the salt-tolerant A. altissima has an increased O2
•− radical scavenging ability(Hu et al., 2012). Contrarily, the direct exposure to severe salinity(300 mM NaCl) and drought stress caused excessive accumulationof H2O2, followed by elevated lipid peroxidation due to the poorenzymatic antioxidant response. It is possible that the excess lev-els of H2O2 during water stress or severe salt stress conditionsmight have inhibited or down-regulated the A. altissima antioxi-dant enzymes (Selote and Khanna-Chopra, 2010). Similar patterns
to SOD activity regulation were observed for CAT, one of the mostimportant H2O2 scavenging enzymes the regulation of which maydepend on the plant species, the development and metabolic stateof the plant, as well as on the duration and intensity of the stress
8 P. Filippou et al. / Environmental and Experimental Botany 97 (2014) 1– 10
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F activia ere u
(Ab1tsaT
ramnadsdpftsCmaa
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ig. 6. Superoxide dismutase (SOD) (A) and catalase (CAT) (B) specific enzymatic
pplication) (A1, B1) and drought stress treatments (A2, B2). Water treated plants w
Chaparzadeh et al., 2004). Therefore, the high CAT activation in. altissima stressed seedlings (150 mM NaCl) is further supportedy similar results in salt-stressed tomato (Rodriguez-Rosales et al.,999) and salt-tolerant barley cultivars (Seckin et al., 2010). Onhe other hand, lower CAT activity in severe salinity and drought-tressed A. altissima plants could be related to a higher H2O2ccumulation and MDA content as shown in previous reports (e.g.anou et al., 2009).
Plants accumulate compatible solutes, such as proline, inesponse to stresses to facilitate water uptake and protect cellsgainst ROS accumulation (Ashraf and Foolad, 2007). Proline accu-ulation was correlated to a variety of stress conditions and is
ow regarded as a major non-enzymatic antioxidant (Szabadosnd Savouré, 2010). Proline content increased significantly underrought and severe salt stress conditions in A. altissima seedlings,upporting its role as a protective agent under oxidative stress con-itions (De Carvalho et al., 2013). In drought-stressed A. altissimalants, proline content after 14 d drought stress imposition was 2.6-old higher compared with control plants, whereas similar age M.runcatula plants showed a more dramatic effect after 9 d droughttress (∼3.5-fold increase in proline content) (Filippou et al., 2011).onsequently, proline in drought-stressed A. altissima seedlingsay function to indirectly protect PSII (Molinari et al., 2007) as well
s to directly scavenge ROS and prevent further lipid peroxidation,iming to achieve a drought stress tolerance mechanism.
In this mechanism, the induction of antioxidant enzyme activ-
ty does not appear to be the major player under drought stressonditions, as indicated by the less pronounced SOD and CAT induc-ion compared with moderate salt stress-treated plants. Instead,roline accumulation seems to play a key role as an antioxidantties in leaves of A. altissima seedlings subjected to salinity (150 and 300 mM NaClsed as a control (n = 3).
regulatory molecule in drought-stressed plants. Moreover, enzy-matic activity of P5CS, which is the key regulatory and rate limitingstress-inducible enzyme in proline biosynthetic pathway (Vasevaet al., 2012), increased in parallel to the increased proline levelssimilar to previous reports (Chen et al., 2009). Activation of P5CSresulted in free proline accumulation in drought-stressed plants,in accordance with previous reports (e.g. Yamada et al., 2005),thus conferring the osmoprotective role of proline in A. altissimastress tolerance. Conversely, the observed significant increase inproline content under severe salt stress conditions (300 mM NaCl)was not accompanied by significant induction in P5CS activity,although increased levels were recorded. This could be poten-tially explained by the possible involvement and activation of otherenzymes involved in proline biosynthesis, such as P5CR (Szabadosand Savouré, 2010), or the possible down-regulation of enzymesinvolved in proline catabolism.
To our knowledge, this is the first comprehensive approachevaluating the joint effects of key environmental stress factors onthe invasive species A. altissima, ultimately aiming to identify howthey interact with the emergence and early establishment of thisinvasive tree under Mediterranean conditions. Interestingly, theresponse of A. altissima seedlings to drought and salinity was com-parative or even more efficient in comparison with model plantspecies of the same age (but different developmental stage), indi-cating a stronger tolerance mechanism for A. altissima plants, partlydue to the induction of osmoregulatory molecules and antioxidant
apparatus. It was shown that defence against nitro-oxidative stressis organized differentially in A. altissima seedlings according to thedistinct abiotic stress factor and the degree of stress imposition(i.e. salt concentration). The combined effects on the early stages of
P. Filippou et al. / Environmental and Experimental Botany 97 (2014) 1– 10 9
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Fig. 7. Proline content (A) and P5CS (�1-pyrroline-5-carboxylate synthetase) enzymatic activity (B) measurements in leaves after treatment with H O (control), 150 and3 ltissim
tras
A
eoOcn
R
AA
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00 mM NaCl application during 48 h (A1, B1), or 14 d drought stress (A2, B2) in A. a
his invasive species and the interaction between genetic and envi-onmental factors, whether or not they are related to habitat type,re still poorly understood and studies involving a more elaboratecreening of antioxidant responses needs to be further elucidated.
cknowledgements
This study was carried out under the Act “The Technologicalducational Institution of Ionian Islands as an International Polef Education and Innovation” and within the framework of theperational Programme “Education and Lifelong Learning” that iso-funded by the European Union (European Social Fund ESF) andatural resources.
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