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Gas exchange and antioxidant response of sweet pepper to foliar urea spray as affected by ambient temperature
Scientia Horticulturae 127 (2011) 334–340
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Gas exchange and antioxidant response of sweet pepper to foliar urea spray as affected by ambient temperature F.M. del Amor ∗ , P. Cuadra-Crespo Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), C/Mayor s/n, 30150 Murcia, Spain
a r t i c l e
i n f o
Article history: Received 29 July 2010 Received in revised form 29 October 2010 Accepted 29 October 2010 Keywords: Capsicum annuum L. Urea Foliar fertilisation Photosynthesis Reactive oxygen species
a b s t r a c t This paper analyses the effect of different air temperatures (10, 20 and 30 ◦ C) on the response of sweet pepper plants (Capsicum annuum L. cv. Herminio) to foliar urea applications after growing plants for 20 day with and without nitrogen (N) applied to the growing substrate. Leaf CO2 assimilation, chlorophyll fluorescence, root respiration, lipid peroxidation and antioxidative enzymes were analysed. Spraying plants with urea increased leaf CO2 assimilation of N-deficient plants when applied at 20 or 30 ◦ C, compared with non-sprayed plants. When plants were sprayed with urea at 10 ◦ C chlorophyll fluorescence of leaves was similar to that of plants that were supplied with full N in the nutrient solution. Root respiration was not affected by urea sprays whilst leaf NO3 − concentration was increased by urea but only when it was sprayed at 10 or 20 ◦ C. Lipid peroxidation and ascorbate peroxidase in N-deficient plants were reduced significantly by urea sprays, especially when plants were sprayed at 20 ◦ C with N-limitation in the growing substrate. This study shows that N-limitation in the growing substrate induces a temperature-dependant increase in the activities of antioxidant enzymes in leaves of pepper and applications of foliar urea can be optimised, when applied at the appropriate temperature, to partly replace the N supplied to the roots of sweet pepper. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, farmers need to adapt to new constraints and respond to new challenges with major implications for environmental crop management. Thus, they now have to combine several objectives: achieving optimum yield, limiting production costs to maximise profit, conserving natural resources by limiting the negative impact of crop production on the environment and obtaining crop products with the qualities demanded by the market (Jeuffroy et al., 2002). During the 20th century, anthropogenic contributions doubled the global rate of nitrogen (N) fixation of pre-industrial terrestrial ecosystems, with approximately 60% of these new inputs coming from the application of synthetic N fertilisers (Vitousek et al., 1997). The method of N application (to soil or leaves) greatly influences plant quality, through effects on growth and storage of N (Habib et al., 1993). Therefore, foliar application of fertilisers has potential benefits, including the possibility of supplying nutrients to the plant when soil conditions restrict root uptake or during periods of rapid growth, when requirements may exceed root supply. Urea is a low-cost nitrogenous fertiliser that when sprayed
∗ Corresponding author at: Dpto. de Citricultura y Seguridad y Garantía Alimentaria, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), C/Mayor s/n, 30150 Murcia, Spain. Tel.: +34 968 368585; fax: +34 968 366729. E-mail address: [email protected] (F.M. del Amor). 0304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.10.028
on leaves could effectively reduce N application to the roots and therefore diminish nitrate leaching to groundwater and superficial watercourses (del Amor et al., 2009). Most plants absorb foliar applied urea rapidly and hydrolyse the urea in the cytosol (Witte et al., 2002), whilst the effect of foliar application on the reserve N level is dependent on the N status of the plant (Cheng et al., 2001; Lea-Cox and Syvertsen, 1995) and a relatively large part of reduced N in a plant is associated with enzymes that are required for energy metabolism (photosynthesis, respiration) (De Groot et al., 2003). Foliar uptake, metabolism and translocation of urea are rapid (Klein and Weinbaum, 1985) and the ammonium released by the increased activity of urease may be assimilated in the leaves by Glnsynthetase and transported to other plant structures (Nicoulaud and Bloom, 1996). However, urea can cause damage to plant cells and potentially release superoxide radicals (Witte et al., 2002). Additionally, nutritional stress (deficiency) invokes oxidative stress in plants as one of the early rapid responses (Cakmak and Marschner, 1988). Oxidative stress results in production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide and the hydroxyl radical. These ROS can cause lipid peroxidation and consequently membrane injury, protein degradation, enzyme inactivation and disruption of DNA strands (Allen, 1995). Thus, in order to prevent or alleviate the damage that reactive oxygen species (ROS) may cause in plant tissues, plants utilise a range of antioxidant enzymes that are able to metabolise ROS to less toxic compounds; these include ascorbate peroxidase and catalase
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(Polle, 2001). The alteration of metabolism and growth by N may require the presence of additional defence mechanisms against ROS (Mullineaux and Creissen, 1997). Foliar fertilisation requires higher leaf area index for absorbing applied nutrient solution in sufficient amount and nutrient concentration and day temperature should be optimal to avoid leaf burning (Fageria et al., 2009). Foliar uptake of applied substances can increase with temperature (Baur and Schönerr, 1995). Thus, penetration through cuticles is affected by the temperature at which urea is applied (Knoche et al., 1994). Photosynthetic activity is especially sensitive to stresses caused by unfavourable temperatures; thus, temperature effects on photosynthesis may occur through increased oxygenase activity of Rubisco, as well as impaired photochemical activity due to membrane injury and damage to electron transport chain components (Berry and Björkman, 1980). The interest in studying the optimal application of urea and the plant response, as affected by the growth temperature, stems from the previously reported advantages of foliar application methods, such as rapid and efficient response to the plant demand, a lesser amount of product needed and independence of soil conditions (Yildirim et al., 2007). Thus, this work is aimed especially at the high-energy-demand crops of the greenhouse industry, where it is essential to define an optimal strategy to save fertiliser, under the most-adequate climate conditions, whilst minimising N leaching. Additionally, one aspect still little studied, is the regulation of the activity of the antioxidant enzymes, by the supply of nitrogen to the plant. Therefore, the objectives of this study were (i) to compare the effects of high and low air temperatures on leaf CO2 assimilation and root respiration of sweet pepper with low N-availability in the growing substrate (N-deficiency); (ii) to identify the capability of foliar urea sprays at different application temperatures to compensate for N-limitation in the growing substrate; and (iii) to examine the role of the antioxidant system (catalase and ascorbate peroxidase) under the combined effect of temperature and foliar fertilisation.
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2.2. Gas exchange measurements Net CO2 assimilation was measured at the end of the experimental period, in the youngest fully expanded leaf of each plant, using a CIRAS-2 (PP System, Amesbury, MA, USA) with a PLC6 (U) Automatic Universal Leaf Cuvette. Data were collected (12:00–13:00) from each leaf until a stable reading was seen, and 2 h after last irrigation. Soil (root) respiration was measured after photosynthesis readings, with the CIRAS-2 and the SRC-1 Soil Respiration Chamber. The chamber (volume: 1171 cm3 and area: 78.5 cm2 ) was placed on the substrate at the top of each container, after holding chamber in air to flush out with the ambient CO2 concentration (15 s). Calibration was performed before each measurement, and readings were taken after CO2 stabilization (maximum 60 s). 2.3. Chlorophyll fluorescence Chlorophyll fluorescence was determined with a pulsemodulated fluorometer (model ADC Fim 1500; Analytical Development Company Ltd., U.K.) and the ratio of the variable fluorescence from a dark-adapted leaf (Fv ) to the maximal fluorescence from a dark-adapted leaf (Fm ) was determined. This ratio is the one mostwidely used in research employing the fluorescence technique and is correlated directly with the photochemical efficiency of photosystem II (PSII) (Netto et al., 2002). A special leaf clip holder was allocated to each leaf (similar size and age to those used to measure photosynthesis), to maintain dark conditions, at least 30 min before the reading (one reading per plant). Chlorophyll fluorescence was measured at the end of each temperature treatment. Chlorophyll fluorescence was determined before photosynthesis readings. 2.4. NO3 − concentration
2. Materials and methods
Pepper leaves (similar size and age to those selected for photosynthesis determination) were dried at 65 ◦ C in a heater for 72 h. NO3 − was extracted from ground material (0.4 g) with 20 mL of deionized water. NO3 − was analysed in an ion chromatograph (METROHM 861 Advanced Compact IC; METROHM 838 Advanced Sampler) and the column used was a METROHM Metrosep A Supp7 250/4.0 mm.
2.1. Plant material and growth conditions
2.5. Determination of lipid peroxidation
Sweet pepper plants (Capsicum annuum L. cv. Herminio) were grown in 12-L black containers filled with coconut coir fibre. Plants were irrigated with a modified Hoagland solution (control) with the following composition in mequiv. L−1 ; NO3 − : 12.0; H2 PO4 − : 1.0; SO4 2− : 7.0; K+ : 7.0; Ca2+ : 9.0; Mg2+ : 4.0. Irrigation was supplied by self-compensating drippers (2 L h−1 ) and fresh nutrient solution was applied to avoid salt accumulation, with a minimum of 35% drainage (del Amor and Gómez-López, 2009). Plants were grown in a climate chamber designed by our department specifically for plant research proposes (del Amor et al., 2010), with fully controlled environmental conditions: 70% RH, 16/8 h day/night photoperiod and a photosynthetically active radiation (PAR) of 250 mol m−2 s−1 provided by a combination of fluorescent lamps (Philips TL-D Master reflex 830 and 840) and high-pressure sodium lamps (Philips Son-T Agro). The experiment was carried out at 10, 20 and 30 ◦ C, each time for 20 day. The experiments were initialised with 36-day-old plants. Plants were supplied with the control solution (+N) or with N-deficient solution (0N) with the following composition: mequiv. L−1 : 3.0 H2 PO4 − ; 20.9 SO4 2− ; 8.6 K+ ; 11.0 Ca2+ ; and 4.9 Mg2+ . Leaves on one-half of the plants grown with 0N solution were sprayed completely and homogeneously with foliar urea (15 g L−1 ) two times during the experiment (once a week).
From each of the six plants per treatment, leaves similar in size and age as those used for photosynthesis and chlorophyll fluorescence readings were collected to analyse lipid peroxidation and enzyme activities. Lipid peroxidation was measured as the amount of thiobarbituric acid-reactive substances (TBARS), as determined by the thiobarbituric acid (TBA) reaction (Heath and Packer, 1968). Lyophilised samples (0.1 g) were homogenised in 3 cm3 of 20% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 3500 × g for 20 min. To a 1.5-cm3 aliquot of the supernatant, 1.5 cm3 of 20% (w/v) TCA containing 0.5% (w/v) TBA and 0.15 cm3 of 4% (w/v) BHT in ethanol were added. The mixture was heated at 95 ◦ C for 30 min and then quickly cooled on ice. The contents were centrifuged at 10,000 × g for 15 min, and the absorbance was measured at 532 nm. The value for non-specific absorption at 600 nm was subtracted. The concentration of TBARS was calculated using an extinction coefficient of 155 mM−1 cm−1 (Balestrasse et al., 2006). 2.6. Catalase and ascorbate peroxidase activities Samples (50 g) of fresh leaves (similar in size and age to those used for photosynthesis readings) from each plant were lyophilised and the catalase (CAT) and ascorbate peroxidase (APOX) activities
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Fig. 1. Effect of temperature and foliar urea on net leaf CO2 assimilation (A) and the maximal efficiency of PSII (Fv /Fm ) (B) of sweet pepper plants grown with control (12.5 mM NO3 − ) or N-deficient (0 mM NO3 − ) nutrient solution. Error bars represent the mean ± SE; n = 6, columns with the same letter are not significantly different at P ≤ 0.05 (Tukey’s multiple range test).
were determined from 0.1 g of lyophilised leaves homogenised in 3 cm3 of extraction buffer containing 50 mM phosphate buffer (pH 7.4), 1.0 mM EDTA, 1.0 g PVP and 0.5% (v/v) Triton X-100, at 4 ◦ C. The homogenates were centrifuged at 10,000 × g for 20 min, and the supernatant was used for the assays. The CAT activity was determined in the homogenates by measuring the decrease in absorption at 240 nm in a reaction medium containing 50 mM potassium phosphate buffer (pH 7.2) and 2.0 mM H2 O2 (Cakmak et al., 1993). The APOX activity was measured immediately in lyophilised extracts, as described by Nakano and Asada (1981), using 1.0 cm3 of a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM H2 O2 , 0.5 mM ascorbate and 0.1 mM EDTA. The H2 O2 dependent oxidation of ascorbate was monitored as the decrease in the absorbance at 290 nm (e: 2.8 mM−1 cm−1 ). One unit of APOX forms 1 mol of oxidised ascorbate per minute under the assay conditions. 2.7. Statistical analysis The variables were judged to be distributed normally and to be homocedastic before analysis of variance by the Shapiro–Wilks and Levene tests, respectively. The statistical package Statgraphics centurion v.15 was used. The differences between means were assessed by Tukey’s test, in a multiple comparison procedure at a significance level of 0.05. Data were analysed using a one-way analysis of variance (ANOVA) with six plants per treatment (9 treatments in total): controls (+N nutrient solution, no urea spray, and 3 temperatures), non-sprayed N-deficient plants (0N nutrient solution and 3 temperatures), and urea-sprayed N-deficient plants (0N nutrient solution and 3 temperatures). 3. Results and discussion 3.1. Gas exchange and chlorophyll fluorescence The leaf CO2 assimilation of control plants (non-sprayed and fully supplied with N by irrigation) was reduced dramatically at 10 ◦ C compared with 20 or 30 ◦ C (Fig. 1A). Thus, for control plants, leaf CO2 assimilation was reduced by 38% and 77.5% at 20 ◦ C and 10 ◦ C, respectively, compared with plants grown at 30 ◦ C. This inhi-
bition of photosynthesis at low temperature has been associated with the inactivation of regulatory, thioredoxin-activated enzymes of the Calvin cycle (Sassenrath et al., 1990), the irreversible loss of Rubisco protein (Brüggemann et al., 1992), selective inhibition of end-product synthesis, which results in the accumulation of phosphorylated metabolites and phosphate limitation (Sharkey et al., 1986), and inhibition of phloem export, producing a rapid accumulation of soluble sugars which represses the expression of photosynthetic genes (Jeong et al., 2002). Urea application did not fully compensate for the complete absence of N supply to the roots, as photosynthesis was significantly reduced compared to plants grown with 12 mM NO3 − . However spraying N-deficient plants with urea increased photosynthesis. Castle et al. (2007) found in white clover plants that foliar urea applications increased photosynthesis and leaf area even compared to plants supplied with N to the roots. The withdrawal of N supply to the roots affected photosynthesis when the temperature was 20 ◦ C or higher. When foliar urea was sprayed onto N-deficient plants, photosynthesis was increased by 48.9% and 30.2% at 20 ◦ C and 30 ◦ C, respectively, compared with the non-sprayed plants. Yamasaki et al. (2002) reported that an increase in growth temperature results in an increase in the optimal temperature for photosynthesis. Orbovic et al. (2001) reported that increasing the temperature from 19 to 28 ◦ C resulted in an increased total amount of urea, which diffused through the cuticles in the first 24 h after application of foliar urea; this agrees with our results where foliar fertilisation produced a significant response of photosynthesis, especially at moderate temperature (20 ◦ C). Low temperature (10 ◦ C) reduced the maximal efficiency of photosystem II (Fv /Fm ) (Fig. 1B) compared with the values observed at 20 ◦ C or 30 ◦ C. Our results agree with those of Camejo et al. (2010), who observed no effect on the efficiency of PSII in tomato when leaf temperature was increased to 35 ◦ C. At 10 ◦ C, the Fv /Fm of control leaves was greater than that of N-deficient plants that were not sprayed with urea. Spraying N-deficient plants with urea did not significantly change the Fv /Fm of pepper leaves. Thus, Zilkah et al. (1996) observed that urea-treated leaves of avocado were 2.5-times more tolerant of freezing (−2 ◦ C, 24 h) than untreated leaves, at the same level of senescence.
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Fig. 2. Effect of temperature and foliar urea on root respiration of sweet pepper plants grown with control (12.5 mM NO3 − ) or N-deficient (0 mM NO3 − ) nutrient solution. Error bars represent the mean ± SE; n = 6, columns with the same letter are not significantly different at P ≤ 0.05 (Tukey’s multiple range test).
Urea had no effect on root respiration at any temperature (Fig. 2). Root respiration was reduced dramatically at 10 ◦ C compared to plants grown at 20 ◦ C and 30 ◦ C. Additionally, at 20 and 30 ◦ C root respiration of control plants was greater than for plants sprayed with urea. Various factors affect soil respiration rate, such as soil temperature, soil moisture, root nitrogen concentrations, soil texture and substrate quantity and quality (Buchmann, 2000). Although Luo and Zhou (2006) found that soil respiration increased with N supply, responses of soil respiration to N fertilisation are extremely variable and depend on fertiliser type, loading level and site conditions; this supports our observations that foliar fertilisation did not provide enough N to the roots to maintain respiration at the same level as when N was supplied fully by irrigation.
3.2. NO3 − concentration The leaf NO3 − concentration was increased in control plants when temperature increased from 10 to 20 ◦ C but no effect was found when temperature increased from 20 to 30 ◦ C (Fig. 3). Additionally, when urea was sprayed to N-deficient plants at 10 ◦ C the leaf NO3 − concentration was higher than in control plants, this effect being attenuated as the temperature increased. Thus, at 30 ◦ C no significant difference in this parameter was observed compared with N-limited and non-sprayed plants. Temperature dependence of nitrate uptake has been demonstrated clearly in higher plants (Glass et al., 1990). Temperature stress may lead to direct inhibition of nutrient uptake in plant tissues (Zhao et al., 2008). Thus, low temperature (10 ◦ C) could have limited N uptake by the roots in control plants, compared with plants grown at 20 ◦ C or 30 ◦ C. But foliar urea, despite its relatively low diffusion at low temperature (Orbovic et al., 2001), could have increased leaf nitrate concentration compared to the control plants that received N through the roots. At higher temperature (20 ◦ C), although urea diffused better through the cuticles, the dilution effect (growth) could have reduced nitrate concentration significantly compared to spraying at 10 ◦ C. Zilkah et al. (1987) also reported that leaf N content can be increased rapidly by the foliar application of urea, whilst Bi et al. (2007) found that urea applied in autumn can improve N uptake efficiency, increase N storage and optimise growth. Additionally, Zilkah et al. (1996) observed that N enrichment through the canopy might be preferable to soil application during the winter and early spring, when uptake and mobilisation through the roots are still limited due to water saturation and low soil temperature. At 30 ◦ C, the effect of urea on NO3 − concentration was not significant compared with the non-sprayed plants. This could be due to the rapid decrease of the drying time of droplets with the increase of temperature. High temperature may increase volatilisation of ammonia from urea, impairing penetration: hence, in order to achieve greater effects of urea sprays, very high temperatures should be avoided.
3.3. Lipid peroxidation Lipid peroxidation was increased significantly when temperature was reduced from 30 ◦ C to 10 ◦ C (Fig. 4A). When temperature was reduced from 20 ◦ C to 10 ◦ C, lipid peroxidation was increased by 34.8% in control plants, but no significant effect was found when temperature was reduced from 30 ◦ C to 20 ◦ C. When no N was applied to the roots, lipid peroxidation was significantly increased at 20 ◦ C, compared with control plants, but not at 30 ◦ C. Additionally, foliar urea produced a significant reduction in lipid peroxidation when it was applied at 20 ◦ C, compared with nonsprayed plants under N-limitation. Low temperatures can induce free radical production, and reactive oxygen species and the degree of membrane degradation can be assessed by thiobarbituric acid (TBA), which reacts with the products of lipid degradation, such as malondialdehyde (MDA) (Boonsiri et al., 2007). Therefore, changes in lipid peroxidation levels in a tissue can be a good indicator of the structural integrity of the membranes of plants subjected to low temperature (Posmyk et al., 2005). Our data show that temperature affected lipid peroxidation, due to the increased levels of TBARS, as temperature decreased, the effect of urea being more evident when it was applied at 20 ◦ C. As reported by Gülen et al. (2008), low temperatures induce oxidative stress in tissues and consequently increase lipid peroxidation: this is indicative of some cellular damage in the leaves of sweet pepper grown at 10 ◦ C. Deficiency of N decreased plant growth, biomass production, photosynthesis and the concentrations of chloroplastic pigments in sweet pepper (del Amor, 2006). Our results also demonstrate that lipid peroxidation increased in N-deficient pepper plants. Tewari et al. (2007) suggested that lipid peroxidation was increased in the leaves of plants deficient in N and the relationship between macronutrient deficiency and oxidative stress is obvious. At a moderate temperature (20 ◦ C), foliar urea lessened lipid peroxidation significantly but did not do so at an elevated temperature (30 ◦ C). 3.4. Enzyme activities Increasing temperature decreased CAT and APOX activities in control plants (Fig. 4B and C). These decreases were significant when temperature was increased from 10 ◦ C to 20 ◦ C but no further reductions were observed at 30 ◦ C. In general, the activities of these two enzymes were less affected by temperature than lipid peroxidation (TBARS), and the effect of increasing temperature on the activity of these enzymes in N-root deficient plants was not significant (both sprayed or non-sprayed plants). Consequently, the observed reduction in TBARS (10 ◦ C compared with 30 ◦ C) in these treatments had no influence on the response of the activities of CAT or APOX. However, at 20 ◦ C a differential response was observed and urea significantly reduced APOX activity com-
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Fig. 3. Effect of temperature and foliar urea on leaf NO3 − concentration of sweet pepper plants grown with control (12.5 mM NO3 − ) or N-deficient (0 mM NO3 − ) nutrient solution. Error bars represent the mean ± SE; n = 6, columns with the same letter are not significantly different at P ≤ 0.05 (Tukey’s multiple range test).
pared to N-deficient plants that were not sprayed. At 20 ◦ C, TBARS was effectively reduced compared with the response at 10 ◦ C or 20 ◦ C. The exposure of plants to different adverse environmental conditions causes oxidative stress and, under these conditions, the mechanisms that contribute to de-energisation of photosystems, such as photorespiration and the Mehler reaction, increase the production of H2 O2 and, in some cases, O2 −2 (Perl-Treves and Perl, 2002). Thus, to cope with oxidative stress, plants produce several antioxidant compounds and enzymatic activities; these have been used to evaluate stress responses in plants. Under conditions of prolonged stress, enhanced generation of ROS disturbs the normal redox environment of cells (Apel and Hirt, 2004) and plants subjected to adverse conditions, such as high or low tempera-
ture or nutrient imbalance, lose their function and the balance between producing and quenching active oxygen species can be disturbed, resulting in oxidative damage. O’Kane et al. (1996) found that for Arabidopsis subjected to low temperatures, H2 O2 accumulated in the cells and the APX activities increased. Polesskaya et al. (2004) found that the N form, as well as N deficiency, affected the levels and the time-course of antioxidant enzyme activities in response to low-temperature treatment, and consequently the activity of APOX increased under this N-deficiency. But, in general, CAT and APOX were not affected clearly by the studied range of temperatures; foliar urea had a significant effect on the activity of APOX only at 20 ◦ C. Recently, del Amor et al. (2009) found that the frequency of foliar urea application did not affect APOX activity compared with the control values, but urea application
Fig. 4. Effect of temperature and foliar urea on lipid peroxidation and catalase and ascorbate peroxidase activity in the leaves of sweet pepper plants grown with control (12.5 mM NO3 − ) or N-deficient (0 mM NO3 − ) nutrient solution. Mean ± SE, n = 6, values with the same letter are not significantly different at P ≤ 0.05 (Tukey’s multiple range test).
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significantly reduced APOX activity compared with deficient and non-sprayed plants. Moreover, Tewari et al. (2007) also found that N-deficiency increased APOX activities and these increases correlated well with the increased H2 O2 concentration in N-deficient plants. Our study shows that APOX, but not CAT, activities enhanced the scavenging of H2 O2 , inhibiting the accumulation of reactive oxygen species (ROS) and protecting leaves from peroxidation of lipid membranes and oxidative damage under N stress. The increased antioxidative enzyme activity was particularly effective at counteracting ROS when foliar urea was applied at moderate temperature (20 ◦ C). 4. Conclusion This work has evaluated the feasibility of using foliar urea to partly mitigate the stressful conditions imposed by growth under N-starvation at different temperatures. Our study shows that, for N-deficient plants, foliar urea was especially effective for both increasing leaf NO3 − concentration and reducing lipid peroxidation (membrane degradation) when applied at 20 ◦ C. In conclusion, if foliar urea is used as a crop nutrient-management practice, the ambient temperature should be taken into account to optimise its effectiveness. Acknowledgements This work has been supported by the Instituto Nacional de Investigaciones Agrarias (INIA) through the project RTA200500087-C02. The authors thank Dr. D.J. Walker for revision of the ˜ for his technical written English in the manuscript, and G. Ortuno assistance. Part of this work was also funded by the European Social Fund. References Allen, R.D., 1995. Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol. 107, 1049–1054. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–379. Balestrasse, K.B., Gallego, S.M., Tomaro, M.L., 2006. Aluminium stress affects nitrogen fixation and assimilation in soybean (Glycine max L.). Plant Growth Reg. 48, 271–281. Baur, P., Schönerr, J., 1995. Temperature dependence of the diffusion of organic compounds across plant cuticles. Chemosphere 30, 1331–1340. Berry, J., Björkman, O., 1980. Photosynthetic response and adaptation to temperature in higher plants. Annu. Rev. Plant Physiol. 31, 491–543. Bi, G., Scagel, C.F., Fuchigami, L.H., Regan, R.P., 2007. Rate of nitrogen application during the growing season alters the response of container-grown rhododendron and azalea to foliar application of urea in the Autumn. J. Hortic. Sci. Biotechnol. 82, 753–763. Boonsiri, K., Ketsa, S., Van Doorn, W.G., 2007. Seed browning of hot peppers during low temperature storage. Postharvest Biol. Technol. 45, 358–365. Brüggemann, W., Kooij, T.A.W., Hasselt, P.R., 1992. Long-term chilling of young tomato plants under low light. II. Chlorophyll a fluorescence, carbon metabolism and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. Planta 186, 179–187. Buchmann, N., 2000. Biotic and abiotic factors controlling soil respiration rates in Picea abies stands. Soil Biol. Biochem. 32, 1625–1635. Cakmak, I., Marschner, H., 1988. Enhanced superoxide radical production in roots of zinc-deficient plants. J. Exp. Biol. 39, 1449–1460. Cakmak, I., Strbac, D., Marschner, H., 1993. Activities of hydrogen peroxide scavenging enzymes in germinated wheat seeds. J. Exp. Bot. 44, 127–132. Camejo, D., Nicolás, E., Torres, W., Alarcón, J.J., 2010. Differential heat-induced changes in the CO2 assimilation rate and electron transport in tomato (Lycopersicon esculentum Mill.). J. Hortic. Sci. Biotechnol. 85, 137–143. Castle, M.L., Crush, J.R., Rowarth, J.S., 2007. Effects of foliar and root applied nitrogen on nitrogen uptake and movement in white clover at low temperature. New Zealand J. Agric. Res. 50, 463–472. Cheng, L., Dong, S., Guak, S., Fuchigami, L.H., 2001. Effects of nitrogen fertigation on reserve nitrogen and carbohydrate status and regrowth performance of pear nursery plants. Acta Hortic. 564, 51–62. De Groot, C.C., Van den Boogaard, R., Marcelis, L.F.M., Harbinson, J., Lambers, H., 2003. Contrasting effects of N and P deprivation on the regulation of photosynthesis in tomato plants in relation to feedback limitation. J. Exp. Bot. 54, 1957–1967.
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Gas exchange and antioxidant response of sweet pepper to foliar urea spray as affected by ambient temperature F.M. del Amor ∗ , P. Cuadra-Crespo Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), C/Mayor s/n, 30150 Murcia, Spain
a r t i c l e
i n f o
Article history: Received 29 July 2010 Received in revised form 29 October 2010 Accepted 29 October 2010 Keywords: Capsicum annuum L. Urea Foliar fertilisation Photosynthesis Reactive oxygen species
a b s t r a c t This paper analyses the effect of different air temperatures (10, 20 and 30 ◦ C) on the response of sweet pepper plants (Capsicum annuum L. cv. Herminio) to foliar urea applications after growing plants for 20 day with and without nitrogen (N) applied to the growing substrate. Leaf CO2 assimilation, chlorophyll fluorescence, root respiration, lipid peroxidation and antioxidative enzymes were analysed. Spraying plants with urea increased leaf CO2 assimilation of N-deficient plants when applied at 20 or 30 ◦ C, compared with non-sprayed plants. When plants were sprayed with urea at 10 ◦ C chlorophyll fluorescence of leaves was similar to that of plants that were supplied with full N in the nutrient solution. Root respiration was not affected by urea sprays whilst leaf NO3 − concentration was increased by urea but only when it was sprayed at 10 or 20 ◦ C. Lipid peroxidation and ascorbate peroxidase in N-deficient plants were reduced significantly by urea sprays, especially when plants were sprayed at 20 ◦ C with N-limitation in the growing substrate. This study shows that N-limitation in the growing substrate induces a temperature-dependant increase in the activities of antioxidant enzymes in leaves of pepper and applications of foliar urea can be optimised, when applied at the appropriate temperature, to partly replace the N supplied to the roots of sweet pepper. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, farmers need to adapt to new constraints and respond to new challenges with major implications for environmental crop management. Thus, they now have to combine several objectives: achieving optimum yield, limiting production costs to maximise profit, conserving natural resources by limiting the negative impact of crop production on the environment and obtaining crop products with the qualities demanded by the market (Jeuffroy et al., 2002). During the 20th century, anthropogenic contributions doubled the global rate of nitrogen (N) fixation of pre-industrial terrestrial ecosystems, with approximately 60% of these new inputs coming from the application of synthetic N fertilisers (Vitousek et al., 1997). The method of N application (to soil or leaves) greatly influences plant quality, through effects on growth and storage of N (Habib et al., 1993). Therefore, foliar application of fertilisers has potential benefits, including the possibility of supplying nutrients to the plant when soil conditions restrict root uptake or during periods of rapid growth, when requirements may exceed root supply. Urea is a low-cost nitrogenous fertiliser that when sprayed
∗ Corresponding author at: Dpto. de Citricultura y Seguridad y Garantía Alimentaria, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), C/Mayor s/n, 30150 Murcia, Spain. Tel.: +34 968 368585; fax: +34 968 366729. E-mail address: [email protected] (F.M. del Amor). 0304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.10.028
on leaves could effectively reduce N application to the roots and therefore diminish nitrate leaching to groundwater and superficial watercourses (del Amor et al., 2009). Most plants absorb foliar applied urea rapidly and hydrolyse the urea in the cytosol (Witte et al., 2002), whilst the effect of foliar application on the reserve N level is dependent on the N status of the plant (Cheng et al., 2001; Lea-Cox and Syvertsen, 1995) and a relatively large part of reduced N in a plant is associated with enzymes that are required for energy metabolism (photosynthesis, respiration) (De Groot et al., 2003). Foliar uptake, metabolism and translocation of urea are rapid (Klein and Weinbaum, 1985) and the ammonium released by the increased activity of urease may be assimilated in the leaves by Glnsynthetase and transported to other plant structures (Nicoulaud and Bloom, 1996). However, urea can cause damage to plant cells and potentially release superoxide radicals (Witte et al., 2002). Additionally, nutritional stress (deficiency) invokes oxidative stress in plants as one of the early rapid responses (Cakmak and Marschner, 1988). Oxidative stress results in production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide and the hydroxyl radical. These ROS can cause lipid peroxidation and consequently membrane injury, protein degradation, enzyme inactivation and disruption of DNA strands (Allen, 1995). Thus, in order to prevent or alleviate the damage that reactive oxygen species (ROS) may cause in plant tissues, plants utilise a range of antioxidant enzymes that are able to metabolise ROS to less toxic compounds; these include ascorbate peroxidase and catalase
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(Polle, 2001). The alteration of metabolism and growth by N may require the presence of additional defence mechanisms against ROS (Mullineaux and Creissen, 1997). Foliar fertilisation requires higher leaf area index for absorbing applied nutrient solution in sufficient amount and nutrient concentration and day temperature should be optimal to avoid leaf burning (Fageria et al., 2009). Foliar uptake of applied substances can increase with temperature (Baur and Schönerr, 1995). Thus, penetration through cuticles is affected by the temperature at which urea is applied (Knoche et al., 1994). Photosynthetic activity is especially sensitive to stresses caused by unfavourable temperatures; thus, temperature effects on photosynthesis may occur through increased oxygenase activity of Rubisco, as well as impaired photochemical activity due to membrane injury and damage to electron transport chain components (Berry and Björkman, 1980). The interest in studying the optimal application of urea and the plant response, as affected by the growth temperature, stems from the previously reported advantages of foliar application methods, such as rapid and efficient response to the plant demand, a lesser amount of product needed and independence of soil conditions (Yildirim et al., 2007). Thus, this work is aimed especially at the high-energy-demand crops of the greenhouse industry, where it is essential to define an optimal strategy to save fertiliser, under the most-adequate climate conditions, whilst minimising N leaching. Additionally, one aspect still little studied, is the regulation of the activity of the antioxidant enzymes, by the supply of nitrogen to the plant. Therefore, the objectives of this study were (i) to compare the effects of high and low air temperatures on leaf CO2 assimilation and root respiration of sweet pepper with low N-availability in the growing substrate (N-deficiency); (ii) to identify the capability of foliar urea sprays at different application temperatures to compensate for N-limitation in the growing substrate; and (iii) to examine the role of the antioxidant system (catalase and ascorbate peroxidase) under the combined effect of temperature and foliar fertilisation.
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2.2. Gas exchange measurements Net CO2 assimilation was measured at the end of the experimental period, in the youngest fully expanded leaf of each plant, using a CIRAS-2 (PP System, Amesbury, MA, USA) with a PLC6 (U) Automatic Universal Leaf Cuvette. Data were collected (12:00–13:00) from each leaf until a stable reading was seen, and 2 h after last irrigation. Soil (root) respiration was measured after photosynthesis readings, with the CIRAS-2 and the SRC-1 Soil Respiration Chamber. The chamber (volume: 1171 cm3 and area: 78.5 cm2 ) was placed on the substrate at the top of each container, after holding chamber in air to flush out with the ambient CO2 concentration (15 s). Calibration was performed before each measurement, and readings were taken after CO2 stabilization (maximum 60 s). 2.3. Chlorophyll fluorescence Chlorophyll fluorescence was determined with a pulsemodulated fluorometer (model ADC Fim 1500; Analytical Development Company Ltd., U.K.) and the ratio of the variable fluorescence from a dark-adapted leaf (Fv ) to the maximal fluorescence from a dark-adapted leaf (Fm ) was determined. This ratio is the one mostwidely used in research employing the fluorescence technique and is correlated directly with the photochemical efficiency of photosystem II (PSII) (Netto et al., 2002). A special leaf clip holder was allocated to each leaf (similar size and age to those used to measure photosynthesis), to maintain dark conditions, at least 30 min before the reading (one reading per plant). Chlorophyll fluorescence was measured at the end of each temperature treatment. Chlorophyll fluorescence was determined before photosynthesis readings. 2.4. NO3 − concentration
2. Materials and methods
Pepper leaves (similar size and age to those selected for photosynthesis determination) were dried at 65 ◦ C in a heater for 72 h. NO3 − was extracted from ground material (0.4 g) with 20 mL of deionized water. NO3 − was analysed in an ion chromatograph (METROHM 861 Advanced Compact IC; METROHM 838 Advanced Sampler) and the column used was a METROHM Metrosep A Supp7 250/4.0 mm.
2.1. Plant material and growth conditions
2.5. Determination of lipid peroxidation
Sweet pepper plants (Capsicum annuum L. cv. Herminio) were grown in 12-L black containers filled with coconut coir fibre. Plants were irrigated with a modified Hoagland solution (control) with the following composition in mequiv. L−1 ; NO3 − : 12.0; H2 PO4 − : 1.0; SO4 2− : 7.0; K+ : 7.0; Ca2+ : 9.0; Mg2+ : 4.0. Irrigation was supplied by self-compensating drippers (2 L h−1 ) and fresh nutrient solution was applied to avoid salt accumulation, with a minimum of 35% drainage (del Amor and Gómez-López, 2009). Plants were grown in a climate chamber designed by our department specifically for plant research proposes (del Amor et al., 2010), with fully controlled environmental conditions: 70% RH, 16/8 h day/night photoperiod and a photosynthetically active radiation (PAR) of 250 mol m−2 s−1 provided by a combination of fluorescent lamps (Philips TL-D Master reflex 830 and 840) and high-pressure sodium lamps (Philips Son-T Agro). The experiment was carried out at 10, 20 and 30 ◦ C, each time for 20 day. The experiments were initialised with 36-day-old plants. Plants were supplied with the control solution (+N) or with N-deficient solution (0N) with the following composition: mequiv. L−1 : 3.0 H2 PO4 − ; 20.9 SO4 2− ; 8.6 K+ ; 11.0 Ca2+ ; and 4.9 Mg2+ . Leaves on one-half of the plants grown with 0N solution were sprayed completely and homogeneously with foliar urea (15 g L−1 ) two times during the experiment (once a week).
From each of the six plants per treatment, leaves similar in size and age as those used for photosynthesis and chlorophyll fluorescence readings were collected to analyse lipid peroxidation and enzyme activities. Lipid peroxidation was measured as the amount of thiobarbituric acid-reactive substances (TBARS), as determined by the thiobarbituric acid (TBA) reaction (Heath and Packer, 1968). Lyophilised samples (0.1 g) were homogenised in 3 cm3 of 20% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 3500 × g for 20 min. To a 1.5-cm3 aliquot of the supernatant, 1.5 cm3 of 20% (w/v) TCA containing 0.5% (w/v) TBA and 0.15 cm3 of 4% (w/v) BHT in ethanol were added. The mixture was heated at 95 ◦ C for 30 min and then quickly cooled on ice. The contents were centrifuged at 10,000 × g for 15 min, and the absorbance was measured at 532 nm. The value for non-specific absorption at 600 nm was subtracted. The concentration of TBARS was calculated using an extinction coefficient of 155 mM−1 cm−1 (Balestrasse et al., 2006). 2.6. Catalase and ascorbate peroxidase activities Samples (50 g) of fresh leaves (similar in size and age to those used for photosynthesis readings) from each plant were lyophilised and the catalase (CAT) and ascorbate peroxidase (APOX) activities
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Fig. 1. Effect of temperature and foliar urea on net leaf CO2 assimilation (A) and the maximal efficiency of PSII (Fv /Fm ) (B) of sweet pepper plants grown with control (12.5 mM NO3 − ) or N-deficient (0 mM NO3 − ) nutrient solution. Error bars represent the mean ± SE; n = 6, columns with the same letter are not significantly different at P ≤ 0.05 (Tukey’s multiple range test).
were determined from 0.1 g of lyophilised leaves homogenised in 3 cm3 of extraction buffer containing 50 mM phosphate buffer (pH 7.4), 1.0 mM EDTA, 1.0 g PVP and 0.5% (v/v) Triton X-100, at 4 ◦ C. The homogenates were centrifuged at 10,000 × g for 20 min, and the supernatant was used for the assays. The CAT activity was determined in the homogenates by measuring the decrease in absorption at 240 nm in a reaction medium containing 50 mM potassium phosphate buffer (pH 7.2) and 2.0 mM H2 O2 (Cakmak et al., 1993). The APOX activity was measured immediately in lyophilised extracts, as described by Nakano and Asada (1981), using 1.0 cm3 of a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM H2 O2 , 0.5 mM ascorbate and 0.1 mM EDTA. The H2 O2 dependent oxidation of ascorbate was monitored as the decrease in the absorbance at 290 nm (e: 2.8 mM−1 cm−1 ). One unit of APOX forms 1 mol of oxidised ascorbate per minute under the assay conditions. 2.7. Statistical analysis The variables were judged to be distributed normally and to be homocedastic before analysis of variance by the Shapiro–Wilks and Levene tests, respectively. The statistical package Statgraphics centurion v.15 was used. The differences between means were assessed by Tukey’s test, in a multiple comparison procedure at a significance level of 0.05. Data were analysed using a one-way analysis of variance (ANOVA) with six plants per treatment (9 treatments in total): controls (+N nutrient solution, no urea spray, and 3 temperatures), non-sprayed N-deficient plants (0N nutrient solution and 3 temperatures), and urea-sprayed N-deficient plants (0N nutrient solution and 3 temperatures). 3. Results and discussion 3.1. Gas exchange and chlorophyll fluorescence The leaf CO2 assimilation of control plants (non-sprayed and fully supplied with N by irrigation) was reduced dramatically at 10 ◦ C compared with 20 or 30 ◦ C (Fig. 1A). Thus, for control plants, leaf CO2 assimilation was reduced by 38% and 77.5% at 20 ◦ C and 10 ◦ C, respectively, compared with plants grown at 30 ◦ C. This inhi-
bition of photosynthesis at low temperature has been associated with the inactivation of regulatory, thioredoxin-activated enzymes of the Calvin cycle (Sassenrath et al., 1990), the irreversible loss of Rubisco protein (Brüggemann et al., 1992), selective inhibition of end-product synthesis, which results in the accumulation of phosphorylated metabolites and phosphate limitation (Sharkey et al., 1986), and inhibition of phloem export, producing a rapid accumulation of soluble sugars which represses the expression of photosynthetic genes (Jeong et al., 2002). Urea application did not fully compensate for the complete absence of N supply to the roots, as photosynthesis was significantly reduced compared to plants grown with 12 mM NO3 − . However spraying N-deficient plants with urea increased photosynthesis. Castle et al. (2007) found in white clover plants that foliar urea applications increased photosynthesis and leaf area even compared to plants supplied with N to the roots. The withdrawal of N supply to the roots affected photosynthesis when the temperature was 20 ◦ C or higher. When foliar urea was sprayed onto N-deficient plants, photosynthesis was increased by 48.9% and 30.2% at 20 ◦ C and 30 ◦ C, respectively, compared with the non-sprayed plants. Yamasaki et al. (2002) reported that an increase in growth temperature results in an increase in the optimal temperature for photosynthesis. Orbovic et al. (2001) reported that increasing the temperature from 19 to 28 ◦ C resulted in an increased total amount of urea, which diffused through the cuticles in the first 24 h after application of foliar urea; this agrees with our results where foliar fertilisation produced a significant response of photosynthesis, especially at moderate temperature (20 ◦ C). Low temperature (10 ◦ C) reduced the maximal efficiency of photosystem II (Fv /Fm ) (Fig. 1B) compared with the values observed at 20 ◦ C or 30 ◦ C. Our results agree with those of Camejo et al. (2010), who observed no effect on the efficiency of PSII in tomato when leaf temperature was increased to 35 ◦ C. At 10 ◦ C, the Fv /Fm of control leaves was greater than that of N-deficient plants that were not sprayed with urea. Spraying N-deficient plants with urea did not significantly change the Fv /Fm of pepper leaves. Thus, Zilkah et al. (1996) observed that urea-treated leaves of avocado were 2.5-times more tolerant of freezing (−2 ◦ C, 24 h) than untreated leaves, at the same level of senescence.
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Fig. 2. Effect of temperature and foliar urea on root respiration of sweet pepper plants grown with control (12.5 mM NO3 − ) or N-deficient (0 mM NO3 − ) nutrient solution. Error bars represent the mean ± SE; n = 6, columns with the same letter are not significantly different at P ≤ 0.05 (Tukey’s multiple range test).
Urea had no effect on root respiration at any temperature (Fig. 2). Root respiration was reduced dramatically at 10 ◦ C compared to plants grown at 20 ◦ C and 30 ◦ C. Additionally, at 20 and 30 ◦ C root respiration of control plants was greater than for plants sprayed with urea. Various factors affect soil respiration rate, such as soil temperature, soil moisture, root nitrogen concentrations, soil texture and substrate quantity and quality (Buchmann, 2000). Although Luo and Zhou (2006) found that soil respiration increased with N supply, responses of soil respiration to N fertilisation are extremely variable and depend on fertiliser type, loading level and site conditions; this supports our observations that foliar fertilisation did not provide enough N to the roots to maintain respiration at the same level as when N was supplied fully by irrigation.
3.2. NO3 − concentration The leaf NO3 − concentration was increased in control plants when temperature increased from 10 to 20 ◦ C but no effect was found when temperature increased from 20 to 30 ◦ C (Fig. 3). Additionally, when urea was sprayed to N-deficient plants at 10 ◦ C the leaf NO3 − concentration was higher than in control plants, this effect being attenuated as the temperature increased. Thus, at 30 ◦ C no significant difference in this parameter was observed compared with N-limited and non-sprayed plants. Temperature dependence of nitrate uptake has been demonstrated clearly in higher plants (Glass et al., 1990). Temperature stress may lead to direct inhibition of nutrient uptake in plant tissues (Zhao et al., 2008). Thus, low temperature (10 ◦ C) could have limited N uptake by the roots in control plants, compared with plants grown at 20 ◦ C or 30 ◦ C. But foliar urea, despite its relatively low diffusion at low temperature (Orbovic et al., 2001), could have increased leaf nitrate concentration compared to the control plants that received N through the roots. At higher temperature (20 ◦ C), although urea diffused better through the cuticles, the dilution effect (growth) could have reduced nitrate concentration significantly compared to spraying at 10 ◦ C. Zilkah et al. (1987) also reported that leaf N content can be increased rapidly by the foliar application of urea, whilst Bi et al. (2007) found that urea applied in autumn can improve N uptake efficiency, increase N storage and optimise growth. Additionally, Zilkah et al. (1996) observed that N enrichment through the canopy might be preferable to soil application during the winter and early spring, when uptake and mobilisation through the roots are still limited due to water saturation and low soil temperature. At 30 ◦ C, the effect of urea on NO3 − concentration was not significant compared with the non-sprayed plants. This could be due to the rapid decrease of the drying time of droplets with the increase of temperature. High temperature may increase volatilisation of ammonia from urea, impairing penetration: hence, in order to achieve greater effects of urea sprays, very high temperatures should be avoided.
3.3. Lipid peroxidation Lipid peroxidation was increased significantly when temperature was reduced from 30 ◦ C to 10 ◦ C (Fig. 4A). When temperature was reduced from 20 ◦ C to 10 ◦ C, lipid peroxidation was increased by 34.8% in control plants, but no significant effect was found when temperature was reduced from 30 ◦ C to 20 ◦ C. When no N was applied to the roots, lipid peroxidation was significantly increased at 20 ◦ C, compared with control plants, but not at 30 ◦ C. Additionally, foliar urea produced a significant reduction in lipid peroxidation when it was applied at 20 ◦ C, compared with nonsprayed plants under N-limitation. Low temperatures can induce free radical production, and reactive oxygen species and the degree of membrane degradation can be assessed by thiobarbituric acid (TBA), which reacts with the products of lipid degradation, such as malondialdehyde (MDA) (Boonsiri et al., 2007). Therefore, changes in lipid peroxidation levels in a tissue can be a good indicator of the structural integrity of the membranes of plants subjected to low temperature (Posmyk et al., 2005). Our data show that temperature affected lipid peroxidation, due to the increased levels of TBARS, as temperature decreased, the effect of urea being more evident when it was applied at 20 ◦ C. As reported by Gülen et al. (2008), low temperatures induce oxidative stress in tissues and consequently increase lipid peroxidation: this is indicative of some cellular damage in the leaves of sweet pepper grown at 10 ◦ C. Deficiency of N decreased plant growth, biomass production, photosynthesis and the concentrations of chloroplastic pigments in sweet pepper (del Amor, 2006). Our results also demonstrate that lipid peroxidation increased in N-deficient pepper plants. Tewari et al. (2007) suggested that lipid peroxidation was increased in the leaves of plants deficient in N and the relationship between macronutrient deficiency and oxidative stress is obvious. At a moderate temperature (20 ◦ C), foliar urea lessened lipid peroxidation significantly but did not do so at an elevated temperature (30 ◦ C). 3.4. Enzyme activities Increasing temperature decreased CAT and APOX activities in control plants (Fig. 4B and C). These decreases were significant when temperature was increased from 10 ◦ C to 20 ◦ C but no further reductions were observed at 30 ◦ C. In general, the activities of these two enzymes were less affected by temperature than lipid peroxidation (TBARS), and the effect of increasing temperature on the activity of these enzymes in N-root deficient plants was not significant (both sprayed or non-sprayed plants). Consequently, the observed reduction in TBARS (10 ◦ C compared with 30 ◦ C) in these treatments had no influence on the response of the activities of CAT or APOX. However, at 20 ◦ C a differential response was observed and urea significantly reduced APOX activity com-
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Fig. 3. Effect of temperature and foliar urea on leaf NO3 − concentration of sweet pepper plants grown with control (12.5 mM NO3 − ) or N-deficient (0 mM NO3 − ) nutrient solution. Error bars represent the mean ± SE; n = 6, columns with the same letter are not significantly different at P ≤ 0.05 (Tukey’s multiple range test).
pared to N-deficient plants that were not sprayed. At 20 ◦ C, TBARS was effectively reduced compared with the response at 10 ◦ C or 20 ◦ C. The exposure of plants to different adverse environmental conditions causes oxidative stress and, under these conditions, the mechanisms that contribute to de-energisation of photosystems, such as photorespiration and the Mehler reaction, increase the production of H2 O2 and, in some cases, O2 −2 (Perl-Treves and Perl, 2002). Thus, to cope with oxidative stress, plants produce several antioxidant compounds and enzymatic activities; these have been used to evaluate stress responses in plants. Under conditions of prolonged stress, enhanced generation of ROS disturbs the normal redox environment of cells (Apel and Hirt, 2004) and plants subjected to adverse conditions, such as high or low tempera-
ture or nutrient imbalance, lose their function and the balance between producing and quenching active oxygen species can be disturbed, resulting in oxidative damage. O’Kane et al. (1996) found that for Arabidopsis subjected to low temperatures, H2 O2 accumulated in the cells and the APX activities increased. Polesskaya et al. (2004) found that the N form, as well as N deficiency, affected the levels and the time-course of antioxidant enzyme activities in response to low-temperature treatment, and consequently the activity of APOX increased under this N-deficiency. But, in general, CAT and APOX were not affected clearly by the studied range of temperatures; foliar urea had a significant effect on the activity of APOX only at 20 ◦ C. Recently, del Amor et al. (2009) found that the frequency of foliar urea application did not affect APOX activity compared with the control values, but urea application
Fig. 4. Effect of temperature and foliar urea on lipid peroxidation and catalase and ascorbate peroxidase activity in the leaves of sweet pepper plants grown with control (12.5 mM NO3 − ) or N-deficient (0 mM NO3 − ) nutrient solution. Mean ± SE, n = 6, values with the same letter are not significantly different at P ≤ 0.05 (Tukey’s multiple range test).
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significantly reduced APOX activity compared with deficient and non-sprayed plants. Moreover, Tewari et al. (2007) also found that N-deficiency increased APOX activities and these increases correlated well with the increased H2 O2 concentration in N-deficient plants. Our study shows that APOX, but not CAT, activities enhanced the scavenging of H2 O2 , inhibiting the accumulation of reactive oxygen species (ROS) and protecting leaves from peroxidation of lipid membranes and oxidative damage under N stress. The increased antioxidative enzyme activity was particularly effective at counteracting ROS when foliar urea was applied at moderate temperature (20 ◦ C). 4. Conclusion This work has evaluated the feasibility of using foliar urea to partly mitigate the stressful conditions imposed by growth under N-starvation at different temperatures. Our study shows that, for N-deficient plants, foliar urea was especially effective for both increasing leaf NO3 − concentration and reducing lipid peroxidation (membrane degradation) when applied at 20 ◦ C. In conclusion, if foliar urea is used as a crop nutrient-management practice, the ambient temperature should be taken into account to optimise its effectiveness. Acknowledgements This work has been supported by the Instituto Nacional de Investigaciones Agrarias (INIA) through the project RTA200500087-C02. The authors thank Dr. D.J. Walker for revision of the ˜ for his technical written English in the manuscript, and G. Ortuno assistance. Part of this work was also funded by the European Social Fund. References Allen, R.D., 1995. Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol. 107, 1049–1054. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–379. Balestrasse, K.B., Gallego, S.M., Tomaro, M.L., 2006. Aluminium stress affects nitrogen fixation and assimilation in soybean (Glycine max L.). Plant Growth Reg. 48, 271–281. Baur, P., Schönerr, J., 1995. Temperature dependence of the diffusion of organic compounds across plant cuticles. Chemosphere 30, 1331–1340. Berry, J., Björkman, O., 1980. Photosynthetic response and adaptation to temperature in higher plants. Annu. Rev. Plant Physiol. 31, 491–543. Bi, G., Scagel, C.F., Fuchigami, L.H., Regan, R.P., 2007. Rate of nitrogen application during the growing season alters the response of container-grown rhododendron and azalea to foliar application of urea in the Autumn. J. Hortic. Sci. Biotechnol. 82, 753–763. Boonsiri, K., Ketsa, S., Van Doorn, W.G., 2007. Seed browning of hot peppers during low temperature storage. Postharvest Biol. Technol. 45, 358–365. Brüggemann, W., Kooij, T.A.W., Hasselt, P.R., 1992. Long-term chilling of young tomato plants under low light. II. Chlorophyll a fluorescence, carbon metabolism and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. Planta 186, 179–187. Buchmann, N., 2000. Biotic and abiotic factors controlling soil respiration rates in Picea abies stands. Soil Biol. Biochem. 32, 1625–1635. Cakmak, I., Marschner, H., 1988. Enhanced superoxide radical production in roots of zinc-deficient plants. J. Exp. Biol. 39, 1449–1460. Cakmak, I., Strbac, D., Marschner, H., 1993. Activities of hydrogen peroxide scavenging enzymes in germinated wheat seeds. J. Exp. Bot. 44, 127–132. Camejo, D., Nicolás, E., Torres, W., Alarcón, J.J., 2010. Differential heat-induced changes in the CO2 assimilation rate and electron transport in tomato (Lycopersicon esculentum Mill.). J. Hortic. Sci. Biotechnol. 85, 137–143. Castle, M.L., Crush, J.R., Rowarth, J.S., 2007. Effects of foliar and root applied nitrogen on nitrogen uptake and movement in white clover at low temperature. New Zealand J. Agric. Res. 50, 463–472. Cheng, L., Dong, S., Guak, S., Fuchigami, L.H., 2001. Effects of nitrogen fertigation on reserve nitrogen and carbohydrate status and regrowth performance of pear nursery plants. Acta Hortic. 564, 51–62. De Groot, C.C., Van den Boogaard, R., Marcelis, L.F.M., Harbinson, J., Lambers, H., 2003. Contrasting effects of N and P deprivation on the regulation of photosynthesis in tomato plants in relation to feedback limitation. J. Exp. Bot. 54, 1957–1967.
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