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Superoxide dismutase, catalase and peroxidase activities do not confer protection against oxidative damage in salt‐stressed cowpea leaves
2004; Wiley; Volume: 163; Issue: 3 Linguagem: Inglês
10.1111/j.1469-8137.2004.01139.x
ISSN1469-8137
AutoresF. R. Cavalcanti, José T.A. Oliveira, A. S. Martins-Miranda, Ricardo Almeida Viégas, Joaquim Albenísio Gomes da Silveira,
Tópico(s)Plant responses to water stress
ResumoNew PhytologistVolume 163, Issue 3 p. 563-571 Free Access Superoxide dismutase, catalase and peroxidase activities do not confer protection against oxidative damage in salt-stressed cowpea leaves Fabio Rossi Cavalcanti, Fabio Rossi Cavalcanti Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, Ceará, BrazilSearch for more papers by this authorJosé Tadeu Abreu Oliveira, José Tadeu Abreu Oliveira Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, Ceará, BrazilSearch for more papers by this authorAparecida Simone Martins-Miranda, Aparecida Simone Martins-Miranda Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, Ceará, BrazilSearch for more papers by this authorRicardo Almeida Viégas, Ricardo Almeida Viégas Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, Ceará, BrazilSearch for more papers by this authorJoaquim Albenísio Gomes Silveira, Corresponding Author Joaquim Albenísio Gomes Silveira Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, Ceará, BrazilAuthor for correspondence: Joaquim Silveira Tel: +55 21 85 2889821 Fax: +55 21 85 2889821 Email: silveira@ufc.brSearch for more papers by this author Fabio Rossi Cavalcanti, Fabio Rossi Cavalcanti Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, Ceará, BrazilSearch for more papers by this authorJosé Tadeu Abreu Oliveira, José Tadeu Abreu Oliveira Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, Ceará, BrazilSearch for more papers by this authorAparecida Simone Martins-Miranda, Aparecida Simone Martins-Miranda Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, Ceará, BrazilSearch for more papers by this authorRicardo Almeida Viégas, Ricardo Almeida Viégas Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, Ceará, BrazilSearch for more papers by this authorJoaquim Albenísio Gomes Silveira, Corresponding Author Joaquim Albenísio Gomes Silveira Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, CP 6020, CEP 60451-970, Fortaleza, Ceará, BrazilAuthor for correspondence: Joaquim Silveira Tel: +55 21 85 2889821 Fax: +55 21 85 2889821 Email: silveira@ufc.brSearch for more papers by this author First published: 03 August 2004 https://doi.org/10.1111/j.1469-8137.2004.01139.xCitations: 192AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary • The aim of this study was to determine whether guaiacol peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT) activities are effective in the protection and recovery of cowpea (Vigna unguiculata (L.) Walp.) leaves exposed to a salt-induced oxidative stress. The salt treatment (200 mm NaCl) was imposed during six consecutive days and the salt withdrawal after 3 d (recovery treatment). Control plants received no NaCl treatment. • The salt treatment caused almost complete cessation of leaf relative growth rate in parallel with the transpiration rate. The restriction in leaf growth was associated with a progressive increase in membrane damage, lipid peroxidation and proline content. Salt withdrawal induced a significant recovery in both leaf growth rate and transpiration. Surprisingly, these prestressed/recovered plants showed only a slight recovery in leaf lipid peroxidation and membrane damage. • Leaf CAT activity experienced a twofold decrease only after 1 d NaCl treatment, and salt withdrawal had no effect on its recovery. SOD activity did not change compared with control plants. By contrast, POX activity significantly increased after 1 d NaCl treatment and showed a significant recovery to levels near to those of control. • In conclusion, it appears that the ability of cowpea plants to survive under high levels of salinity is not caused by an operating antioxidant system involving SOD, POX and CAT activities in mature leaves. Introduction Cowpea (Vigna unguiculata) is a species widely cultivated in the semi-arid regions of Africa and Brazil. Fortunately, some varieties of this crop are adapted to cope with several abiotic stresses such as drought, salinity and high levels of temperature and radiation (Silveira et al., 2003b) which, alone or in combination, can induce oxidative damage in the plant (Foyer & Noctor, 2000). The injurious osmotic effects and ionic toxicity caused by salt stress lead to the generation of oxidative stress and/or to inhibition of the system that operates on the re-establishment of homeostasis to control stress damage, repair and detoxification (Hernández & Almansa, 2002). The oxidative stress is characterized by the overproduction of highly active oxygen species (AOS) represented predominantly by superoxide anion (O2−), hydrogen peroxide (H2O2), hydroxyl radical (·OH), and singlet oxygen (1O2). Overproduction of AOS in chloroplasts of plants under drought and salt stress has been described and is suggested to be the major factor responsible for oxidative damage in leaves (Foyer & Noctor, 2003). Plants have defensive mechanisms and utilize several biochemical strategies to avoid damage caused by AOS. Plant enzymatic defences include antioxidant enzymes such as the phenol peroxidase (POX; EC 1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.1), glutathione peroxidase (GPX; EC 1.11.1.9), superoxide dismutase (SOD; EC 1.15.1.1), and catalase (CAT; EC 1.11.1.6) which, together with other enzymes of the ascorbate–glutathione cycle, promote the scavenging of AOS (Hernández et al., 2001). POX is widely distributed in all higher plants and protects cells against the destructive influence of H2O2 by catalysing its decomposition through oxidation of phenolic and endiolic cosubstrates (Dionisio-Sese & Tobita, 1998; Sudhakar et al., 2001; Lin & Kao, 2002). SOD catalyses the dismutation of O2− to H2O2 and molecular oxygen. CAT is present in the peroxisomes of nearly all aerobic cells and virtually absent from chloroplast (Dionisio-Sese & Tobita, 1998). It can protect the cell from H2O2 by catalysing its decomposition into O2 and H2O (Foyer & Noctor, 2000). The biochemical defence system also includes carotenoids, ascorbate, glutathione and tocopherols (Noctor & Foyer, 1998). Furthermore, the amino acid proline, an osmo-solute and cellular protector largely accumulated in several plant species in response to salt and water stress (Silveira et al., 2003a), might act as an AOS scavenger (Alia et al., 2001). Although several works have provided evidence for an effective protector role of the POX–CAT–SOD system against oxidative stress in diverse plant species (Mittler, 2002; Vaidyanathan et al., 2003; Jung, 2004), some physiological aspects remain unknown. First, it is questionable whether the increase in guaiacol-POX activity is related to elimination of H2O2per se, or whether it promotes a higher lignification process which leads to a stunted plant growth as an acclimation process to salt stress (Zheng & Van Huystee, 1992; Dionisio-Sese & Tobita, 1998). Second, the response of CAT activity to osmotic stress has frequently been contradictory. Accordingly, some works have shown enhanced CAT activity (Grosset et al., 1994; Vaidyanathan et al., 2003), whereas others reported a salt-induced downregulation (Foyer & Noctor, 2000; Shim et al., 2003). Another controversial point is the lack of conclusive information on the efficiency of oxidative enzymes during recovery from osmotic stress (Jung, 2004). Cowpea is a salt-tolerant species capable of surviving under high-salinity conditions, mainly because of its ability to exclude Na+ from leaves (Silveira et al., 2001a) and to maintain high leaf water potential associated with an effective stomatal closure mechanism (Souza et al., 2004). It has been demonstrated that stomatal closure reduces the CO2/O2 ratio in leaves and inhibits CO2 fixation, conditions that increase the rate of AOS formation via enhanced leakage of electrons to oxygen (Foyer & Noctor, 2003). Activated oxygen species attack proteins, lipids and nucleic acids, and the degree of damage depends on the balance between the formation of an AOS and its removal by the antioxidative scavenging systems (Hernández & Almansa, 2002). We have recently demonstrated that the photosynthetic apparatus of cowpea leaves, as indicated by the fluorescence parameters associated with photosystem II activity, is less affected by water deficit (Souza et al., 2004). In addition, when subjected to severe water stress its leaves presented significant energy dissipation by nonphotochemical quenching. These observations suggest that cowpea plants provide an excellent model for studying the underlying mechanisms of salt and drought tolerance related to AOS production and antioxidant enzymatic protection. In this work we tested the hypothesis that the activities of POX, SOD and CAT have a role in the protection and recovery of leaves from salt-induced oxidative stress in a salt-tolerant cowpea cultivar (Pitiúba). The activities of the antioxidant enzymes studied showed distinct patterns of response during salt stress and recovery, suggesting that they play different physiological protective roles. Nevertheless, they apparently did not confer significant protection to or recovery from membrane damage and lipid peroxidation. Materials and Methods Plant material and growth conditions Seeds of cowpea [Vigna unguiculata (L.) Walp.] cv. Pitiúba (salt-tolerant), from Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Brazil, were surface-sterilized in 1% (v/v) ethanol for 3 min and 0.1% (w/v) sodium hypochlorite for 1 min, thoroughly rinsed with distilled water and germinated in 1.0 l pots containing sand. After emergence the seedlings were watered daily with quarter-strength Hoagland's nutrient solution (Hoagland & Arnon, 1950) for 12 d. Plants were grown in a glasshouse under natural conditions for the semiarid region of Brazil, with means of day/night temperature 31/26°C; 43/85% relative humidity; 12 h photoperiod; and average maximum photon flux density 870 µmol m−2 s−1 measured at plant level (190SA quantum sensor, Li-Cor, Lincoln, NE, USA). Salt stress and harvest Cowpea plants at the first fully expanded trifoliate leaf stage were subjected to three treatments. (1) 200 mm NaCl prepared in the nutrient solution daily, applied during six consecutive days (salt treatment). (2) 3 d 200 mm NaCl treatment, followed by intensive pot washings with tap water until the electric conductivity of percolate reached the same level of control, and subsequent irrigation with nutrient solution up to 70% of pot capacity, similar to control plants (recovery treatment). (3) Watering with nutrient solution up to 70% of pot capacity during 6 d (control). Control and salt-treated plants were harvested daily during six consecutive days; those under the recovery treatment were harvested at 1, 2 and 3 d after NaCl withdrawal. Before each harvesting (at 08:00 h), plants were transferred to a controlled growth room (27°C, 72% RH, 230 µmol m−2 s−1 photon flux density) for 2 h. Next the leaves (mature trifoliate leaf) were sampled, frozen in liquid nitrogen and immediately utilized for biochemical determinations. Determination of leaf relative growth rate, relative water content, transpiration, membrane damage and Na+ content in leaves Leaf relative growth rate (RGR) was calculated as (W2 − W1)/W1, where W2 − W1 represents the difference between two consecutive leaf dry weight daily measurements. Leaf relative water content (RWC) was determined as described previously (Silveira et al., 2003a). Thirty leaf discs (1.0 cm diameter) were sampled and immediately weighed (FW). Next they were immersed in distilled water in Petri dishes for 7 h at 25°C under a photon flux density of 40 µmol m−2 s−1, blotted on filter paper, and the turgid weight (TW) determined. Discs were dried in an oven at 80°C for 48 h and the dry weight (DW) obtained. The RWC was calculated using the equation: RWC = (FW − DW)/(TW − DW) × 100. Transpiration was measured as the water lost from daily weighing of each pot. Comparable pots, but without plants, were used to correct evaporation. The transpiration rate was expressed as g water g−1 of leaf dry matter per day. Leaf membrane damage was estimated by recording K+ leakage. Twenty leaf discs (1.0 cm diameter) were placed in test tubes containing 10 ml deionized water. Tubes were incubated in a shaking water bath at 25°C for 24 h and the K+ concentration of the medium (K1) measured. Samples were boiled at 100°C for 60 min to release all electrolytes, cooled to 25°C, and the final K+ (K2) measured by flame photometry. The relative K leakage (percentage of membrane damage, MD) was calculated using the formula: MD(%) = K1/K2 × 100. The leaf and root Na+ concentration was determined by flame photometry as previously described (Silveira et al., 2001a). Determination of leaf lipid peroxidation and proline content The level of lipid peroxidation was determined in terms of thiobarbituric acid-reactive substances (TBARS) concentration as described by Carmak & Horst, 1991), with minor modifications. Fresh leaf (1 g) was homogenized in 3 ml 1.0% (w/v) trichloroacetic acid (TCA) at 4°C. The homogenate was centrifuged at 20 000 g for 15 min and 0.5 ml of the supernatant obtained was added to 3 ml 0.5% (v/v) thiobarbituric acid (TBA) in 20% TCA. The mixture was incubated at 95°C in a shaking water bath for 50 min, and the reaction stopped by cooling the tubes in an ice water bath. Then samples were centrifuged at 9000 g for 10 min, and the absorbance of the supernatant read at 532 nm. The value for nonspecific absorption at 600 nm was subtracted. The concentration of TBARS was calculated using the absorption coefficient 155 mm−1 cm−1 (Carmak & Horst, 1991). Proline concentration was determined according to the method of Bates et al. (1973). Enzyme assays Leaf fresh material (1.0 g) was homogenized with a mortar and pestle in 3 ml ice-cold 100 mm K-phosphate buffer pH 6.8 containing 0.1 mm EDTA for 5 min. After filtration through cheesecloth the homogenate was centrifuged at 16 000 g for 15 min and the supernatant used as the source of enzymes. All the steps were carried out at 0–4°C. The addition of serine and cysteine proteinase inhibitors (1 mm phenylmethylsulfonyl fluoride +1 µg ml−1 aproptinin) in the extracting buffer did not alter the results of enzyme activities measured in both control and salt-treated leaves when 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) was determined by adding 25 µl of the crude enzyme preparation to 2 ml of a solution containing 50 mm potassium phosphate buffer pH 6.8, 20 mm guaiacol and 20 mm H2O2. After incubation at 30°C for 10 min, the reaction was stopped by adding 0.5 ml 5% (v/v) H2SO4 and the absorbance was read at 480 nm (Urbanek et al., 1991). One POX unit was defined as the change of 1.0 absorbance unit per ml enzymatic extract, and expressed as units of enzyme activity per g fresh matter per min (UA g−1 FW min−1). Catalase (CAT) activity was determined by adding 50 µl enzymatic extract to 3 ml of a solution containing 50 mm potassium phosphate buffer pH 7.0 and 20 mm H2O2 and measuring the decrease in absorbance at 240 nm and 30°C (Havir & McHale, 1987). Enzyme activity was calculated using the molar extinction coefficient 36 × 103 mm−1 m−1 and expressed as µmol H2O2 oxidized g−1 FW min−1. The activity of SOD was determined by adding 50 µl of the enzymatic extract to a solution containing 13 mm l-methionine, 75 µmp-nitroblue tetrazolium chloride (NBT), 100 µm EDTA and 2 µm riboflavin in a 50 mm potassium phosphate buffer pH 7.8. The reaction took place in a chamber under illumination of a 30 W fluorescent lamp at 25°C. The reaction was started by turning the fluorescent lamp on, and stopped 5 min later by turning it off (Van Rossun et al., 1997). The blue formazane produced by NBT photoreduction was measured as increase in absorbance at 560 nm. The control reaction mixture had no enzyme extract. The blank solution had the same complete reaction mixture, but was kept in the dark. One SOD unit was defined as the amount of enzyme required to inhibit 50% of the NBT photoreduction in comparison with tubes lacking the plant extract, and expressed as units of enzyme activity (AU) g−1 FW min−1. Experimental design and statistical analysis A completely randomized design was used with three principal treatments (salt treatment, recovery, control) and six harvesting times for the salt and control and three harvesting for recovery treatment. Six replicates per treatment were utilized and an individual plant represented a replicate. Data were analysed by anova and means compared by the least significant difference test at the 0.05 level of confidence. Results Cowpea plants were initially exposed to an osmotic shock induced by 200 mm NaCl, followed or not by recovery after 3 d treatment. The salt treatment rapidly caused a cessation of leaf growth, as indicated by the RGR which reached values near to zero after only 1 d treatment (Fig. 1a), paralleled by the transpiration rate (Fig. 1b). Also, leaf RWC decreased significantly after 1 d treatment (Fig. 1c), associated with mild leaf wilting. In contrast, the leaf dry mass production of plants irrigated with an NaCl-free nutrient solution (control) increased linearly as indicated by the almost constant RGR over the experimental period (Fig. 1a). Figure 1Open in figure viewerPowerPoint (a) Leaf dry mass accumulation; (b) leaf transpiration; and (c) leaf relative water content of salt-stressed (closed squares), salt-recovered (closed circles) and control (open circles) cowpea (Vigna unguiculata (L.) Walp.) plants during the experimental period. Arrow, NaCl withdrawal. Bars, SD (n = 6). Although leaf Na+ accumulation occurred progressively as a function of time of exposure to NaCl (Fig. 2a), it reached only approx. 50 mmol Na+ kg−1 tissue water (approx. 50 mm), a value four times lower than that of the root medium. This Na+ concentration in leaf tissue is considered low to induce any toxic effect (Tester & Davenport, 2003). Thus an osmotic shock, rather than ionic toxicity, appears to be the major component of salt stress in cowpea leaves. The root Na+ concentration was higher than that of leaves, and reached a maximum of 85 mmol kg−1 tissue water at day 3 (Fig. 2b). Curiously, afterwards the root Na+ content experienced a slight but significant decrease, suggesting the existence of an effective Na+-exclusion mechanism. Figure 2Open in figure viewerPowerPoint (a) Leaf Na+ content; (b) root Na+ content; and (c) leaf proline content of salt-stressed (closed squares), salt-recovered (closed circles) and control (open circles) cowpea (Vigna unguiculata (L.) Walp.) plants during the experimental period. Arrow, NaCl withdrawal. Bars, SD (n = 6). The recovery treatment applied on the third day was able to promote a complete withdrawal of NaCl from the root medium, as indicated by the electric conductivity values which were similar (1.9 ± 0.2 dS m−1) to those of control plants (2.1 ± 0.2 dS m−1). Moreover, the complete withdrawal of NaCl from the external medium induced a significant recovery in the leaf RGR (Fig. 1a) in parallel with a pronounced recovery in both transpiration (Fig. 1b) and leaf RWC (Fig. 1c) after 3 d salt withdrawal. The recovery treatment also caused a rapid and prominent decrease in the Na+ concentration of both leaf (Fig. 2a) and root (Fig. 2b), reaching values near to that of control plants. Thus the recovery treatment was able to remove the osmotic pressure in the root medium as well eliminating excess Na+ ions within the plant. The osmotic shock induced by NaCl caused a progressive increase in leaf proline concentration up to 4 d treatment, followed by a slight decrease afterwards (Fig. 2c), similar to what was observed in the root Na+ concentration (Fig. 2b). A remarkable increase in leaf membrane damage was observed after 2 d NaCl treatment (Fig. 3a) in parallel with the increase in leaf Na+ concentration (Fig. 2a). Indeed, the K+ leakage reached values approximately 3.6 times higher than control after 6 d treatment. Withdrawal of salt induced a rapid and prominent decrease in leaf proline content parallel to the leaf and root Na+ concentrations and a slight recovery of leaf membrane damage. Figure 3Open in figure viewerPowerPoint (a) Leaf membrane damage; and (b) leaf lipid peroxidation of salt-stressed (closed squares), salt-recovered (closed circles) and control (open circles) cowpea (Vigna unguiculata (L.) Walp.) plants during the experimental period. Arrow, NaCl withdrawal. Bars, SD (n = 6). A similar, though proportionately smaller, trend to that observed for membrane damage was noticed for the membrane lipid peroxidation estimated as the contents of TBARS (Fig. 3b). This increase in membrane lipid peroxidation occurred steadily from 1 d onwards when compared with control plants. Within the experimental period (6 d) it changed from 52.53 to 80.15 ηmol TBARS g−1 FM. The increase in TBARS levels occurred before the changes in membrane damage and leaf Na+ accumulation. Although NaCl withdrawal was of great effectiveness in decreasing the endogenous Na+ level, this effect was not to any marked degree able to promote restoration of both membrane integrity (Fig. 3a) and leaf lipid peroxidation (Fig. 3b). Leaf guaiacol peroxidase (POX) activity increased slightly in control plants during the experimental period. However, under salinity there was a remarkable increase in POX activity from the third day onward as compared with the control. Withdrawal of NaCl diminished leaf POX activity to levels near to those of control at day 6 (Fig. 4a). The decline of POX activity in prestressed/recovered plants was associated with the recovery of both leaf RGR (Fig. 1a) and transpiration (Fig. 1b) that occurred from day 3 onwards, suggesting that POX activity has a bearing on stunted leaf growth. In contrast, CAT activity was significantly decreased (approximately twofold) after only 1 d treatment compared with controls, and remained nearly constant until the end of the experimental period (Fig. 4b). Furthermore, this behaviour was independent of NaCl withdrawal. Activity of leaf SOD did not change significantly (P < 0.05) in the salt-treated and recovered plants compared with controls during the experimental period (Fig. 4c). Thus, similarly to CAT, SOD activity did not show any change after NaCl withdrawal. Figure 4Open in figure viewerPowerPoint Changes in activities of (a) guaiacol-POX; (b) catalase; and (c) SOD in leaves of salt-stressed (closed squares), salt-recovered (closed circles) and control (open circles) cowpea (Vigna unguiculata (L.) Walp.) plants during the experimental period. Arrow, NaCl withdrawal. Bars, SD (n = 6). The NaCl osmotic treatment severity greatly restricted leaf RGR in cowpea (Fig. 1a). However, levels of chlorophylls a and b (data not shown) were not altered; nor was the appearance of any typical visual symptom of salt toxicity noticed, such as necrotic spots and chlorosis in the oldest leaves. These observations suggest that the photosynthetic apparatus of cowpea plants was apparently preserved during the hyperosmotic treatment. Likewise, the fluorescence parameters associated with photosystem II activity were less affected in cowpea plants submitted to water deficit (Souza et al., 2004). Discussion The data presented in the present study suggest that the physiological strategies with regard to leaf growth and water relations exhibited by cowpea plants in response to NaCl-induced osmotic stress were similar to those presented in response to drought stress (Silveira et al., 2001b; Silveira et al., 2003b; Souza et al., 2004). The great similarities of the plant responses to both drought and salt stress have been reported by several authors (for review see Munns, 2002), and the mechanisms overlap (Zhu & Xiong, 2002). Under these stress conditions cowpea leaves rapidly close their stomata leading, in turn, to a severe reduction in dry matter accumulation caused by restriction in CO2 photosynthetic assimilation (Souza et al., 2004). Indeed, under salt treatment the leaf RGR declined rapidly to values near to zero after only 1 d treatment (Fig. 1a). As expected, in the salt-treated plants a prominent decrease was observed in the transpiration rate (Fig. 1b), resulting from the increase in stomatal resistance (Souza et al., 2004). These changes were associated with reduction in the leaf RWC (Fig. 1c), possibly related to a salt-induced water deficit in the leaf tissues. These results support the hypothesis that the major effect of salt stress on cowpea plants is a salt-induced water deficit associated with the oxidative stress caused by the osmotic component of salinity. Indeed, although the leaf Na+ concentration increased almost linearly during the entire experimental period of cowpea exposure to 200 mm NaCl (Fig. 2a), its concentration did not reach toxic levels according to the recent review by Tester & Davenport (2003). As demonstrated previously, cowpea plants appear to employ a strategy of Na+ exclusion that avoids direct toxic effects on their leaves (Silveira et al., 2001a). NaCl withdrawal from day 3 removed practically all the excess of leaf Na+ (Fig. 2a,b) and restored leaf turgor (Fig. 1c). As a consequence, the cowpea leaves significantly recovered their transpiration and RGR (Fig. 1a,b). Surprisingly, leaf membrane integrity, as indicated by the increase in both K+ leakage and leaf TBARS production (Fig. 3a,b), recovered poorly after NaCl withdrawal, indicating that salt-induced oxidative damage to cowpea leaves was quite irreparable, at least at day 3 of recovery. It should be emphasized that all measurements in this present study were performed on mature leaves. In a previous report we have observed that the restoration of leaf growth in drought-prestressed/recovered cowpea plants occurred almost exclusively in new young leaves (Silveira et al., 2001b). These findings suggest that the major recovery strategy triggered in cowpea plants under salt-induced oxidative stress might be related to a rapid growth of the newly developing trifoliate leaves. In this context, our hypothesis is that the recovery and membrane integrity in young leaves might be greater than in mature leaves. This assumption is reinforced by results obtained in leaves of Arabidopsis thaliana exposed to cycles of water deficit/recovery, which showed that mature leaves suffered more oxidative stress than young ones (Jung, 2004). Proline had rapidly and prominently accumulated in the leaves of salt-stressed cowpea plants. However, a complete recovery was observed in prestressed/recovered cowpea leaves, to levels similar to control (Fig. 2c). Similar results were noticed previously when cowpea was exposed to water stress followed by recovery (Silveira et al., 2003b). Interestingly, these changes in proline content occurred in parallel with the changes observed in the root Na+ concentration (Fig. 2b). These results suggest, at least in part, that proline accumulation in cowpea leaves is related to both the injuries caused by the salt-induced osmotic stress and growth restriction (Silveira et al., 2001a). Moreover, the rapid decrease of proline content after recovery might reflect an intense utilization of this amino acid for de novo protein synthesis and utilization as C and N sources for growth recovery (Silveira et al., 2003b). A number of workers have reported that osmotic stress causes severe damage to the membrane integrity of various tissue types in different plant species (Dionisio-Sese & Tobita, 1998; Hernández et al., 2001; Sudhakar et al., 2001). Moreover, membrane integrity is frequently correlated with plant tolerance to salt and osmotic stress. Lipid peroxidation is an effective indicator of cellular oxidative damage, and was estimated here by levels of TBARS. The observed increase in TBARS concentration in stressed plants might indicate extensive lipid peroxidation of cell-membrane components caused by AOS generated by the oxidative stress (Sairam et al., 2002). In cowpea and other plant species, a rise in AOS production may result from stomatal closure, causing a decrease in CO2 concentration inside the chloroplasts. This, in turn, causes a decrease in NADP+ concentration with the concomitant generation of AOS (Foyer & Noctor, 2003; Souza et al., 2004). The results presented here strongly suggest that the biochemical machinery of cowpea leaf tissue was unable to prevent the harmful effects of AOS on membrane integrity. In addition there was poor recovery from these harmful effects after NaCl withdrawal from the root medium, suggesting that the membrane damage in mature leaves is relatively irreversible. Superoxide dismu
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