Roots and leaves display contrasting oxidative response during salt stress and recovery in cowpea

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1 Journal of Plant Physiology 164 (27) Roots and leaves display contrasting oxidative response during salt stress and recovery in cowpea Fabio Rossi Cavalcanti a, João Paulo Matos Santos Lima a, Sérgio Luiz Ferreira-Silva a, Ricardo Almeida Viégas b, Joaquim Albenisio Gomes Silveira a, a Laboratório de Metabolismo de Plantas, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 633, CEP , Fortaleza, Ceará, Brazil b Universidade Federal de Campina Grande, Paraíba, Brazil Received 7 March 26; accepted 2 March 26 KEYWORDS Osmotic stress; Oxidative damage; Oxidative response; Salinity; Vigna unguiculata Summary In this study, we compare some antioxidative responses of leaves and roots associated to growth reduction in cowpea plants (Vigna unguiculata) during shortterm salt stress and recovery. The salt treatment was imposed (2 mm NaCl) for six consecutive days and the salt withdrawal after 3 d. The salt treatment caused an almost complete cessation in the relative growth rate of both leaves and roots. Although NaCl withdrawal has induced an intense reduction in the Na + content from the leaves and roots, the growth recovery was slight, after 3 d. The leaf lipid peroxidation was increased in salt-stressed plants and slightly reduced in recovered plants after 3 d. Surprisingly, in the salt-stressed roots it decreased markedly after 3 d treatment and in the pre-stressed/recovered roots it was restored to levels near to the control. In leaves, catalase (CAT) activity showed a rapid and prominent decrease after 1 d of NaCl treatment and salt withdrawal had no effect on its recovery. In contrast, the root CAT activity was not changed by effects of both NaCl and salt withdrawal, over time interval. Leaf superoxide dismutase (SOD) activity did not change in all treatments, whereas in roots it significantly decreased after 3 d of salt treatment and recovered after NaCl withdrawal. Contrasting to the other enzymes, the guaiacol-peroxidase activity increased in leaves and roots, reaching almost 2% of control values and it significantly decreased in both organs from the pre-stressed/recovered plants. In conclusion, cowpea roots and leaves present distinct mechanisms of response to lipid peroxidation and CAT and SOD activities during salt stress and recovery. However, these responses and/or the oxidative Abbreviations: CAT, catalase; DM, dry matter; POX, guaiacol peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, Thiobarbituric acid-reactive substances Corresponding author. Tel./fax: address: silveira@ufc.br (J.A.G. Silveira) /$ - see front matter & 26 Elsevier GmbH. All rights reserved. doi:1.116/j.jplph

2 592 F.R. Cavalcanti et al. damages caused by reactive oxygen species were not related with the growth reduction. & 26 Elsevier GmbH. All rights reserved. Introduction One of the most important abiotic factors limiting plant productivity is water stress induced by drought or salinity. This is especially acute in arid and semi-arid regions, like Brazilian Northeast, where cowpea (Vigna unguiculata) is a widely cultivated species (Souza et al., 24). This crop has adapted to cope with several abiotic stresses such as drought, salinity, and high levels of temperatures and radiation, which, alone or in combination, can induce oxidative damage to the plant (Foyer and Noctor, 2). Most of the environmental stresses effected in plant metabolism are mediated by the enhanced production of reactive oxygen species (ROS), such as superoxide (O 2 ), hydrogen peroxide (H 2 O 2 ), singlet oxygen and hydroxyl radical (Foyer and Noctor, 23). These ROS are extremely cytotoxic and can seriously disrupt normal metabolism through oxidative damage to lipids (Alscher et al., 22), nucleic acids and proteins (Herbette et al., 22). As a consequence, a series of cellular degenerative processes are triggered, including peroxidation of membrane lipids and programmed cell death (Gechev et al., 22). In order to avoid the damage caused by ROS compounds, plants have evolved molecular defence systems that both limit the formation of ROS and promote its removal (Alscher et al., 22). The plant enzymatic defences include antioxidant enzymes such as the phenol peroxidase (POX), ascorbate peroxidase (APX), glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT), which together with other enzymes of the ascorbate glutathione cycle promote the scavenging of ROS (Cavalcanti et al., 24). POX is widely distributed in all higher plants and protects cells against the destructive influence of H 2 O 2 by catalyzing its decomposition through oxidation of phenolic and enodiolic co-substrates (Lin and Kao, 22). SOD, considered the first line of defence against ROS (Gomez et al., 24), catalyzes the dismutation of O 2 to H 2 O 2 and molecular oxygen. CAT is present in the peroxisomes of nearly all aerobic cells, and, to a lesser extent, in mitochondria (Shigeoka et al., 22); it is virtually absent in chloroplast (Dionisio-Sese and Tobita, 1998). It can protect the cell from H 2 O 2 by catalyzing its decomposition into O 2 and H 2 O. Some physiological aspects still remain unknown regarding the effectiveness of the POX CAT SOD system as a protective mechanism (Cavalcanti et al., 24). Rather than seeing increased guaiacol- POX activity as merely related to elimination of toxic H 2 O 2 per se (Zheng and Van Huystee, 1992; Dionisio-Sese and Tobita, 1998), some authors have raised the question that perhaps the increased guaiacol-pox activity is more intrinsically associated with a higher lignification process leading to stunted plant growth in an acclimation process to salt stress. Furthermore, the response of CAT activity under osmotic stress has been frequently contradictory. Accordingly, some works have shown enhanced CAT activity (Grosset et al., 1994; Vaidyanathan et al., 23), whereas others have reported a salt-induced down-regulation (Foyer and Noctor, 2; Shim et al., 23). Another controversial point is the lack of conclusive information on the efficiency of oxidative enzymes during the recovery from osmotic stress (Jung, 24). In leaves of salt-stressed plants, chloroplasts are especially prone to generate ROS because of an imbalance between O 2 production during the photochemical phase and the low rates of NADP + generated by Calvin cycle reactions due to increases in stomata resistance to CO 2 assimilation. At this point, the over-reduction of the overall photosynthesis process becomes inevitable (Badawi et al., 24) and the formation of ROS is exceptionally favored. Moreover, in C3 plants, these oxygen species are generated in the photorespiration process, which is significantly enhanced under salt stress conditions (Foyer and Noctor, 2). This is supported by reports demonstrating that the oxygenase activity of Rubisco is increased remarkably under these conditions (Sivakumar et al., 2; Vaidyanathan et al., 23). On the other hand, photorespiration can also generate metabolites such as glycine that can be used in other metabolic pathways, such as a provision for the synthesis of glutathione (Noctor et al., 1997, 1998, 1999). Also, it has been suggested that photorespiration is important in electron flow maintenance, which prevents photoinhibition under stress conditions (Wingler et al., 2) and is a physiologically advantageous process in water stress conditions, where stomatal closure may decrease the availability of intracellular CO 2 (Noctor et al., 22).

3 Salt induced oxidative response in cowpea roots and leaves 593 Although the oxidative response of leaves has been well studied, root response is quite unexplored in the literature. Also, there is a notable lack of studies devoted to understanding oxidative responses (signalizing pathways, mechanisms of damage and repair). The coordination among enzymatic activities, antioxidant substrate flux, and gene expression in roots might be different from that of leaves (Ren et al., 1999), even though these two organs share almost the same enzymatic machinery. In addition, it is widely known that oxidative metabolism is essentially redundant in higher plants (Mittler, 22). Thus, little is known about the integration and functioning of root organelles and the cytosolic antioxidative apparatus, especially under oxidative stress conditions induced by salinity or a water deficit. Under salt stress conditions, the root mitochondrial electron transport might be disrupted, promoting O 2 accumulation in a manner similar to that from hypoxia conditions. This reactive anion is rapidly converted to H 2 O 2 by spontaneous dismutation or by mitochondrial SOD activity. Even though the activity of the alternative oxidase enzyme might alleviate this H 2 O 2 production, these species could diffuse the mitochondrial membrane and act as a signalling molecule or cause cellular damage (Fukao and Bailey-Serres, 24). ROS are also produced by a plasma membrane NAD(P)H oxidase that is involved in ROS-mediated cell growth. These enzymes generate superoxide anions that are transformed into H 2 O 2 by apoplastic SODs. Thus, hydroxyl radicals can be formed from H 2 O 2 in the presence of transition metals such as Cu 2+ or Fe 2+. This radical is extremely reactive, interacting directly with most target biomolecules (Foreman et al., 23). Cowpea is a salt-resistant species capable of surviving under high-salinity conditions, due to its ability to exclude salt from leaves (Silveira et al., 21; Cavalcanti et al., 24) and its ability to maintain high leaf water potential associated with an effective stomatal closure mechanism. We have recently demonstrated that the photosynthetic apparatus of cowpea leaves, as indicated by fluorescence parameters associated with photosystem II activity, is less affected by water deficit. Moreover, severe water stress promotes significant energy dissipation by non-photochemical quenching (Souza et al., 24). In our recent studies, we have established cowpea as an excellent model for studying the underlying mechanisms of salt and drought resistance related to ROS production and antioxidant enzymatic protection. In this work, we have studied a part of the plant enzymatic antioxidant system (SOD, CAT, and POX activities) associated with lipid peroxidation from cowpea leaves and roots subjected to osmotic shock induced by high NaCl level (2 mm NaCl) followed or not by recovery. We hypothesized that POX, CAT and SOD enzymes present distinct responses in roots and leaves during salt stress and recovery. The possible mechanisms of effectiveness and protection against the ROS production by enzymatic action in each organ are discussed. Material and methods Plant material and growth conditions Cowpea seeds [Vigna unguiculata (L.) Walp.] cv. Pitiuba, from Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Brazil, were surface sterilized in 1% (v/v) ethanol for 3 min and.1% (w/v) sodium hypochlorite for 1 min, thoroughly rinsed with distilled water and germinated in pots of 1. L containing sand. After emergence, the seedlings were irrigated daily with 1 4 strength Hoagland s nutrient solution (Hoagland and Arnon, 195) during 12 d. Plants were grown in a greenhouse under natural conditions of a semi-arid region of Brazil, with means of day/night temperature 31/26 1C, 43/85% relative humidity, 12-h photoperiod, and an average of maximum photon flux density of 5 mmol m 2 s 1 measured at plant level (19SA quantum sensor, LI-COR, USA). Treatments and harvest A set of homogenous plants (n ¼ 96), at the first fully expanded trifoliate leaf stage, was distributed randomly and divided in three groups (control, salttreated and recovery). These plants were submitted to following treatments: (1) 2 mm NaCl prepared in the nutrient solution daily applied during six consecutive days (salt treatment); (2) 3-d 2 mm NaCl treatment followed by intensive pot washings with tap water until the electric conductivity (E.C) of percolate reached the same level of control and subsequent irrigation with the nutrient solution up to 7% of pot capacity similar to control plants (recovery treatment); (3) watering with the nutrient solution up to 7% of pot capacity during 6 d (control). At each time, a sample of the plants (six plants per treatment), was randomly taken for analysis. Control and salttreated plants were harvested daily during six consecutive days whereas those under the recovery treatment at 1, 2 and 3 d after NaCl withdrawal. Prior to each harvesting, at 8: a.m, the plants

4 594 were transferred to a controlled growth room (27 1C, 72% relative humidity and 23 mmol m 2 s 1 photon flux density) for 2 h. Next, the leaves (the mature trifoliate leaf) and terminal root segments (3 5 cm length) were sampled, frozen in liquid nitrogen and immediately utilized for biochemical determinations. Determination of leaf and root relative growth and Na + content Leaf and root relative growth were daily calculated as the dry mass produced by stressed and recovered plants in relation to that of untreated control all harvested at the same time (% of control). The leaf and root Na + concentration was determined by flame photometry as previously described (Silveira et al., 21). Determination of leaf and roots lipid peroxidation (thiobarbituric acid-reactive substances TBARS) The level of lipid peroxidation was determined in terms of TBARS concentration as described in Cakmak and Horst (1991), with minor modifications. One gram of fresh leaf or root segment was homogenized in 3 ml of 1.% (w/v) TCA at 4 1C. The homogenate was centrifuged at 2,g for 15 min and.5 ml of the supernatant obtained was added to 3 ml of.5% (v/v) TBA in 2% TCA. The mixture was incubated at 95 1C in a shaking water bath for 5 min, and the reaction stopped by cooling the tubes in ice water bath. Then, the samples were centrifuged at 9g, 1 min, and the absorbance of the supernatant read at 532 nm. The value for non-specific absorption at 66 nm was subtracted. The concentration of TBARS was calculated using the absorption coefficient of 155 mm 1 cm 1 (Cakmak and Horst, 1991). The results were expressed as percentage (%) of control. Enzyme assays Leaf and roots fresh material (1. g) was homogenized with a mortar and pestle in 3 ml of ice-cold 1 mm K-phosphate buffer ph 6.8, containing.1 mm EDTA for 5 min. After filtration in cheesecloth the homogenate was centrifuged at 16,g for 15 min and the supernatant used as the source of enzymes. All the steps were carried out at 4 1C. Addition of serine- and cysteine proteinase inhibitors (1 mm PMSF+1 mg/ml aproptinin) in the extracting buffer did not alter the results of enzyme activities measured in both control and F.R. Cavalcanti et al. salt-treated leaves when the soluble proteins were extracted in the absence of these proteinase inhibitors. Thus, the inhibitors were omitted from the extracting buffer. The activity of guaiacol peroxidase (POX, EC ) was determined by adding 25 ml of the crude enzyme preparation to 2 ml of a solution containing 5 mm potassium phosphate buffer, ph 6.8, 2 mm guaiacol and 2 mm H 2 O 2. After incubation at 3 1C for 1 min, the reaction was stopped by adding.5 ml of 5% (v/v) H 2 SO 4 and the absorbance was read at 48 nm (Urbanek et al., 1991). One POX activity unit (AU) was defined as the change of 1. absorbance unit per milliliter of enzymatic extract and expressed as unit of enzyme activity per gram fresh matter per minute (AU g 1 FM min 1 ). CAT (EC ) activity was determined by adding 5 ml of enzymatic extract to 3 ml of a solution containing 5 mm potassium phosphate buffer, ph 7. and 2 mm H 2 O 2 and by measuring the decrease in absorbance at 24 nm, at 3 1C (Havir and Mchale, 1987). The enzyme activity was calculated using the molar extinction coefficient of mm 1 cm 1 and expressed as mmol H 2 O 2 oxidized g 1 FM min 1. The activity of SOD (EC ) was determined by adding 5 ml of the enzymatic extract to a solution containing 13 mm L-methionine, 75 mm p-nitro blue tetrazolium chloride (NBT), 1 mm EDTA and 2 mm riboflavin in a 5 mm potassium phosphate buffer, ph 7.8. The reaction took place in a chamber under illumination of a 3 W fluorescent lamp at 25 1C. The reaction was started turning the fluorescent lamp on and stopped 5 min later turning it off (Van Rossun et al., 1997). The blue formazane produced by NBT photoreduction was measured by the increase in the absorbance at 56 nm. Control reaction mixture had no enzyme extract. The blank solution had the same complete reaction mixture but it was kept in the dark. One SOD AU was defined as the amount of enzyme required to inhibit 5% of the NBT photoreduction in comparison with tubes lacking the plant extract. Because the soluble protein values did not show significant alterations, between the different treatments during the whole experimental period, the enzymatic activities were expressed in dry matter (DM) basis (AU g 1 DM min 1 ). For better analysis comprehensibility, all the enzymatic activity data were plotted as percentage (%) of control. Experimental design and statistic analysis A completely randomized design was used with three principal treatments (salt treatment, recovery

5 Salt induced oxidative response in cowpea roots and leaves 595 and control) and six harvesting time for the salt and control and three harvesting for recovery treatment. Six replicates per treatment were utilized and an individual plant represented a replicate. The original data were analyzed for significance by ANOVA and the data means7sd representation in the figures at each time harvesting being in terms of control per cent. The experiment was replicated twice and the significant difference between means was compared by the Tuckey test at 5% probability. Results In the present study, cowpea plants were exposed daily to a high NaCl level (2 mm) on the root medium, followed or not by recovery (salt withdrawal) after 3 d of salt treatment. Under the experimental conditions, leaf and root DM from untreated plants increased linearly over the experimental period (data not shown). However, the relative growth rate of both leaves and roots was almost ceased in salt-stressed plants just after 1 d of NaCl exposure, which can be observed by the linear decrease in the dry mass expressed as percent of control (Fig. 1A, B). The recovery treatment (complete NaCl withdrawal from the root medium) restored the leaf and root turgor, as indicated by visual appearance and recovery in the relative water content (data not shown), but induced only small increases in DM after 3 d, when contrasted to the salt-stressed plants (Fig. 1A, B). Moreover, when the root and leaf DM of the recovery treatment were compared to untreated plants (in % of control basis), it corresponded to just about 6% and 55%, respectively, after 3 d of NaCl withdrawal. Thus, the relative effects of NaCl treatment on the reduction and recovery in leaf and root growth, during the experimental period, were proportionally similar. The Na + accumulation pattern between leaves and roots of salt-treated plants differed considerably. Although leaf Na + have accumulated progressively Figure 1. Relative changes in (A) leaf dry mass accumulation; (B) root dry mass accumulation; (C) leaf Na + content; and (D) root Na + content of salt-stressed (K K) and salt-recovered (J J) cowpea plants during the experimental period. Salt withdrawal was performed at 3 d after salt treatment (2 mm NaCl). All results are expressed as % of salt untreated control. The ranges of control during the 6 d were: Leaf DM ¼ g DM plant 1 ; root DM ¼ g DM plant 1 ; leaf Na + content ¼ mmol Na + kg 1 DM; and root Na + content ¼ mmol Na + kg 1 DM. Bars indicate SD (n ¼ 6). % of control % of control % of control % of control Leaf DM (A) Root DM (B) Leaf Na* (C) Root Na* (D) Days

6 596 F.R. Cavalcanti et al. with the plant exposure to NaCl, it reached only 5 mmol Na + per kg of tissue water (5 mm); this was a 4-fold lesser concentration than the one supplied to the rooting medium. The root Na + concentration values were higher than those from leaves, reaching a maximum value of 87 mmol per kg of tissue water on the third day of treatment (Fig. 1D); afterward, the root Na + content slightly decreased. The recovery treatment (salt withdrawal) applied on the third day provoked a complete removal of Na + and Cl from the root medium, as indicated by the E.C values ( ds m 1 ). This E.C value was similar to that from the control plants ( ds m 1 ). Moreover, this recovery treatment induced a rapid and prominent decrease in Na + concentration in both leaves and roots, reaching values close to those of control plants. Thus, the recovery was able to relieve the osmotic pressure from the root medium and to reduce the excess of Na + ions within the plant. The extreme decrease in the relative growth rate of salt-treated leaves (expressed as % of control, Fig. 1A) was inversely related to an increase in the lipid peroxidation levels, measured by the TBARS production (Fig. 3A). Actually, the leaf TBARS content increased progressively, between the second and the sixth day of salt-treatment, when it reached a 1.5-fold higher value than that of control plants. The NaCl withdrawal did not provide significant (po:5) restoration in the leaf lipid peroxidation (Fig. 2A). In contrast, the TBARS level of the salt-treated roots was slightly reduced until the fourth day, and then abruptly decreased to only 36% of the control values at the end of the experiment (Fig. 2B). Interestingly, after 1 d of salt withdrawal, the root TBARS content of the prestressed/recovered plants was restored to similar control level (Fig. 2B). The SOD activity has showed distinct responses between leaves and roots. Indeed, in leaves, the SOD activity did change neither by NaCl presence nor after NaCl withdrawal, in the whole experimental period (Fig. 2C). In contrast, the root SOD activity was progressively reduced, in NaCl treatment, reaching 45% of control at day 6. On the other hand, in a similar Figure 2. Relative changes in (A) leaf lipid peroxidation; (B) root lipid peroxidation; (C) leaf SOD activity; and (D) root SOD activity of salt-stressed (K K) and salt-recovered (J J) cowpea plants during the experimental period. Salt withdrawal was performed at 3 d after salt treatment (2 mm NaCl). All results are expressed as % of saltuntreated control. The ranges of control during the 6 d were: Leaf TBARS ¼ nmol g 1 DM; root TBARS ¼ nmol g 1 DM; leaf SOD ¼ AU g 1 DM min 1 ; and root SOD ¼ AU g 1 DM min 1.BarsindicateSD(n ¼ 6). % of control % of control % of control % of control Leaf TBARS (A) Root TBARS (B) Leaf SOD (C) Root SOD (D) Days

7 Salt induced oxidative response in cowpea roots and leaves 597 manner to the root TBARS response, the root SOD activity exhibited a rapid and remarkable recovery after salt withdrawal, attaining 8% of the control values (Fig. 2D). Leaves and roots also presented distinct responses to salinity in relation to CAT activity (Fig. 3A, B). In leaves, the salt stress caused a rapid and prominent decrease in CAT activity. Indeed, after 2 d of salt treatment, leaf CAT activity was reduced to 52% of the control values. The CAT activity levels of the salt-treated plants did not present any changes during the rest of the experimental period. Furthermore, salt withdrawal from the root medium did not induce any recovery in leaf CAT activity (Fig. 3B). In contrast to the leaves, root CAT activity did not show salt-dependent alterations at any time. As a consequence, the NaCl withdrawal did not impact enzyme activity. In contrast to the TBARS content and SOD and CAT activities, POX activity response from leaves and roots of salt-treated plants were similar (Fig. 3C, D). In both organs, NaCl significantly induced POX activity, mainly after the third day of treatment. It is important to note that the NaCl withdrawal rapidly reversed these increases in the POX activity of both leaves and roots. Thus, taken together, these results strongly suggest that roots and leaves of cowpea plants differed greatly in their responses to oxidative stress when exposed to a high NaCl level. % of control % of control Leaf POX (A) Root POX (B) Leaf CAT (C) Discussion The accumulation of harmful ROS depends on an imbalance between the rates of production and elimination through several biochemical and chemical reactions. To date, it is still not known which is the critical level (threshold) for a specific ROS type (H 2 O 2,O 2 or d OH), in a particular cell, tissue or organ, of a determined plant species or cultivar. This threshold would be sufficient to disturb the ROS homeostasis, either modulating stress defence pathways or causing cellular damages (Mittler Figure 3. Relative changes in activities of (A) leaf POX; (B) root POX; (C) leaf CAT; and (D) root CAT of saltstressed (K K) and salt-recovered (J J) cowpea plants during the experimental period. Salt withdrawal was performed at 3 d after salt treatment (2 mm NaCl). All results are expressed as % of salt untreated control. The ranges of control during the 6 d were: Leaf POX ¼ UA g 1 DM min 1 ; root POX ¼ AU g 1 DM min 1 ; leaf CAT ¼ mmol H 2 O 2 g 1 DM min 1 ; and root CAT ¼ mmol H 2 O 2 g 1 DM min 1. Bars indicate SD (n ¼ 6). % of control % of control Root CAT (D) Days

8 598 et al., 24). In this study, the growth rates of both cowpea leaves and roots exposed to a NaCl-induced osmotic shock were intensely affected. Also, the complete salt withdrawal from the root medium only induced a slight recovery in the DM accumulation of both cowpea organs (Fig. 1A, B). In spite of the similar effect of NaCl and recovery treatments on the growth of leaves and roots, however, the oxidative response presented by these organs was extraordinarily divergent. In fact, taking the TBARS level as a good indicator of oxidative damage to a particular tissue (Dudda et al., 1996; Gechev et al., 22), our data clearly indicate that the responses presented by these organs were very distinct. The cowpea leaves presented a common response to salt-induced oxidative damages, i.e. a continued increase in the TBARS level in function of exposure time (Cavalcanti et al., 24). Overproduction of ROS in chloroplasts and plant peroxisomes under osmotic and salt stress has been widely suggested as the major contributor to oxidative damage in leaves (Willekens et al., 1997; Foyer and Noctor, 23). It is primarily associated with the stress-induced stomatal closure that, in turn, causes a decrease in the CO 2 /O 2 ratio in the chloroplasts (Wingler et al., 2; Cavalcanti et al., 24). It has been demonstrated that this CO 2 /O 2 ratio reduction in leaves inhibits CO 2 fixation, increasing the rate of ROS formation by enhancing electron leakage to oxygen molecules, and also, therefore, increasing the photorespiration process (Wingler et al., 2; Foyer and Noctor, 23). We have recently reported that salt and water stress induce a rapid stomata closure in cowpea leaves (Souza et al., 24). This increased ROS production in salt-stressed cowpea leaves then may have caused an intense lipid peroxidation, as indicated by the increase in the TBARS level (Fig. 2A). The slight recovery tendency in the TBARS content after 3 d of salt withdrawal was reasonably unexpected, since cowpea is a water-stress-resistant species in photosynthetic apparatus stability terms (Souza et al., 24). Given the fact that the leaf K + leakage levels (considered an indirect estimate of the membrane damage) were also slightly recovered (data not shown), it is possible to speculate that most of the oxidative damage took place on the leaf plasmallema, but not in the chloroplast membranes. Since there must be a peroxisomal H 2 O 2 accumulation due to the increased photorespiration associated with a saltinduced down-regulation of the CAT activity (Fig. 3C), it might be possible that a significant portion of this ROS from the peroxisomes migrated to the leaf cytosol, contributing to the generation F.R. Cavalcanti et al. of hydroxyl radicals and causing lipid peroxidation in plasmallema (Dhindsa et al., 1981). In contrast to the leaves, the salt-treated plant roots presented an uncommon response in terms of lipid peroxidation levels, assessed by the TBARS content. During the first 3 d of the salt treatment, the root TBARS level had suffered minor alterations (decrease of 15%), while between the third and the sixth days it has abruptly decreased, from 86% to 36% of the respective control values (Fig. 2B). Usually, salt or osmotic stress induces increases in the TBARS content (Chaparzadeh et al., 24). Although the abiotic-stress-induced TBARS reduction is poorly reported in the literature, there are some reports that show decreases in root lipid peroxidation under salt stress conditions (Azevedo- Neto et al., 25). Moreover, the root TBARS level presented a rapid and prominent recovery after 2 d of salt removal (compared to the stressed roots), in contrast to that observed on the leaves (Fig. 2A). It is important to note that the visual morphological aspects of the cowpea salt-stressed roots (wilting and turgor lost) indicate that they were severely injured. However, on the third day of the recovery treatment, the emission of young hairy roots was observed (data not shown). The osmotic and/or ionic effects of the Na + and Cl might have directly affected the SOD protein integrity and subsequently caused reduction in the root SOD activity as demonstrated by Hernandez et al. (1994), working with cowpea leaf protoplasms submitted to different NaCl concentrations. Besides, the virtual cessation in transpiration of saltstressed cowpea (Cavalcanti et al., 24) might have caused a transient hypoxia into root tissues due to excess water in the root medium and a consequent limitation in O 2 availability. Hypoxia conditions severely reduce respiratory activity and, in consequence, mitochondrial ROS production in plants (Foyer and Noctor, 2). However, some important questions concerning to the root oxidative metabolism remain unanswered. Why did the root TBARS level abruptly decrease only after the fourth day of salt treatment? Why did the prestressed/recovered roots present a rapid and prominent recovery in the TBARS level when compared to control? It could be speculated that on the fourth day, there were profound metabolic alterations in the membrane fatty acid composition, which are the main targets of lipid peroxidation and TBARS formation. Unfortunately, these are quite unexplored approaches in the current literature and nothing was found about these questions. The contrasting CAT activities between roots and leaves of salt-stressed and recovered cowpea

9 Salt induced oxidative response in cowpea roots and leaves 599 plants might be explained by the marked differences found among the types of cellular and organelle metabolism existing in those organs. Moreover, the widely studied CAT isoform is localized in the peroxisomes, and there is still an open question about the existence of mitochondrial and cytosolic isoforms of this enzyme (Mfller, 21). In salt-stressed leaves, the higher amount of H 2 O 2 production is mainly localized in peroxisomes, especially in C3 plants (Willekens et al., 1997) like cowpea, due to high rates of photorespiration. The peroxisomal CAT protein is very sensitive to salt and high temperatures stress (Hertwig et al., 1992; Foyer and Noctor, 2) probably because an imbalance that occur between its synthesis and degradation. Some reports have demonstrated that leaf CAT is also sensitive to high radiation levels (Polidoros and Scandalios, 1997) such as the one imposed on cowpea plants in this study. On the other hand, the absolute absence of recovery in leaf CAT activity in the pre-stressed/ recovered plants, even after 3 d, suggests that the enzyme suffered irreversible damage to its structure and/or that very low rates of de novo synthesis occurred. No salt-induced alterations were observed on root CAT activity, even after salt withdrawal (Fig. 3D). These are surprising and unexpected results. Given that the total SOD activity had suffered a salt-induced decrease, a lower H 2 O 2 production would have been expected. Therefore, the in vitro total CAT activity, determined in the present work (under a saturating concentration of H 2 O 2 substrate in reaction medium), might reflect a constitutive level of the enzyme not dependent on salt treatment or of a salt-induced gene expression. In this context, our results are consistent, showing a salt-independent root CAT activity, even under a recovery treatment, contrasting with the observed response of leaves, where the CAT activity was very sensitive to salt exposure. In conclusion, cowpea roots and leaves present distinct mechanisms of response to lipid peroxidation and CAT and SOD activities against the oxidative stress during salt stress and recovery. However, these differential responses and/or oxidative damages were not related to the growth reduction. Acknowledgments To Fundac-ão Cearense de Apoio à Pesquisa (FUNCAP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support. J.A.G. Silveira is a CNPq fellowship honored researcher and F.R. Cavalcanti is a CNPq/ FUNCAP fellowship honored researcher. References Alscher RG, Erturk N, Heath LS. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot 22;53: Azevedo-Neto AD, Prisco JT, Eneas-Filho J, Rolim Medeiros JV, Gomes-Filho E. Hydrogen peroxide pretreatment induces salt-stress acclimation in maize plants. J Plant Physiol 25;162: Badawi GH, Yamauchi Y, Shimada E, Sasaki R, Kawano N, Tanaka K, et al. Enhanced tolerance to salt stress and water deficit by overexpressing superoxide dismutase in tobacco (Nicotiana tabacum) chloroplasts. Plant Sci 24;166: Cakmak I, Horst WJ. Effect of aluminum on lipidperoxidation, superoxide-dismutase, catalase, and peroxidase-activities in root-tips of soybean (Glycine max). Physiol Plant 1991;83: Cavalcanti FR, Oliveira JTA, Martins-Miranda AS, Viegas RA, Silveira JAG. Superoxide dismutase, catalase and peroxidase activities do not confer protection against oxidative damage in salt-stressed cowpea leaves. New Phytol 24;163: Chaparzadeh N, D Amico ML, Khavari-Nejad RA, Izzo R, Navari-Izzo F. Antioxidative responses of Calendula officinalis under salinity conditions. Plant Physiol Biochem 24;42: Dhindsa RS, Plumbdhindsa P, Thorpe TA. Leaf senescence correlated with increased levels of membranepermeability and lipid-peroxidation, and decreased levels of superoxide-dismutase and catalase. J Exp Bot 1981;32: Dionisio-Sese ML, Tobita S. Antioxidant responses of rice seedlings to salinity stress. Plant Sci 1998;135:1 9. Dudda A, Herold M, Holzel C, Loidl-Stahlhofen A, Jira W, Mlakar A. Lipid peroxidation, a consequence of cell injury? S Afr J Chem 1996;49: Foreman J, Demidchik V, Bothwell JHF, Mylona P, Miedema H, Torres MA, et al. Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 23;422: Foyer CH, Noctor G. Oxygen processing in photosynthesis: regulation and signalling. New Phytol 2;146: Foyer CH, Noctor G. Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiol Plant 23; 119: Fukao T, Bailey-Serres J. Plant responses to hypoxia is survival a balancing act? Trends Plant Sci 24;9: Gechev T, Gadjev I, Van Breusegem F, Inze D, Dukiandjiev S, Toneva V, et al. Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes. Cell Mol Life Sci 22;59:78 14.

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