Does grafting provide tomato plants an advantage against H2O2 production under conditions of thermal shock

Does grafting provide tomato plants an advantage against H2O2 production under conditions of thermal shock
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  PHYSIOLOGIA PLANTARUM 117: 44–50. 2003  Copyright C Physiologia Plantarum 2003 Printed in Denmark – all rights reserved   ISSN 0031-9317  Does grafting provide tomato plants an advantage against H 2 O 2 production under conditions of thermal shock? Rosa M. Rivero*, Juan M. Ruiz, Esteban Sa´nchez and Luis Romero* Department of Plant Biology, Faculty of Sciences, University of Granada 18071- Granada, Spain*Corresponding author, e-mail: lromero / Received 11 February 2002; revised 16 May 2002 Non-grafted tomato plants ( Lycopersicon esculentum  L. cv.Tmknvf  2 ) and grafted tomato plants ( L.esculentum  L. cv.Tmknvf  2 ¿ L.esculentum  L. cv. RX-335) were grown for 30days at three different temperatures (10 æ C, 25 æ C and 35 æ C).In the leaves of these plants, the enzymatic activities of SOD,GPX, CAT, APX, DHAR and GR were analysed, as were theconcentrations of total H 2 O 2 , ascorbate and glutathione aswell as foliar DW. Regardless of whether the plant was graftedor not, our results indicate that the thermal stress occurred Introduction Many crops are cultivated in areas where the climaticconditions are not always ideal and where temperaturesmay periodically fall far below or rise substantiallyabove optimal levels. Such conditions can trigger oxida-tive metabolism in plants, brought on by an overpro-duction of active oxygen species (AOS), such as superox-ide radicals ( ¡ O 2 –  ), hydroxyl ( ¡ OH), singlet oxygen ( 1 O 2 ),and hydrogen peroxide (H 2 O 2 ) (Elstner and Oswald1994, Prasad et al. 1994, Queiroz et al. 1998). For theirchemical properties, the AOS are highly reactive and candamage proteins, chlorophylls, membrane lipids andnucleic acids, upsetting the homeostasis of the organism(Shaaltiel and Gressel 1986, Scandalios 1993). To pre-vent or alleviate these damages, plants have developedvarious mechanisms based on the production of defenceantioxidants (Jahnke et al. 1991, Walker and McKersie1993, Hodges et al. 1997).The electrons generated by the activity of PSII giverise to the formation of different AOS, the radical  ¡ O 2 –  being one of the most important (Salin 1989, Bowler Abbreviations  – AOS, active oxygen species; APX, ascorbate peroxidase; AsA, ascorbate; BSA, bovine serum albumin; CAT, catalase; DHA, dehy-droascorbate; DHAR, dehydroascorbate reductase; ETDA, ethylenediaminetetraacetic acid; Fe-EDDHA, ethylenediamine-di( o -hydroxyphenylaceticacid); GPX, guaiacol peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; H 2 O 2 , hydrogen peroxide; NTB,nitroblue tetrazolium; ¡ O 2 –  , superoxide radical; ¡ OH, hydroxyl radical; PPDF, photosynthetic photon flux density; PSII, photosystem II; SOD, superox-ide dismutase. Physiol. Plant. 117, 2003 44 mainly at 35 æ C, with the following effects: (1) high SOD ac-tivity; (2) H 2 O 2  accumulation; (3) foliar-biomass reduction;(4) low GPX, CAT, APX, DHAR and GR activities; and (5)high concentrations of ascorbate and glutathione. In addition,our data show these effects to be much weaker in grafted thanin non-grafted plants, directly reflected in greater biomassproduction. Therefore, the use of grafted plants under excess-ively high temperatures may offer an advantage over non-grafted plants in terms of resistance against thermal shock. et al. 1992, Scebba et al. 1998). The enzyme SOD bringsabout the dismutation of the radical  ¡ O 2 –  to H 2 O 2 , forwhich it is commonly considered as the first line of celldefence (Halliwell and Gutteridge 1989). In fact, H 2 O 2 can be detoxified from the different cell compartmentsby different enzymes, either on the one hand by the ac-tivity of GPX or CAT, which bring about the automaticcatalysis of H 2 O 2  to H 2 O (Peters et al. 1989, Takahamaand Oniki 1992), or on the other hand, by an oxidation-reduction system of antioxidant metabolites (ascorbateand glutathione) which involves other enzymes (APX,DHAR and GR) called the ascorbate/glutathione cycleor route of Halliwell-Asada (Asada and Takahashi 1987,Halliwell and Gutteridge 1989, Salin 1989, Ushimaruet al. 2000).The exposure of plants to stress situations generallytriggers antioxidant defence systems, since the regulationof the coding genes for these enzymes appears to behighly sensitive to the rise in cellular levels of AOS pro-duced under these conditions (del Rı´o et al. 1991, Palma  et al. 1991, Scandalios 1993, Allen 1995, Rao et al.1996). In this way, higher cellular levels of H 2 O 2  underthermal stress indicate a clear rise in AOS production(Lafuente and Martı´nez-Te´llez 1997, Paolacci et al.1997). Both high as well as low temperatures can promptH 2 O 2  accumulation in different plant tissues resultingfrom stronger SOD activity and by a partial inhibitionof the enzymes in charge of detoxification (Yamakawa1983, Campa 1990, Gaspar et al. 1991). In this way,H 2 O 2  accumulation in plants subjected to thermal stressis considered the prime cause for the reduction in plantbiomass (Queiroz et al. 1998).The cultivation of grafted plants began in late 1920primarily to counteract or diminish attacks by soilpathogens such as  Fusarium oxysporum  (Yamakawa1983, Lee 1994). However, the current applications of grafting involve virtually all fields of plant physiology.For example, grafted plants were used to induce re-sistance against low root temperatures (Bulder et al.1990), against iron chlorosis in calcareous soils (Romeraet al. 1991, Shi et al. 1993), greater nutrient uptake (Ba-varesco et al. 1991, Ruiz et al. 1996, 1997), increasedsynthesis of endogenous hormones (Proebsting et al.1992), greater growth and productivity of the aerial part(Hussein and Slack 1994, Ruiz and Romero 1999) andimproved fruit quality (Autio 1991).Given that plant species differ in sensitivity to heatfluctuations, grafting onto root bases more resistant tothese changes could encourage growth and developmentof the above-ground part and thereby improve plant ad-aptation to thermal stress. In this light, the aim of thepresent work was to compare the advantage of usinggrafted tomato plants under conditions of thermal stressand, in this regard, to investigate the role of oxidativemetabolism (both enzymes and antioxidant compounds)in the resistance to stress in these plants. Materials and methods Experimental design Two varieties of tomato plants ( Lycopersicon esculentum L.) were used: cv. Tmknvf  2 , and cv. RX-335. Seeds of both varieties were germinated and grown for 30 days ina growth chamber under controlled conditions of hu-midity and photoperiod, at a constant temperature of 23–26 æ C (optimal growth temperature for these plants)(Maroto 1995). Afterwards, the two varieties were graft-ed together (by needle graft), using RX-335 as the root-stock and Tmknvf  2  as the scion.After 30 days the grafts were inspected to confirmproper healing of the joint, and the experiments werebegun. Three different experiments were conducted inwhich the only variable was temperature. In each experi-ment, 12 non-grafted tomato plants ( L.esculentum  L. cv.Tmknvf  2 ) and 12 grafted ones ( L.esculentum  L. cv.Tmknvf  2 ¿ L.esculentum  L. cv. RX-335) were grown ina growth chamber. The first experiment was conductedat 10 æ C (day/night), the second at 25 æ C (day/night) and Physiol. Plant. 117, 2003  45 the third at 35 æ C (day/night). Temperature was measuredby CR21X sensors (Campbell Scientific) placed in themiddle and upper shoot zone of the plants as well as inthe root zone at 12 cm in depth. In all cases, the tem-perature difference between shoot and the root zone was ∫ 2 æ C, a margin of error small enough to be disregardedin the interpretation of the results.Each experiment lasted 30 days; that is, from day 60– 90 after germination. In all cases, the growth-chamberconditions were maintained constant with a relative hu-midity of 60–80% and 16 h or photoperiod (PPDF of 350  m mol m ª 2 s ª 1 measured in the highest part of theplants with a 190 SB quantum sensor, LI-COR, Inc.,NE, USA).All plants were grown in individual pots (25 cm indiameter and 25 cm in height) completely filled with ver-miculite. For 90 days, all the plants received a completenutrient solution composed of: 2 m M   KNO 3 , 4 m M  (NO 3 ) 2 Ca, 1.5 m M   NaH 2 PO 4 , 2 m M   CaCl 2 , 3 m M  SO 4 K 2 , 1.25 m M   MgSO 4 , 5  m M   Fe-EDTA, 2  m M  MnSO 4 , 1  m M   ZnSO 4 , 0.25  m M   CuSO 4 , 0.05  m M  (NH 4 ) 6 Mo 7 O 24  and 2.5 m M   H 3 BO 3  (Van Zinderen 1986).This solution was renewed every 3 days and the pH wasmaintained at between 6.0 and 6.1. Plant sampling Plants were sampled on day 60 after sowing, all sampledleaves being in the mature state. The material was rinsedthree times in H 2 O after disinfecting with 1% non-ionicdetergent (Decon 90) (Wolf 1982), and then blotted onfilter paper. Of each treatment, half of the plants wereused for the analysis of SOD, CAT, GPX, APX, DHAR,GR, H 2 O 2 , AsA, DHA, total ascorbate, GSH, GSSGand total glutathione (triplicate assays for each extrac-tion). The other half of the treated plants were used todetermine shoot DW. Leaves of these plants were driedin a force-air oven at 70 æ C for 24 h. DW was recordedand expressed as g DW shoot ª 1 . Metabolite assays The methods used to extraction and of total H 2 O 2  werethose of McNervin and Uron (1953) and Brennan andFrenkel (1977). Hydroperoxides form a specific complexwith titanium (Ti 4 π ), which can be measured by col-ourimetry at 415 nm. The concentration of peroxide inthe extracts was determined by comparing the absorb-ance against a standard curve representing a titanium-H 2 O 2  complex from 0.1 to 1 m M  . The hydroperoxidesrepresent the total peroxides.Reduced ascorbate (AsA), dehydroascorbate (DHA),and total ascorbate (AsA π DHA) were determined fol-lowing Gosset et al. 1994). From the same extract, AsAand total ascorbate were assayed. Ascorbate standardsof between 0.001 and 0.5  m mol ml ª 1 ascorbate in  m -phosphoric acid were analysed in the same manner asextracts. For each sample, DHA was estimated from thedifference of total ascorbate and AsA.  The GSSG, GSH and total glutathione (GSSG π GSH) amounts were determined following Gosset et al.(1994). From the same extract, GSSG and total glutathi-one were assayed. A standard curve was developed bypreparing solutions of 0.002–0.0001 g ml ª 1 GSH in 60ml ª 1 m -phosphoric acid (pH2.8) containing 1 m M  EDTA, diluting 1:2000 with 50 ml l ª 1 Na 2 PO 4 , and ana-lysing in the same manner as the extracts. Levels of GSHwere estimated as the difference between total glutathi-one and GSSG. Enzyme assays SOD activity was assayed by monitoring the inhibitionof photochemical reduction of NBT, according to themethods of Giannopolitis and Ries (1977) and Beyerand Fridovitch (1987), with some modifications (Yu 1998). A 5-ml reaction mixture was used, containing50 m M   HEPES (pH7.6), 0.1 m M   EDTA, 50 m M  Na 2 CO 3  (pH 10.0), 13 m M   methionine, 0.025% (v/v)Triton X-100, 63  m M   NTB, 1.3  m M   riboflavin and anappropriate aliquot of enzyme extract. The reactionmixtures were illuminated for 15 min; PPFD was 380 m mol m ª 2 s ª 1 . Identical reaction mixtures that not wereilluminated were used to correct for background absorb-ance. One unit of SOD activity was defined as theamount of enzyme required to cause 50% inhibition of the reduction of NTB as monitored at 560 nm.CAT activity was determined as described Bandianiet al. (1990) by following the consumption of H 2 O 2  (ex-tinction coefficient, 39.4 m M  ª 1 cm ª 1 ) at 240 nm for 3minutes.GPX activity was determined as described Kalir et al.(1984) and Ruiz et al. (1998) by the oxidation of guai-acol in the presence of H 2 O 2  (extinction coefficient, 26.6m M  ª 1 cm ª 1 ) at 470 nm.APX activity was determined according to Gossettet al. (1994) by following the decrease in A 290  of an assaymixture containing 0.5 m M   ascorbate (extinction coef-ficient, 2.8 m M  ª 1 cm ª 1 ). Corrections were made fornon-enzymatic rates and for interfering oxidations.DHAR activity was determined following Ushimaruet al. (1992) and GR activity was assayed as describedby Ushimaru et al. (2000).In our enzyme assays activities rates were determinedat substrate saturation. All of the activities were ex-pressed as a function of the oxidized or reduced sub-strate per milligram of protein per minute. The proteinconcentration was determined by the method of Brad-ford (1976) using BSA as standard. Statistical analysis Analysis of variance was used to assess the significanceof treatment. Results shown are mean values ∫  . A cor-relation analysis was also conducted to determine therelationship between the different variables. Levels of significance are represented by at * P  0.05, ** P  0.01,*** P  0.001 and NS, not significant by    at  P Ω Physiol. Plant. 117, 2003 46 0.05. Differences between the two temperatures and sep-arated   s were tested for each variable. The  t -testfor each variable was then carried out and a Bonferronicorrection was made. Results and discussion Plants subjected to thermal stress tend to overproduceAOS in different plant tissues (Levine et al. 1994), andin response the enzyme SOD constitutes the first line of cellular defence, detoxifying  ¡ O 2 –  radicals and giving riseto H 2 O 2  production. Figure1 shows that SOD activitysignificantly differed with respect to the three tempera-tures applied ( P  0.001), regardless of the whether theplant was grafted or not. The SOD activity provedhighest at 35 æ C (superoptimal temperature), wherevalues were 262% higher in grafted plants and 310%higher in non-grafted plants than at 25 æ C (the optimaltemperature for tomato plants) (Maroto 1995), the tem-perature that gave the lowest SOD values. When theplants were grown at 10 æ C, SOD activity was higher thanat 25 æ C, although less notably than when plants weregrown at 35 æ C. Some works indicate that thermal stresscan induce SOD activity by an overproduction of AOSunder such conditions (Smirnoff 1993). Figure1 reflectslower SOD activity in grafted plants than in non-graftedones, irrespective of the growth temperature. However,the greatest differences in SOD activity were found be-tween grafted and non-grafted plants at 35 æ C ( P  0.001), for which, in principle, we might assume that theproduction of AOS under thermal-stress conditions (inour case 35 æ C) remained lower in grafted than in non-grafted plants.To test the above hypothesis we determined the con-centration of the foliar H 2 O 2 , this being the first com-pound resulting from the detoxification of the  ¡ O 2 –  rad- Fig.1. SOD activity in grafted and non-grafted tomato plants atthree temperatures. SOD activity expressed as units of SOD (mgprot) ª 1 (min) ª 1 .  Table1. Effect of temperature on H 2 O 2  concentrations and DW in grafted and non-grafted tomato plants. H 2 O 2  expressed as m mol of H 2 O 2 (g FW) ª 1 . Shoot DW expressed g per plant.TEMP H 2 O 2A DW B Grafted Non-grafted Grafted Non-grafted Signif. A Signif. B 10 æ C 32.09 ∫ 1.01 42.19 ∫ 1.02 47.12 ∫ 1.37 24.86 ∫ 1.18 * ***25 æ C 29.03 ∫ 0.98 24.77 ∫ 0.89 54.54 ∫ 1.44 43.40 ∫ 1.25 * *35 æ C 43.95 ∫ 1.12 71.83 ∫ 1.29 36.21 ∫ 1.12 15.88 ∫ 1.09 *** ***Signif. ** *** ** *** icals by SOD, and we found significant differences withrespect to the different temperatures applied. Table1 in-dicates that the H 2 O 2  concentration at the different tem-peratures applied had a behaviour similar to that of SOD activity, with 35 æ C registering the highest concen-trations of this compound, regardless of whether theplant was grafted or not. When the plants were grownat 25 æ C, the H 2 O 2  concentration fell by 65% with respectto 35 æ C in non-grafted plants, but by 34% in graftedones. Many authors have reported that, under thermalstress, H 2 O 2  can accumulate in different plant tissuesdue to an overproduction of SOD (Paolacci et al. 1997,Willenkens et al. 1997, Wendehenne et al. 1998), thisstrengthening the results found in the present work. Thecorrelation coefficients between SOD activity and theH 2 O 2  concentration in all cases proved positive andhighly significant (non-grafted: SOD-H 2 O 2 , r Ω 0.969***; grafted: SOD-H 2 O 2 , r Ω 0.929***), this ac-counting for the proportionally direct relationship foundbetween grafted and non-grafted plants in our experi-ment.On the other hand, Table1 shows that at 35 æ C theH 2 O 2  concentration in non-grafted plants was almostdouble that in grafted plants, and thus we conjecturethat in the latter the AOS production was less than inthe non-grafted plants.Willenkens et al. (1997) demonstrated that an H 2 O 2 accumulation in the different tissues of a plant couldresult in reduced biomass. In fact, Table1 shows lowerfoliar biomass both in grafted and non-grafted plants at35 æ C, the temperature at which the highest concen-trations of H 2 O 2  were found. Furthermore, the relation-ship between the two parameters proved negative andsignificant in all cases (non-grafted: DW-H 2 O 2 , r Ωª 0.922***; grafted: DW-H 2 O 2 , r Ωª 0.924***), support-ing the hypothesis of Willenkens et al. (1997).As indicated above, the accumulation of foliar H 2 O 2 is lower in grafted plants than in non-grafted, and thusit might be expected that the reduction in foliar biomasswould also be lower, as reflected in Table1. In this sense,the use of grafted plants implies an advantage with re-spect to non-grafted plants, since under the same con-ditions greater biomass production resulted in plantsgrafted over a more resistant rootstock.The fact that our experiment reflected a lower H 2 O 2 concentration in grafted plants could be due either tolower AOS production or to more efficient detoxificationof this compound owing to the activity of a number of  Physiol. Plant. 117, 2003  47 enzymes involved in the ascorbate/glutathione cycle(APX, DHAR, GR) which act in the breakdown of H 2 O 2  into H 2 O and O 2 , through a system of oxidation/reduction of antioxidant compounds such as ascorbateand glutathione (Queiroz et al. 1998, Ushimaru et al.2000). Therefore, in this study, we included not only theactivities of CAT and GPX, but also of the antioxidantenzymes and compounds of the ascorbate/glutathionecycle.As revealed in Fig.2A,B, the activities of GPX andCAT showed similar behaviour, responding significantlyto the different temperatures applied ( P  0.001). Thehighest GPX and CAT activities (Fig.2A,B, respectively)resulted when the plants were grown at 25 æ C, while at10 æ C and especially at 35 æ C the activities of both en-zymes declined considerably, irrespective of whether theplant was grafted or not. This decline at 35 æ C, with re-spect to 25 æ C, for GPX activity was 66% (Fig.2A) innon-grafted plants and 48% in grafted plants. For theCAT activity (Fig.2B), this reduction was 40% both ingrafted plants as well as non-grafted ones. Some re-searchers hold that conditions of thermal stress can pro-mote enzymatic activity of GPX and CAT (Lafuente andMartı´nez-Te´llez 1997, Paolacci et al. 1997). Neverthe-less, our results appear to indicate the contrary, leadingus to conclude that excessively high temperatures mightpartially inhibit these enzymes, giving rise to H 2 O 2  ac-cumulation in different plant tissues. The correlation co-efficients between the H 2 O 2  concentration and the activ-ities of GPX and CAT in all cases proved negative (non-grafted: GPX-H 2 O 2 , r Ωª 0.750**; CAT-H 2 O 2 , r Ωª 0.816**; grafted: GPX-H 2 O 2 , r Ωª 0.959***; CAT-H 2 O 2 , r Ωª 0.889***), implying that H 2 O 2  is not detoxi-fied by either of the two enzymes and that this com-pound accumulates in the different plant tissues.On the other hand, Fig.2A,B reflect that in graftedplants the activities of both enzymes were significantlyhigher than in non-grafted plants ( P  0.001), and there-fore we might deduce that in grafted plants the detoxi-fication of H 2 O 2  by these enzymes is somewhat moreefficient than in non-grafted plants.With respect to the ascorbate/glutathione cycle, the ac-tivities of APX, DHAR and GR (Fig.3A,B,C, respec-tively) varied significantly with respect to the differenttemperatures applied. As in the case of the two pre-viously discussed enzymes, the activities of these latterthree were highest at 25 æ C (optimal temperature forgrowing these plants), while in plants grown at 10 æ C,  and especially 35 æ C, these activities diminished sharply,this decline being far more drastic when the plants werenot grafted. According to some researchers, excessivelyhigh temperatures partially inhibit the activities of theenzymes APX, DHAR and GR (Lafuente and Martı´-nez-Te´llez 1997, Queiroz et al. 1998), conclusions whichsupport our results. Figure.3A,B,C) show that the mostsignificant differences between grafted and non-graftedplants appear at 35 æ C ( P  0.001 in all cases). Figure4reveals that grafting promoted APX activity up to thepoint that, at both 10 æ C and 35 æ C, the activity of thisenzyme was greater than in non-grafted plants grown at25 æ C, a situation similar with respect to the activities of the enzymes DHAR (Fig.3B) and GR (Fig.3C).The correlation coefficients between the activities of these three enzymes and the H 2 O 2  concentration provednegative in all cases, although the statistical significancewas much stronger in non-grafted than in grafted plants(non-grafted: APX-H 2 O 2 , r Ωª 0.978***; DHAR-H 2 O 2 , r Ωª 0.878***; GR-H 2 O 2 , r Ωª 0.928***; graft- Fig.2. (A) GPX activity in grafted and non-grafted tomato plantsat three temperatures. GPX activity expressed as  m mol guaiacoloxidized (mg prot) ª 1 (min) ª 1 . (B) CAT activity in grafted and non-grafted tomato plants at three temperatures. CAT activity expressedas m mol H 2 O 2  reduced (mg prot) ª 1 (min) ª 1 . Physiol. Plant. 117, 2003 48 ed: APX-H 2 O 2 , r Ωª 0.521*; DHAR-H 2 O 2 , r Ωª 0.672**; GR-H 2 O 2 , r Ωª 0.716**). This could explainthe foliar concentrations of H 2 O 2  found in graftedplants, which were far less than in non-grafted ones. Fig.3. (A) APX activity in grafted and non-grafted tomato plantsat three temperatures. APX activity expressed as  m mol ascorbateoxidized (mg prot) ª 1 (min) ª 1 . (B) DHAR activity in grafted andnon-grafted tomato plants at three temperatures. DHAR activityexpressed as  m mol dehydroascorbate reduced (mg prot) ª 1 (min) ª 1 .(C) GR activity in grafted and non-grafted tomato plants at threetemperatures. GR activity expressed as m mol NADPH oxidized (mgprot) ª 1 (min) ª 1 .
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