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Plant Physiology and Biochemistry 56 (2012) 79e96

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Biochemical dissection of diageotropica and Never ripe tomato mutantsto Cd-stressful conditions

Priscila L. Gratão a, Carolina C. Monteiro b, Rogério F. Carvalho a, Tiago Tezotto c, Fernando A. Piotto b,Lázaro E.P. Peres d, Ricardo A. Azevedo b,*

aDepartamento de Biologia Aplicada à Agropecuária, Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), 14884-900 Jaboticabal, SP, BrazilbDepartamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo (USP), Av. Pádua Dias, n. 11, 13418-900 Piracicaba, SP, BrazilcDepartamento de Produção Vegetal, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo (USP), 13418 900 Piracicaba, SP, BrazildDepartamento de Ciências Biológicas, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo (USP), 13418-900 Piracicaba, SP, Brazil

a r t i c l e i n f o

Article history:Received 10 February 2012Accepted 13 April 2012Available online 25 April 2012

Keywords:Hormonal mutantsAntioxidant enzymesOxidative stressCadmiumHeavy metalsEthyleneAuxin

Abbreviations: Nr, Never ripe; dgt, diageotropica; MROS, reactive oxygen species; O2�, superoxide radicahydrogen peroxide; OH�, hydroxyl radical; SOD, sascorbate peroxidase; CAT, catalase; GPX, glutathionetype peroxidase; GSH, glutathione; AsA, ascorbic acidGR, glutathione reductase; NADPH, reduced nicotinPCD, programmed cell death (PCD); TBARS, thiobarbitMDA, malondialdehyde; PAGE, polyacrylamide gel ele* Corresponding author. Tel.: þ55 19 3429 4475; fa

E-mail address: [email protected] (R.A. Azevedo).

0981-9428/$ e see front matter � 2012 Elsevier Masdoi:10.1016/j.plaphy.2012.04.009

a b s t r a c t

In order to further address the modulation of signaling pathways of stress responses and their relation tohormones, we used the ethylene-insensitive Never ripe (Nr) and the auxin-insensitive diageotropica (dgt)tomato mutants. The two mutants and the control Micro-Tom (MT) cultivar were grown over a 40-dayperiod in the presence of Cd (0.2 mM CdCl2 and 1 mM CdCl2). Lipid peroxidation, leaf chlorophyll,proline content, Cd content and antioxidant enzyme activities in roots, leaves and fruits were deter-mined. The overall results indicated that the MT genotype had the most pronounced Cd damage effectswhile Nr and dgt genotypes might withstand or avoid stress imposed by Cd. This fact may be attributed,at least in part, to the fact that the known auxin-stimulated ethylene production is comprised in dgtplants. Conversely, the Nr genotype was more affected by the Cd imposed stress than dgt, which may beexplained by the fact that Nr retains a partial sensitivity to ethylene. These results add further infor-mation that should help unraveling the relative importance of ethylene in regulating the cell responses tostressful conditions.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Heavy metals have become one of the main classes of abioticstress agents for living organisms because of their increasing use inthe developing fields of industry and high bioaccumulation andtoxicity [1,2]. High heavy metals concentrations may disturbcellular signaling and cause irreversible damage to biologicalsystems [1,3]. Cadmium (Cd) is probably the most damaging heavymetal to plant species and in high concentrations can causeoxidative stress by favoring the production or enhancing avail-ability of reactive oxygen species (ROS), disrupting the plant

T, Micro-Tom; Cd, cadmium;l; 1O2, singlet oxygen; H2O2,uperoxide dismutase; APX,peroxidase; GPOX, guaiacol-; GSSG, oxidized glutathione;amide adenine dinucleotide;uric acid reactive substances;ctrophoresis.x: þ55 19 3447 8620.

son SAS. All rights reserved.

defense system [4,5]. The oxidative stress induced by heavy metalsin plants can generate ROS such as superoxide radicals (O2

��),singlet oxygen (1O2), hydrogen peroxide (H2O2) and hydroxylradicals (OH�) [6,7], which are continuously produced in bothunstressed and stressed plants cells [8,9]. As a consequence, theregulation of plant development in response to the stress requiresadjustments in redox state [10,11] through the evolution ofa complex pathway of cellular responses, such as the production ofstress proteins, non-enzymatic and enzymatic detoxificationmechanisms and accumulation of metabolites [12e14].

The antioxidant defense system comprises antioxidant enzymessuch as superoxide dismutase (SOD, EC 1.15.1.1), which dismutatesO2

�� to H2O2 which may be detoxified to H2O by ascorbateperoxidase (APX, EC 1.11.1.11), catalase (CAT, 1.11.1.6), glutathioneperoxidase (GPX, EC 1.11.1.9), guaiacol-type peroxidases (GPOX, EC1.11.1.7), as well as non-enzymatic antioxidant such as metabolismsof glutathione (GSH), ascorbic acid (AsA), carotenoids, organicacids, stress proteins and proline [15e17]. The regeneration ofglutathione (GSH) from oxidized glutathione (GSSG) is catalyzed byglutathione reductase (GR, EC 1.6.4.2) using NAD(P)H as a reducingagent [18]. The metabolite proline is probably the most widespread

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P.L. Gratão et al. / Plant Physiology and Biochemistry 56 (2012) 79e9680

in plants, having an important protective role against heavy metals[19,20]; for instance, free proline has been found to chelate Cd ionin plants and form nontoxic Cd-proline complex [21,22].

Since the coordinated regulation of plant development inresponse to the stress requires a cross-talk between the networkthat integrates cellular responses and signaling pathways [23,24], itis not surprising that the phytohormones are components exten-sively exploited during stress.

Although the involvement of phytohormones in the response ofplants to heavy metals stress have already been studied in differentplant species [25e27], the mechanisms involved in interaction ofphytohormones with heavy metal-stress responses have not beenwell established. For instance, the ethylene phytohormone canregulate multiple stress responses [28] such as the signal trans-duction events during Cd-induced programmed cell death (PCD)[29], induction of plant-specific transcription factors which havebeen linked to Cd-tolerance [30] and regulation of chitinase andheat shock protein 71.2 to protect cells against Cd damage [31]. Onthe other hand, the auxin phytohormone can alleviate toxic effectsof heavy metals on plant and shoot growth [32], involving heavy-metal resistance and accumulation in plants [33].

Although these multiple stress responses are essential for plantsurvival to heavy metal-stress conditions, the exact role of phyto-hormones in these responses is currently not known, mainly thecross-talk among ROS, phytohormones and antioxidant systems.

In order to further address the known interaction betweenphytohormones and stress responses, one can take advantage ofthe plethora of mutants that exist in models such as Arabidopsis[34] and tomato [35]. Through the use of these mutants, an over-view of the modulation of ROS signaling, as well as the cellularcoordination of components can be discussed, providing insightsabout the cross-talk between these pathways and phytohormones[34,36].

In tomato (Solanum lycopersicum L., formely known as Lyco-persicon esculentum Mill.) there are six ethylene receptors (LeETR1e LeETR6), being the LeETR3 receptor correspondent to the muta-tion Never ripe (Nr) [37], whose expression increases duringsenescence and abscission [38]. The Nr mutant lost the capacity torespond to either endogenously or exogenously generated ethylene[39]. The use of Nr mutant has suggested that the ethyleneperception - not production e can regulate developmentalresponses under different stresses [36,40,41]. On the other hand,the DIAGEOTROPICA (DGT) gene encodes a component of a specificauxin signaling pathway [42] and the dgt mutant is relativelyinsensitive to auxin [43]. It is interesting to note that althoughethylene production is not stimulated by auxin in dgt, it can still bedirectly stimulated by flooding stress [44]. Whether or not suchkind of auxin-ethylene cross-talk is relevant for other kinds ofstresses, e.g. Cd, remains to be determined.

For this purpose, throughout the use of the Nr and dgt tomatomutants, the aim of this work was to understand or provideevidence about the modulation of signaling pathways of oxidativestress and their relation with ethylene and auxin. Thus, under-standing the biochemical processes involved with this cross-talkbetween antioxidant responses and both hormones againstoxidative stress induced by Cd, new possibilities about detoxifica-tion strategies can be used to manipulate heavy metal tolerance.

2. Results

2.1. Plant growth and Cd content

Over the 40 days of treatment, MT, Nr and dgt plants cultivatedin CdCl2 exhibited growth reduction of all tissues analyzed (Fig. 1)when compared to control plants (0 CdCl2). This reduction was

more pronounced in 1 mM CdCl2, particularly for the MT genotype(Fig. 1). Although we did not determine the metal accumulation inplants at day zero, when the Cd treatments were initiated, after 1,12 and 40 days, a gradual increase in Cd accumulation occurred forall the genotypes, being more pronounced in roots (Fig. 2A) than inleaves (Fig. 2B). The fruit tissue also exhibited accumulation of Cdfor MT, Nr and dgt at 40 d of treatment (Fig. 2C), but considerablyless when compared to roots (Fig. 2A) or even leaves (Fig. 2B). Aclear pattern was observed among the genotypes for Cd accumu-lation independent of the tissue, Cd concentration used and timelength of exposure to Cd, so that dgt exhibited the highest Cdaccumulation followed by Nr and then MT (Fig. 2).

2.2. Lipid peroxidation

Changes in lipid peroxidation rates (expressed as MDA content)induced by CdCl2 were observed over the three time-periodssampled (1, 12 and 40 days) for MT, Nr and dgt genotypes (Fig. 3).In all tissues, themost pronounced lipid peroxidation changes wereobserved for the MT genotype after 40 d treatment for both Cdconcentrations, whereas, Nr and particularly dgt exhibited lowerperoxidation rates when compared to MT (Fig. 3). As a matter offact, at 40 d the lipid peroxidation trends for all genotypes (Fig. 3)were exactly the opposite encountered for Cd accumulation trends(Fig. 2).

2.3. Chlorophyll content

Although from 1 to 40 d dgt mutant appears to retain morechlorophyll, this pigment did not vary much between Cd concen-trations (Fig. 4). Perhaps the more noticeable changes in chloro-phyll levels were those observed for all three genotypes after 40 dof treatment, particularly at the highest CdCl2 concentration.However, after 50 days in 1 mM CdCl2 the reduction in chlorophyllcontent was quite dramatic for MT, whilst the two mutantsalthough exhibiting reduced chlorophyll contents, they wereclearly less affected by Cd than MT (Fig. 4).

2.4. Proline content

Following treatment with CdCl2, MT, Nr and dgt leaves (Fig. 5A)and fruits (Fig. 5B) exhibited increases in proline content whencompared to the 0 mM treatment at the time points. Cd-inducedproline content increase in both organs, but was morepronounced in the dgt leaves (Fig. 5A) after 12 d treatment with1 mM CdCl2.

2.5. Antioxidant enzymes activities

The activity of SOD in extracts of plants grown over a 40-dayperiod in the presence of CdCl2 was determined based on theseparation of isoenzymes by non-denaturing PAGE (Figs. 6e8, forroots, leaves and fruits, respectively). Three distinct SOD isoen-zymes were detected and characterized as Mn/SOD (SOD I) andCueZn/SOD (SOD II and III) (isoenzyme classification is not shown)independent of organ, stage of development, CdCl2 treatment andgenotype. Althoughwe did not carry out a densitometric analysis, itis possible to observe very consistent results. The only significantchanges observed were dependent upon organ; for instance, SOD IIis predominant in roots (Fig. 6) and fruits (Fig. 8), but in leaves SODII and III exhibit very similar levels of activity (Fig. 7), whereas SOD Iexhibited always lower levels of activity. Apart from the fact that inNr and dgt roots at 40 d of CdCl2 treatment (Fig. 6C) inwhich SOD IIIwas present in 0 mM CdCl2 (Fig. 6C, lanes 5 and 8, Nr and dgt,respectively) and almost disappeared when CdCl2 was added at 0.2

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Fig. 1. Roots (A), leaves (B) and fruits (C) dry mass (g dry wt) of MT, Nr and dgt plants grown over a 40-day period in the presence of CdCl2. Values are the means of three replicates�SEM. Different uppercase letters on top of the columns indicate that means of a same time of Cd exposure are different at p < 0.05, by Tukey test. Different lowercase letters on topof the columns indicate that means of different times and same treatment are different at p < 0.05, by Tukey test. *Statistical analyzes not shown.

P.L. Gratão et al. / Plant Physiology and Biochemistry 56 (2012) 79e96 81

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Fig. 2. Roots (A), leaves (B) and fruits (C) Cd accumulation (mg g�1 dry weight) during MT, Nr and dgt plants grown over a 40-day period in the presence of CdCl2. The plants weresubjected to CdCl2 treatment and samples analyzed after 1, 12 and 40 days. The 0 mM CdCl2 values were all below 0.4 mg g�1 dry weight. Different uppercase letters on top of thecolumns indicate that means of a same time of Cd exposure are different at p < 0.05, by Tukey test. Different lowercase letters on top of the columns indicate that means of differenttimes and same treatment are different at p < 0.05, by Tukey test. *Statistical analyzes not shown.

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Fig. 3. Lipid peroxidation measured as malondialdehyde (MDA) content (nmol g�1 fresh weight) in roots (A), leaves (B) and fruits (C) of MT, Nr and dgt plants grown over a 40-dayperiod in the presence of CdCl2. Values are the means of three replicates �SEM. Different uppercase letters on top of the columns indicate that means of a same time of Cd exposureare different at p < 0.05, by Tukey test. Different lowercase letters on top of the columns indicate that means of different times and same treatment are different at p < 0.05, byTukey test. *Statistical analyzes not shown.



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Fig. 4. Chlorophyll content measured in leaves during MT, Nr and dgt leaves after a period of 47, 58, 85 and 95 d post germination, corresponding to 1, 12, 40 and 50 d of exposure toCdCl2, respectively. A Minolta SPAD-502 chlorophyll meter was used to take readings on forth leaves. Values are the means of four replicates �SEM. Different uppercase letters ontop of the columns indicate that means of a same time of Cd exposure are different at p < 0.05, by Tukey test. Different lowercase letters on top of the columns indicate that meansof different times and same treatment are different at p < 0.05, by Tukey test. *Statistical analyzes not shown.


(Fig. 6C, lanes 6 and 9, Nr and dgt, respectively) and 1 mM (Fig. 6C,lanes 7 and 10, Nr and dgt, respectively) concomitant with a lightdecrease in SOD II activity (as described for SOD II), and a lightincrease in SOD I activity with the increase in CdCl2 for bothmutants, no other changes could be observe that could be attrib-uted to the CdCl2-induced stress or genotype under the conditionstested in this research.

Analysis of CATactivity revealed a complex response (Fig. 9). Yet,some results are clear. For instance, in roots, the lowest CdCl2concentration used induced high levels of CAT activity in both, Nrand dgt roots, beingmore pronounced in Nr roots (Fig. 9A), whereasthe highest CdCl2 treatments caused an increase in CAT activity inMT roots (Fig. 9A). In leaves, the only clear result is that CAT activitywas higher in the Nr mutant when compared to MT and dgt(Fig. 9B), whereas in fruits some increase in CAT activity wasobserved for all three genotypes at 1 mM CdCl2 (Fig. 9C).

Analysis of total GR activity revealed distinct trends that do notclearly indicate specific effect on GR activity for the genotypestested (Fig. 10). Perhaps the most interesting results were thoseobserved for leaves (Fig. 10B) and fruits (Fig. 10C). In leaves, theactivity of GR was lower in dgt when compared to MT and Nr witha tendency to a reduction in activity at 1 mM CdCl2 up to 12 d oftreatment, whilst at 40 d all genotypes exhibited increased GRactivity at 1 mM CdCl2 (Fig. 10B). GR activity in fruits was increasedin both CdCl2 concentrations used and all three genotypes(Fig. 10C).

The analysis of GPOX activity was also determined (Fig. 11) andthe results revealed a very variable response among Cd concen-trations used, genotypes, tissues and time length of exposure, beingdifficult to establish or suggest any clear trends.

Contrary to what was observed for GPOX, APX activity trendswere clear (Fig. 12). For instance, in roots both CdCl2 concentrationsused induced increases in APX activity at 1, 12 and 40d of treatmentand for MT, Nr and dgt (Fig. 12A). On the other hand, in the leaf(Fig. 12B) and fruit (Fig. 12C) tissues the results were not clear andtrends could not be detected among the Cd concentrations used,genotypes, tissues and time length of exposure.

3. Discussion

Plant development is subject to hormonal control and adapta-tion to environmental stresses [45]. So, the use of hormonalmutants has become a promising way to investigate potential plantresponses to environmental stress, with regard to the modulationof signaling pathways affected by the mutations [34,46,47].

We used the Nr and dgt tomato mutants, which show lowethylene and auxin sensitivity, respectively, in order to obtainfurther information about modulation of Cd-induced oxidativestress by ethylene and auxin. So, our experimental design was setup by growing the plants for a 40-day period in the presence ofCdCl2 (0.2 mM or 1 mM CdCl2). The experiment conditions; timeand concentrations; were based on our previous report in whichMT plants acquired tolerance during a chronic treatment byexposing them to a range of CdCl2 concentrations varying from 0.05to 1 mM [48]. Moreover, in another recent work with Nr and dgtmutants published by our group [49] we were able to integrate thestudy of hormonalmutants with cadmium-induced oxidative stressand ultrastructural changes induced by Cd.

According to our results, at 40 d for all genotypes, Cd-inducedoxidative stress detected as an increase in MDA content (Fig. 3) anddecrease in the chlorophyll content (Fig. 4), leading to growthreduction (Fig. 1). These responses seem to be controlled byethylene and auxin since the Nr and dgtmutants exhibited a higherdry weight and smaller induction of the MDA content of all tissuesas well as more retention of leaf chlorophyll compared to MT. Inaccordance with this [36], verified that Nr displays more toleranceto Cd stress, indicating a fundamental involvement of ethylene inthe stress modulation and confirming previous reports [50e52]. Onthe other hand, although it is now becoming clear that auxin canmodulate stress [53], it is still difficult to draw any specific role forthis hormone.

It is known that the low sensitivity to auxin in dgt results in rootspoorly developed and lack lateral root initiation [42,54], whereasCd stress can inhibit root dry weight, root diameter and number oflateral roots [55], although no ultrastructural alterations were

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Fig. 5. Proline content (mmol g�1 fresh weight) in roots (A), leaves (B) and fruits (C) of MT, Nr and dgt plants grown over a 40-day period in the presence of CdCl2. Values are themeans of three replicates �SEM. Different uppercase letters on top of the columns indicate that means of a same time of Cd exposure are different at p < 0.05, by Tukey test.Different lowercase letters on top of the columns indicate that means of different times and same treatment are different at p < 0.05, by Tukey test. *Statistical analyzes not shown.


observed in dgt roots submitted to Cd in our prior study [49]. On theother hand, the inhibition of root growth in Nr can be a result ofreduction in diameter [49], suggesting that the sensitivity of the Nrmutant to severe stress can be modulated by the Nr locus gene [56].

The pronounced reduction of growth in MT plants to Cd showedby all tissues analyzed when compared to Nr and dgt genotypes(Fig. 1), can be related to changes in ROS metabolism integratedwith the ethylene effect on increasing membrane leakage in plants[57] and changes in auxin distribution [58]. As a result of themembrane leakage, phospholipid signaling may lead to increasedproduction of ROS, accumulation of H2O2 and lipid peroxidationproducts (Fig. 3), followed by reduction of roots and leaf growth[59].

As already stated in the introduction, the dominant Never ripe(Nr) tomato mutant is blocked in ethylene perception [37], whilstthe dgt mutant is relatively insensitive to auxin with respect toethylene production [44]. Thus, the pronounced growth reduction

in MT plants (Fig. 1) and MDA amounts (Fig. 3) suggest that theethylene perception - not productione can regulate developmentalresponses under stress condition. It has been showed that theinhibition of ethylene perception can minimize the effect of Cdtoxicity through the increased membrane stability and delay leafsenescence [29]. Moreover, ethylene has been established asa stress hormone, playing an important role as an amplifier for ROSaccumulation, implying a synergistic effect between biosynthesis ofROS and ethylene [60]. The oxidative damage caused by ethylenecan oxidize membrane lipids and protein, making the membranemore permeable [60]. Furthermore, the MDA reduction observedfor dgt under Cd treatment (Fig. 4) indicates a fundamental role ofthe auxin during membrane leakage. However, relatively little isknown about how, for instance, ROS interacts with auxin duringstress. For example, it has been shown that auxin-induced reactionscan be enhanced by oxidative stress [61,62], yet, it was found thatauxin application results in a reduction in the H2O2 level [53].

Fig. 6. Activity staining for superoxide dismutase (SOD) following non-denaturing polyacrylamide gel electrophoresis of extracts of roots isolated from MT, Nr and dgt plants grownover a 1 (A), 12 (B) and 40 (C)-day period in the presence of CdCl2. The lanes listed are: (1) bovine SOD standard; (2) MT, zero; (3) MT, 0.2 mM CdCl2; (4) MT, 1 mM CdCl2; (5) Nr, zero;(6) Nr, 0.2 mM CdCl2; (7) Nr, 1 mM CdCl2; (8) dgt, zero; (9) dgt, 0.2 mM CdCl2 and (10) dgt, 1 mM CdCl2. The SOD isoforms are (I) Mn-SOD; (II and III) Cu/Zn-SODs.


It seems reasonable that alterations in root growth couldmediate root-to-shoot signaling, since the root system is the firsttissue to normally get in contact with the stressor, in this case, Cd.The decrease in water uptake presented by plants submitted to Cdcan be related to photosynthesis inhibition [63], so that for growthmaintenance a coordinated regulation of stomatal conductance toavoid metabolic inhibition of photosynthesis, leading to theoxidative damage and senescence may be required [64].

According to our prior study with these hormonal mutants [49]the stomatal closure and decrease in leaf conductance observed inMT grown in the presence of Cd could reduce photosynthetic CO2uptake and decrease chlorophyll content in MT genotype (Fig. 4), incontrast to the Nr and dgt mutants which tended to present themajority of the stomata open when exposed to Cd [49]. Moreover,the decline in chlorophyll content (Fig. 4) might be partially due tolipid peroxidation of the chloroplast membranes as revealed by thehigher MDA content (Fig. 3B).

Our results revealed that Cd accumulated mainly in the roots(Fig. 2A) showing that Cd is primarily stored at the site of metaluptake, i.e. in the roots of most tomato varieties [65], which can berelated to the restriction of translocation or movement through theroot symplasm by the sequestration of Cd-chelates in vacuoles,production of phytochelatins and the development of extracellularbarriers [66]. The pronounced Cd accumulation in the tomato rootsappears to be related to the more pronounced amount of someamino and organic acids involved in cellular compartmentation ofCd detected in the roots when compared to the shoots [67].

Moreover, oxalic acid secreted from the root apex in tomato plantsmight also reduce Cd uptake as suggested by [68].

Nevertheless, Cd was translocated to the upper parts of theplants (Fig. 2B, C), including the fruits (Fig. 2C), even though inconcentrations considerably reduced when compared to theamount that remained in the roots. The translocation of Cd mightbe related to the adsorption abilities of specific ligands to the metalas indicated by [69]. It is important to mention that a wide range ofprocesses, including immobilization into vacuoles, binding of freemetal cations and different transpiration rates, could be involved inthe Cd transport from roots to shoots [70,71]. Moreover, metalpumps, which can load zinc ions into xylem, might also be relatedto the Cd accumulation in the shoot [70].

The current work showed that less Cd was translocated to theupper parts in MT plants (Fig. 2) when compared to Nr and dgtshoots. This difference could indicate that Cd accumulation in leavescould be driven by transpiration [72,73], based on the positiverelationship between the stomatal closure and decrease in leafconductance observed inMT plants [49]. Furthermore, the presenceof conspicuous intercellular air spaces in the roots of MT plantsexposed to Cd might cause a decrease in water uptake [49], leadingto transpiration decrease. Although we now have an increasedunderstanding of ethylene and auxin effect on transport and accu-mulation of Cd in tomato since enhanced Cd accumulation in alltissues ofNr and dgtmutants was observed (Fig. 2), it is necessary toelucidatewhether this response is due to fact that themajority of thestomata of these mutants open when exposed to Cd [49].

Fig. 7. Activity staining for superoxide dismutase (SOD) following non-denaturing polyacrylamide gel electrophoresis of extracts of leaves isolated fromMT, Nr and dgt plants grownover a 1 (A), 12 (B) and 40 (C)-day period in the presence of CdCl2. The lanes listed are: (1) bovine SOD standard; (2) MT, zero; (3) MT, 0.2 mM CdCl2; (4) MT, 1 mM CdCl2; (5) Nr, zero;(6) Nr, 0.2 mM CdCl2; (7) Nr, 1 mM CdCl2; (8) dgt, zero; (9) dgt, 0.2 mM CdCl2 and (10) dgt, 1 mM CdCl2. The SOD isoforms are (I) Mn-SOD (II and III) Cu/Zn-SODs.


The enhanced accumulation of protective metabolites, such asproline, in plant tissues can occur in response to a stress condition[74e76]. Interesting, we detected an exaggerated accumulation ofproline in leaves (Fig. 5A) and fruits (Fig. 5B) of Nr and dgtmutants,being dose-dependent increasing with the Cd concentrations used.This response, probably, can be associated to the less pronounceddecline in chlorophyll (Fig. 4) and lower MDA contents (Fig. 3) inthese genotypes confirming the protective role of proline againstCd-induced stress. In accordance with this, it has been shown thatethylene [77] and auxin [78] can regulate the proline accumulationin stress condition. As already stated in the introduction, thecellular protection of plant development in response to the Cd-

Fig. 8. Activity staining for superoxide dismutase (SOD) following non-denaturing polyacrylaover a 1 (A), 12 (B) and 40 (C)-day period in the presence of CdCl2. The lanes listed are: (1) bov(6) Nr, 0.2 mM CdCl2; (7) Nr, 1 mM CdCl2; (8) dgt, zero; (9) dgt, 0.2 mM CdCl2 and (10) dgt

stress requires a complex combination of non-enzymatic andenzymatic detoxification mechanisms [12]. In the current work, wehave investigated some key antioxidant enzymes which have beenshown to respond to the oxidative burst induced by Cd, whichvaried considerably among genotypes, tissues, Cd concentrationsand duration of exposure.

Three SOD isoenzymes (SOD I, II, III) were identified followingPAGE (Figs. 6e8) in a similar manner to that reported by [36] for MTand Nr plants. The presence of others SOD isoenzymes bands,which were observed by [36], might be a result of the amount ofprotein used for the gel activity staining. Although only three SODisoenzymes were identified in our current work, they represent the

mide gel electrophoresis of extracts of fruits isolated from MT, Nr and dgt plants grownine SOD standard; (2) MT, zero; (3) MT, 0.2 mM CdCl2; (4) MT, 1 mM CdCl2; (5) Nr, zero;, 1 mM CdCl2. The SOD isoforms are (I) Mn-SOD (II and III) Cu/Zn-SODs.

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Fig. 9. Catalase (CAT) specific activity (mmol min�1 mg�1) in the roots (A), leaves (B) and fruits (C) of MT, Nr and dgt plants grown over a 40-day period in the presence of CdCl2.Values are the means of three replicates �SEM. Different uppercase letters on top of the columns indicate that means of a same time of Cd exposure are different at p < 0.05, byTukey test. Different lowercase letters on top of the columns indicate that means of different times and same treatment are different at p < 0.05, by Tukey test. *Statistical analyzesnot shown.


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Fig. 10. Glutathione reductase (GR) specific activity (mmol min�1 mg�1) in the roots (A), leaves (B) and fruits (C) of MT, Nr and dgt plants grown over a 40-day period in the presenceof CdCl2. Values are the means of three replicates �SEM. Different uppercase letters on top of the columns indicate that means of a same time of Cd exposure are different atp < 0.05, by Tukey test. Different lowercase letters on top of the columns indicate that means of different times and same treatment are different at p < 0.05, by Tukey test.*Statistical analyzes not shown.


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Fig. 11. Guaiacol peroxidase (GPOX) specific activity (U) In the roots (A), leaves (B) and fruits (C) of MT, Nr and dgt plants grown over a 40-day period in the presence of CdCl2. Valuesare the means of three replicates �SEM. Different uppercase letters on top of the columns indicate that means of a same time of Cd exposure are different at p < 0.05, by Tukey test.Different lowercase letters on top of the columns indicate that means of different times and same treatment are different at p < 0.05, by Tukey test. *Statistical analyzes not shown.


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Fig. 12. Ascorbate peroxidase (APX) specific activity (mmol min�1 mg�1) in the roots (A), leaves (B) and fruits (C) of MT, Nr and dgt plants grown over a 40-day period in the presenceof CdCl2. Values are the means of three replicates �SEM. Different uppercase letters on top of the columns indicate that means of a same time of Cd exposure are different atp < 0.05, by Tukey test. Different lowercase letters on top of the columns indicate that means of different times and same treatment are different at p < 0.05, by Tukey test.*Statistical analyzes not shown.



same isoenzyme classification such as observed by Monteiro et al.[36], being SOD I, Mn/SOD and SOD II and III, CueZn/SODs (i.e. SODIV and V).

SOD is related to the dismutation of O2�e into H2O2 and are

located in different cell organelles [12]. As a more general rule forplant SODs, Mn/SODs are located in the mitochondria and peroxi-somes; Fe/SOD in the chloroplast; while the Cu/Zn-SODs arelocated in the cytosol, chloroplasts and peroxisomes [12]. SOD II(CueZn/SOD) was clearly highly active and present in all the tissuestested, whereas SOD I (Mn/SOD) and SOD III (CueZn/SOD) showeddistinct activity patterns in response to Cd in roots (Fig. 6) and fruits(Fig. 8). SOD I may be hormonal dependent, at least in roots (Fig. 6),since there was a specific effect of Cd on SOD I in roots of both Nrand dgt genotypes, which was more accentuated at 40 days ofexposure to the metal (Fig. 6C), indicating that the SOD activityinduced by Cd can involve ethylene and auxin signaling pathway. Ina recent work [79] showed that tobacco transgenic plants withreduced ethylene biosynthesis exhibited increased activity of ROS-detoxifying enzymes, including Mn/SOD and an auxin-stimulatedCuZn-SOD in roots of tomato [53]. However, how these hormonescontrol SOD activity appears to be complex too. SOD I did notappear to be stage dependent since it also appeared in the dgt fruitstreated with 1 mM CdCl2 (Fig. 8). Moreover, while the increase ofSOD I activity observed in Nr and dgt might play an important rolein the cellular protection against Cd toxicity, the decrease of SOD IIIactivity in roots for all genotypes, when compared with theirrespective controls (0 mM CdCl2), could represent a toxic effectof Cd.

When other antioxidant enzymes are concerned, CAT (Fig. 9A),GR (Fig. 10A) and APX (Fig. 12A) appears to be involved in H2O2degradation in the roots, whereas GPOX (Fig. 11A) did not appar-ently participate effectively in such process. From these observa-tions, APX exhibited a similar activity pattern among the genotypesduring metal-stress, while CAT was an important enzyme in thedirect breakdown of H2O2 in MT roots exposed to 1 mM CdCl2 andin Nr roots exposed to 0.2 mM CdCl2. Differently from CAT activity,GR exhibited a clear response in Nr roots for both Cd treatments.Although we have not quantified H2O2 in this study, it is notreasonable to suggest that increases in H2O2 have happened basedon the responses of the antioxidant enzymes we measured. In Nrand dgt roots, the H2O2 generation could be attributed to the dis-mutation of O2

� to H2O2 by SOD I in the highest Cd treatment, buthow ethylene and auxin control the signaling pathway of the dis-mutation cannot yet be fully explained. For instance, it has beenshown that ethylene can interact with H2O2 in a tissue- [36] orconcentration-manner, and that the auxin can increase [80] orreduce [53,81] H2O2. On the other hand, it has also been reportedthat H2O2 can induce auxin [82], making interaction between auxinand H2O2 more complex.

In leaves, a similar SOD activity pattern (Fig. 7) was observed forall periods, genotypes and treatments indicating that H2O2 gener-ation could not be explained by the dismutation of O2

��. Thedecrease of CAT (Fig. 9B) and GPOX (Fig. 11B) activities in MT anddgt genotypes could represent a toxic effect of the highest Cdconcentration for these peroxidases. On the other hand, Nr leavesexhibited increased CAT and GPOX activities for both Cd concen-trations, whereas GR (Fig. 10B) and APX (Fig. 12B) activities weremore pronounced in leaves of Nr plants in 1 mM of CdCl2, whereasdgt leaves showed increased activities in the lowest Cd concen-tration (0.2 mM of CdCl2).

The similar responses for SOD activities to the stressful condi-tions applied in fruits (Fig. 8) also suggest other sources of H2O2generation. Based on the results observed, it appears that CAT(Fig. 9C), GR (Fig. 10C) and GPOX (Fig. 11C) were involved in thecontrol of H2O2 level, and while the response of GR was efficient in

response to Cd stress for all genotypes, CAT and GPOX activitieswere more pronounced in Nr fruits.

The overall results obtained in our research (i.e. growth, Cdcontent, MDA, proline content, chlorophyll, antioxidant enzymes)allow us to suggest that the MT genotype is more sensitive to thestress induced by Cd, whilst the Nr and dgt mutants appear towithstand or avoid more efficiently the oxidative stress imposed byCd. As commented by [83], the inhibition of ethylene perception canalleviate the adverse effects of stress. These authors showed that thetoxic effects of ROS in plants stressed by high temperature (HT)could cause decreases in SOD, CAT and POX activities, whereasplants treated with an inhibitor of ethylene perception (1-MCP)exhibited higher SOD, CAT and POX activities, due to the preventionof stress ethylene perception. Conversely, HT-stressed plantsincreased ethylene production rate leading to premature leafsenescence. However, the application of the inhibitor of ethyleneperception, 1-MCP, reduced premature leaf senescence traits byinhibiting ethylene production [84]. Thus, the defense responseexhibited by Nr mutant could be related to a blockage or limitedethyleneperception,whilst indgt, it could be related to a blockageorlimited ethylene production. In this way, disconnectedly, theinvolvement of ethylene and auxin on antioxidant mechanismsappears to be clear, but it is acceptable to assign that the alterationsin this mechanisms observed in Nr and dgt can involve interactionbetween these hormones. Therefore, it is possible to suggest that thedgtmaywithstandor avoid stress imposed byCd, at least inpart, dueto the fact that the known auxin-stimulated ethylene production iscomprised in dgt plants. Conversely, the Nr genotype was moreaffected by the Cd imposed stress than dgt, whichmay be explainedby the fact that Nr retains a partial sensitivity to ethylene [85].

Here, we summarize this issue specially focusing the involve-ment ofNR receptor andDGTgene in relation to Cd stress responses.However, it cannot rule out the integration of multiple hormonesignaling pathways involved with abiotic stress responses. Theseresults add further information that should help unraveling therelative importance of ethylene in regulating the cell responses tostressful conditions. Moreover, studies on hormone interactionsunder stress and the transcriptional and post-transcriptional regu-lation of stress-responsive gene expression have indicated impor-tant signaling pathways and genes involved with abiotic stress [86].In the case of interaction between ethylene and auxin, we are nowgenerating double mutants such as Nr dgt and Nr epi (epinastic;ethylene-overproducer mutant) for further elucidation of thecontrol of the oxidative stress duringmetal as well as other stresses.

Further analysis is needed for a better understanding about themechanisms connecting antioxidant responses, ROS and phyto-hormones on plant growth processes and stressful conditions.Ongoing research in our laboratories are investigating such aspectsand also using a proteomic approach to identify proteins specifi-cally responding to the effect of Cd, which could significantlycontribute to unravel the possible relationships between proteinprofile changes and plant stress [87e89]. Besides, the expansion ofour knowledge on abiotic stress tolerance and avoidance strategiesin tomato plants may help understanding similar aspects andeventual exploitation of other crops. Such studiesmay contribute tothe modification of hormone biosynthesis pathways for designingof effective strategies for engineering crops that will be tolerant toabiotic stresses [90,91].

4. Materials and methods

4.1. Plant material

Seeds of the tomato (S. lycopersicum L.) cultivar Micro-Tom (MT),and the mutants diageotropica (dgt) and Never ripe (Nr) near


isogenic to MT [35,92] were germinated in boxes containinga mixture of 1:1 (by volume) commercial pot mix (Plantmax HTEucatex, Brazil) and vermiculite, supplemented with 1 g L�1

10:10:10 NPK and 4 g L�1 lime (MgCO3 þ CaCO3) and maintained ina greenhouse with an average mean temperature of 28 �C, 11.5 h/13 h (winter/summer) photoperiod, and 250e350 mmol m�2 s�1

PAR irradiance (natural radiation reduced with a reflecting mesh(AluminetePolysack Industrias Ltda, Leme, Brazil). After the firsttrue leaves appeared, seedlings were transplanted to 1 L Leonardpots [93] (2 seedlings per pot) filled with sand and irregular pelletsof polystyrene (4:3) and Hoagland’s nutrient solution (750 mL).Thirty-day-old plants were selected and further grown in the samesolution, but containing 0 mM (control), 0.2 mM or 1 mM CdCl2.The Hoagland solution with or without CdCl2 was changed weeklyand water was used to complete the total volume once a week.After a period of 47, 58 and 85 d post germination, corresponding to1, 12 and 40 d of exposure to CdCl2, respectively, roots, leaves andfruits were collected, washed in distillededeionized water andstored at �80 �C for further analysis.

4.2. Cd content

Cd concentration in samples of roots, leaves and fruits wasdetermined by digestion with a mixture of nitric and perchloricacids as described by Malavolta et al. [94]. Quantitative Cd analysiswas carried out using a flame atomic absorption spectroscopy witha Perkin Elmer spectrometer model 310. Cd concentrations wereexpressed as mg g�1 DW.

4.3. Lipid peroxidation

Lipid peroxidation was determined by measuring the concen-tration of thiobarbituric acid reactive substances (TBARS) asdescribed by Heath and Packer [95]. Plant tissues were homoge-nized in a pestle and mortar with 20% (w/v) insoluble poly-vynilpyrrolidone (PVPP) and 0.1% trichloroacetic acid (TCA). Thehomogenate was centrifuged at 10,000 g for 5 min. 250 mL of thesupernatant was added to 1 mL 0.5% TBA in 20% TCA. This solutionwas incubated in a water bath at 95 �C for 20 min, and the reactionwas stopped by quickly cooling in an ice-water bath. The absor-bance of the formed TBARS was determined spectrophometricallyat 535 nm. Measurements were corrected for unspecific turbidityby subtracting the absorbance at 600 nm. The concentration ofmalondialdehyde (MDA) equivalents was calculated using theabsorbance coefficient 1.55 � 10�5 mol�1 cm�1.

4.4. Chlorophyll determination

Leaf chlorophyll content was determined using a Minolta SPAD-502 m, which measures leaf transmittance at two wavelengths: red(approximately 660 nm) and near infrared (approximately940 nm). SPAD readings were taken weekly on the terminal leafletof the fourth leaf from the base of the shoot. The SPAD sensor wasplaced randomly on leaf mesophyll tissue only, avoiding the veins.

4.5. Proline content

Free proline content was measured as described by Bates [96].Plant tissues were extracted with 3% sulphosalicylic acid. Thehomogenate was centrifuged at 10,000 g, 15 min, 4 �C, and 2 mL ofthe supernatant was held for 1 h in boiling water by adding 2 mLninhydrin and 2mL glacial acetic acid, to which cold toluene (4 mL)was added. The absorbance was read at 520 nm and calculated asmmol g�1 FW against standard proline.

4.6. Enzyme extraction and protein determination

The following steps were carried out at 4 �C unless statedotherwise. Roots, leaves and fruits samples were homogenized inbuffer volume/fresh weight (2/1) in a mortar with a pestle in100 mM potassium phosphate buffer (pH 7.5) containing 1 mMethylenediaminetetraacetic acid (EDTA), 3 mM DL-dithiothreitoland 5% (w/v) insoluble polyvinylpolypyrrolidone [97]. Thehomogenate was centrifuged at 10,000 g for 30 min, and thesupernatant was stored in separate aliquots at �80 �C prior to SOD,CAT, GR, GPOX and APX analysis. Protein concentration for allsamples was determined by the method of Bradford [98] usingbovine serum albumin as a standard.

4.6.1. Catalase assayCatalase (CAT) activity was assayed at 25 �C in a reaction

mixture containing 1 mL 100 mM potassium phosphate buffer (pH7.5), containing 2.5 mL H2O2 (30% solution) [99]. The reaction wasinitiated by the addition of 25 mL of plant extract, and the activitydetermined by following the decomposition of H2O2 as a change inabsorbance at 240 nm over 1 min. CAT activity is expressed as mmolmin�1 mg�1 protein.

4.6.2. Glutathione reductase assayGlutathione reductase (GR) activity was assayed at 30 �C in

a mixture consisting of 1 mL 100 mM potassium phosphate buffer(pH 7.5) containing 1 mM 5,5”-dithiobis (2-nitrobenzoic acid)(DTNB), 1 mM oxidized glutathione (GSSG) and 0.1 mM NADPH[48]. The reaction was started by the addition of 50 mL of enzymeextract. The rate of reduction of GSSG was followed in a spectro-photometer by monitoring the increase in absorbance at 412 nmover a 1 min period. GR activity is expressed as mmol min�1 mg�1

protein.

4.6.3. Guaiacol peroxidase assayGuaiacol peroxidase (GPOX) activity was determined in a reac-

tion medium contained 250 mL phosphateecitrate buffer (sodiumphosphate dibasic 0.2 M : citric acid 0.1 M) pH 5.0, 15 mL enzymeextract and 25 mL 0.5% guaiacol, which was incubated at 30 �C for15 min [100]. The reaction was stopped in an ice-water bath, fol-lowed by the addition of 25 mL of 2% sodium metabisulphidesolution. The GPOX activity was evaluated by monitoring theabsorbance at 450 nm. One enzyme activity unit (U) of GPOXcorresponds to an increase of 0.001 in absorbance per min per mgprotein.

4.6.4. Ascorbate peroxidase assayAscorbate peroxidase (APX) activity was determined by the

addition of 40 mL plant extract to 1 mL of a medium containing50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbate,0.1 mM EDTA and 0.1 mM H2O2 [101]. APX activity was determinedby monitoring the rate of ascorbate oxidation at 290 nm at 30 �C.APX activity is as nmol ascorbate min�1 mg�1 protein.

4.7. Polyacrylamide gel electrophoresis (PAGE)

Electrophoretic analysis was carried out under non-denaturingcondition in 12% polyacrylamide gels, followed by SOD activitystaining. Electrophoresis buffers and gels were prepared asdescribed by Vitória et al. [102], except that SDS was excluded.

4.8. SOD activity staining

SOD activity staining was carried out as described by Garciaet al. [103]. After non-denaturing-PAGE separation, the gel was


rinsed in distilled-deionized water and incubated in the dark in50 mM potassium phosphate buffer (pH 7.8) containing 0.05 mMriboflavin, 1 mM EDTA, 0.1 mM nitroblue tetrazolium and 0.3%N,N,N0,N0-tetramethylethyllenediamine. After 30min, the gels wererinsed with distillededeionized water and then illuminated inwater until the achromatic bands of SOD activity were visible ona purple-stained gel. Afterward, SOD isoenzymes were distin-guished by their sensitivity to inhibition by 2 mM potassiumcyanide and 5 mM hydrogen peroxide (H2O2).

4.9. Statistical analysis

The experimental designwas randomized with nine plants fromthree replicate pots, being the results expressed as mean andstandard error of mean (�SEM) of three independent replicates ofeach extract for plant growth, Cd accumulation, TBARS, chlorophylland proline contents, CAT, GR, GPOX and APX activities. Thestatistical analysis was performed using the R software version2.14.2. A multiple comparison between means by the Tukey testfollowed an individual ANOVA for each character, at a 0.05 level ofsignificance.

Acknowledgments

This work was funded by Fundação de Amparo à Pesquisa doEstado de São Paulo (FAPESP - Grant n�.09/54676-0). We thankConselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq-Brazil) (R.A.A., P.L.G, F.A.P and L.E.P.P.), and FAPESP (C.C.M.;T.T.) for the fellowships and scholarships granted.

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Biochemical dissection of diageotropica and Never ripe tomato ...

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