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Journal of Plant Research ISSN 0918-9440 J Plant ResDOI 10.1007/s10265-017-0933-5
Exogenous application of urea and a ureaseinhibitor improves drought stress tolerancein maize (Zea mays L.)
Wei Gou, Pufan Zheng, Li Tian, MeiGao, Lixin Zhang, Nudrat Aisha Akram& Muhammad Ashraf
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J Plant Res DOI 10.1007/s10265-017-0933-5
REGULAR PAPER
Exogenous application of urea and a urease inhibitor improves drought stress tolerance in maize (Zea mays L.)
Wei Gou1 · Pufan Zheng1 · Li Tian1 · Mei Gao1 · Lixin Zhang1 · Nudrat Aisha Akram2 · Muhammad Ashraf3
Received: 16 October 2016 / Accepted: 14 December 2016 © The Botanical Society of Japan and Springer Japan 2017
Keywords Urea · N-(n-butyl) thiophosphoric triamide (NBPT) · Drought stress · Zea mays L.
Introduction
Maize (Zea mays L.) is one of the major cereals which can grow in a range of soil and environmental conditions. Due to low rainfall, poor soil, high evapotranspiration and improper water management practices, drought stress could induce severe economic losses in arid and semi-arid regions (Campos et al. 2004; Meng et al. 2013; Xie 2009). Drought caused a multitude of metabolic changes which influenced plant growth and development, includ-ing a marked suppression in plant photosynthetic efficiency (Ashraf and Harris 2013; Pinheiro and Chaves 2011), high rate of reactive oxygen species (ROS) production (Sharma et al. 2012), and plasma membrane depolarization (Talbi et al. 2015), which could bring about macromolecules and a reduction in growth under drought stress (Doupis et al. 2013). Numerous changes in plant morphology and metab-olism process would take place in plants under drought stress condition including activating antioxidant system and accumulating compatible solutes (Blum 2016; Hasanuzza-man et al. 2013; McCue and Hanson 1990; Rasheed et al. 2011; Sharma and Dubey 2005). Therefore, considerable efforts in crop management practices are being encouraged to enhance drought tolerance such as application of various exogenous substances (Akram et al. 2016; Latif et al. 2016; Shafiq et al. 2015; Shu et al. 2013; Yasmeen et al. 2013).
Urea is not only the major nitrogen form supplied as a fertilizer in agriculture, but also an important metabolite in plants (Bollard et al. 1968; Coruzzi and Bush 2001). Appropriate levels of urea-sprayed could cause several physiological and biochemical reactions in plants under
Abstract Drought is believed to cause many metabolic changes which affect plant growth and development. How-ever, it might be mitigated by various inorganic substances, such as nitrogen. Thus, the study was carried out to investi-gate the effect of foliar-applied urea with or without urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) on a maize cultivar under drought stress simulated by 15% (w/v) polyethylene glycol 6000. Foliar-applied urea resulted in a significant increase in plant dry weight, relative water con-tent, and photosynthetic pigments under water stress con-dition. Furthermore, the activities of superoxide dismutase (SOD), peroxidase (POD), and hydrogen peroxidase (CAT), were enhanced with all spraying treatments under drought stress, which led to decreases in accumulation of hydrogen peroxide (H2O2), superoxide anion (O⋅−
2) and malondialde-
hyde (MDA). The contents of soluble protein and soluble sugar accumulated remarkably with urea-applied under drought stress condition. Moreover, a further enhancement in above metabolites was observed by spraying a mixture of urea and urease inhibitor as compared to urea sprayed only. Taken together, our findings show that foliar application of urea and a urease inhibitor could significantly enhance drought tolerance of maize through protecting photosyn-thetic apparatus, activating antioxidant defense system and improving osmoregulation.
* Lixin Zhang [email protected]
1 State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, College of Life Sciences, Northwest A&F University, Yangling, People’s Republic of China
2 Department of Botany, GC University, Faisalabad, Pakistan3 Pakistan Science Foundation, Islamabad, Pakistan
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abiotic stresses (Younis et al. 2009). The improved adapta-tion to drought induced by urea-application was thought to be linked with a combination of physical, physiological and cellular effects, which commonly included enhancement of leaf water potential (Mir et al. 2010), increase in photosyn-thetic efficiency (Del Amor and Cuadra-Crespo 2011) and antioxidase activities (Kaya et al. 2015a, b), and promotion of high accumulation of osmolytes (Zhang et al. 2012a, b). Generally, urea is absorbed by plants as a nitrogen source through NH3 transport (Zhang et al. 2015). An increase in hydrolysis of urea not only disturbed nitrogen availabil-ity but also reduced nitrogen use efficiency (Zaman et al. 2013). So, use of urease inhibitors such as N-(n-butyl) thi-ophosphoric triamide (NBPT) reduced ammonia emissions from urea fertilizer. The NBPT, a structural analog of urea, played a prominent role in reducing urease activity (Juan et al. 2009). It was suggested that NBPT could be taken up by root and translocate to leaves to inhibit urease activities of leaf and root in plants (Artola et al. 2011; Cruchaga et al. 2011; Watson and Miller 1996). However, we ignored the possibility that plants can directly absorb urea molecules into the cell without decomposition. When sprayed with urea and urease inhibitor, the urea content increased signifi-cantly, indicating that urea molecules might be present in the cell without being decomposed (Liu et al. 2003; Witte et al. 2002). It had been reported that urea molecules could regulate downstream transcription factors as signal sub-stances in animals (Cohen 1996), while the precise mecha-nisms involved in metabolism regulation and plant growth are still unclear. Therefore, our study investigated the effects of urea combined with urease inhibitor (NBPT) on drought tolerance in maize by focusing particularly on pho-tosynthesis, antioxidant systems, and osmotic adjustment to supply a promising method to mitigate the adverse effects of drought stress on maize production in semi-arid areas.
Materials and methods
Plant materials and treatments
The experiment was conducted in a glasshouse under con-trolled environmental condition. The seeds of maize cv. Zheng Dan 958 were sterilized with 75% (v/v) ethanol for 5 min and then placed in moistened petri plates in a small germination container (13 × 19 × 12 cm) at 25 °C under dark for germination. Four days later, the seedlings selected at random were planted in a hydroponic system containing half strength Hoagland’s nutrient solution (Hoagland and Arnon 1950) in a growth chamber set at photoperiod 15/9 h and light intensity 250 μmol m−2 s−2. Before initiation of drought stress treatment, plants were allowed to grow for 7 days.
When plants were at three-leaf stage, the seedlings were exposed to 15% (w/v) polyethylene glycol-6000 solu-tion to induce drought stress (DS) and then three foliar-sprayed treatments were conducted with distilled water (DS+W), urea (DS+U) as well as urea and urease inhibitor (DS+U+C) prepared in 0.04% ethanol and 0.125% Triton-X 100 as a surfactant, respectively. The same amount of distilled water along with surfactants as mentioned above applied to leaves under normal irrigation regime, was con-sidered as control. Each treatment included three replica-tions. The leaves of the maize seedlings were collected after 1, 3, 5 and 7 days of growth for determining the fol-lowing attributes.
Leaf relative water content (RWC) and dry weight (DW)
Leaf RWC was estimated according to the method of Cas-tillo (1996). The fresh seedling samples were then placed in paper envelopes and dried at 80 °C for one day and exam-ined for dry weight as proposed by Levin et al. (2002).
Contents of photosynthetic pigments
The chlorophyll and carotenoid contents were extracted from the leaves and estimated following Lichtenthaler and Wellburn (1983). Fresh leaves (0.2 g) samplings were soaked in 10 mL of 80% acetone at 4 °C under dark con-dition. Then, the absorbance of the extraction was meas-ured at 645, 663 and 480 nm against 80% acetone as blank, respectively.
Activities of antioxidant enzymes
Leaf samples (each 0.5 g) were extracted in ice-cold 50 mM Na-phosphate (pH 7.8) containing 2 mM edetate disodium and 1% polyvinyl-polypyrrolidon (w/v). Then the extract was centrifuged at 12,000×g for 15 min at 4 °C and the supernatant was collected for determination of the fol-lowing enzymes:
The superoxide dismutase (SOD) activity was examined following Abedi and Pakniyat (2010). The reaction mix-ture included 162 mL 14.5 μM methionine, 0.6 mL 30 μM edentate disodium, 6 mL 60 μM riboflavin, 6 mL 2.25 mM nitroblue tetrazolium, 5.4 mL 0.05 M sodium phosphate buffer (pH 7.8) and enzyme extract. The mixture was illu-minated with light intensity 300 μmol m−2 s−2 to initiate the reaction for 20 min. The absorbance was read at 560 nm and one unit (U) of SOD activity was expressed as enzyme amount of protein.
The peroxidase (POD) activity was examined follow-ing Muñoz-Muñoz et al. (2009). The reaction mixture con-tained 200 mL 0.2 M sodium phosphate buffer (pH 7.8),
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0.076 mL guaiacol, 0.112 mL 30% H2O2 (v/v) and enzyme extract. An increase in absorbance was measured at 470 nm in 10 min followed by adding 0.2 mL 5% (m/v) metaphos-phate to stop the reaction. One unit (U) of POD activity was calculated as enzyme amount which caused an increase in absorbance of 0.01 per minute.
The hydrogen peroxidase (CAT) activity was examined following Wang (1995) with minor changes. The reac-tion mixture contained 200 mL 0.15 M sodium phosphate buffer (pH 7.0), 0.3092 mL H2O2 (v/v) and enzyme extract. The absorbance was recorded at 240 nm. One unit (U) of CAT activity was defined as the amount of enzyme that used 1 nmol H2O2 per minute.
Contents of hydrogen peroxide (H2O2), superoxide anion (O⋅−
2) and malondialdehyde (MDA)
The H2O2 content was determined following Paździoch-Czochra and Wideńska (2002). Fresh leaves samplings were grinded in a precooling mortar containing 5 mL acetone. Then the sample was centrifuged at 3000×g for 10 min at 4 °C. The supernatant was used for H2O2 deter-mination. The reaction solutions contained 0.1 mL 5% titanium sulfate, 0.2 mL concentrated ammonia and 1 mL crude extract. Then the mixture was centrifuged again at 3000×g for 10 min at 4 °C. The precipitate was washed twice with 5 mL of acetone and dissolved with 5 mL of 2 M sulfuric acid solution. The absorbance was recorded at 415 nm.
Leaf samplings were homogenized in cold 65 mM sodium phosphate buffer (pH 7.8) and centrifuged at 12,000×g for 10 min at 4 °C. The supernatants were col-lected and analyzed for O⋅−
2 generation rate. The reac-
tion mixture contained 2 mL supernatant, 1.5 mL 65 mM sodium phosphate buffer (pH 7.8), 0.5 mL hydroxylamine hydrochloride and then the mixture was incubated in a water bath at 25 °C for 20 min. Thereafter, 2.0 mL 17 mM sulfanilic and 2.0 mL 7 mM α-naphthylamine were added to 2 mL of the reaction mixture and incubated at 30 °C for 30 min again. The absorbance was recorded at 530 nm for appraising O⋅−
2 following Ke et al. (2007).
Malondialdehyde (MDA) content was determined fol-lowing Kumar and Knowles (1993). After different chemi-cal reactions, the absorbance of the supernatant was meas-ured at 450, 532 and 600 nm, respectively.
Content of total soluble protein
The soluble protein content was determined following Mohammadkhani and Heidari (2007). The reaction solu-tion contained 80 μL 0.05 M sodium phosphate buffer (PBS, pH 7.8), 2.9 mL coomassie brilliant blue G-250 and 20 μL crude enzyme extract. After a reaction for 2 min,
the absorbance was recorded at 595 nm taking the blank of 0.1 mL phosphate-buffered saline (PBS) and 2.9 mL coomassie brilliant blue.
Content of soluble sugar
The fresh leaf samples were placed in 100 °C at 15 min and then kept at 70 °C for drying. The dry samples (100 mg) were homogenized with 4 mL ethanol. The extract was heated in a water bath at 80 °C for 30 min and then cen-trifuged at 6000×g for 10 min. The precipitate was again homogenized in 2 mL 80% ethanol (m/v) at 80 °C water bathing for 30 min and then centrifuged again described above. Then merged the supernatant and added 0.5 g acti-carbon to the mixture for decolorizing at 80 °C for 30 min. After that, the volume of the mixture was raised to 10 mL using 80% ethanol solution. The filtered solution was used for the determination of soluble sugar. The supernatants (each 0.2 mL) were taken in test tubes including 5 mL sul-furic acid-anthrone reagent in each and the samples were heated for 10 min at 100 °C in a water bath and then cooled rapidly. Control volume was made by adding deionized water. The absorbance of treated samples was determined at 625 nm (Quilot et al. 2004).
Statistical analysis
One-way analysis of variance and Duncan’s multiple range test were employed to the original data for each vari-able. The values are mean ± SD for three samples in each treatment.
Results
Growth parameters
Drought stress had a detrimental effect on relative water content (RWC) and dry weight (DW) of maize (Fig. 1). Exogenous application of urea under DS condition signifi-cantly increased biomass production and RWC by 11.1 and 3.4% at day 7, respectively. The effect of mixture of urea and urease inhibitor was more pronounced as compared those under only urea sprayed condition. The DW was fur-ther increase by 7.0%, while RWC was slightly increased under DS+U+C condition, respectively.
Photosynthetic pigment contents
The pigment contents after spraying urea and urease inhibi-tor under drought stress were shown in Fig. 2. The pigment contents decreased significantly with prolongation of treat-ment time under DS condition. Chlorophyll content was
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increased by 3.1, 2.9, 1.4, and 11.6% at 1, 3, 5, and 7 days under DS+U condition, respectively. Foliar-spray with urea and urease inhibitor further increased the chlorophyll content by 8.4, 7.1, 19.4, and 12.9% at different days, com-pared with those urea-sprayed only, respectively (Fig. 2a). The carotenoid contents showed the same change pattern. Carotenoid content was increased by 14.8, 22.1, 26.0, and 16.2% at days 1, 3, 5, and 7 under DS+U+C condition, as compared to those in DS+W, respectively (Fig. 2b).
Antioxidant enzyme activities
The activities of SOD, POD and CAT enzymes showed the same change trend with firstly increased and then decreased
in different treatments (Fig. 3). As shown in Fig. 3a, a sub-stantial increase in SOD activity was observed with respect to control regardless of treatments applied. The highest value of SOD activity was noted at day 5 under DS+U+C condition. Similarly, a significant increase was observed for POD activity under DS condition. Multiplied increases caused by urea-sprayed were 8.1, 15.9, 14.1, and 15.9%, and their corresponding increasing rate was 2.7, 30.0, 17.8, and 19.8% with both urea and NBPT sprayed at day 1, 3, 5, and 7 compared to the control group, respectively (Fig. 3b). Correspondingly, CAT activity increased considerably under DS condition. There were increases in CAT activity by 23.4, 8.0, 54.5, and 35.0% at day 1, 3, 5, and 7 with urea-sprayed only, and by 38.2, 36.0, 84.5, and 69.3% with urea and NBPT applied, respectively. The greatest CAT activity
Fig. 1 Leaf relative water content and dry matter weight of maize seedlings fed with urea under drought stress. NDS+W, DS+W, DS+U and DS+U+C represent normal water condition with distilled water spray, drought stress with distilled water spray, drought stress with urea spray, drought stress with spray of urea and urease inhibi-tor, respectively. Different letters following the values indicate signifi-cant differences among the treatment values at P < 0.05. Data repre-sent the means ± SE of three replicates
Fig. 2 Chlorophyll content and carotenoids content of maize seed-lings fed with urea under drought stress. NDS+W, DS+W, DS+U and DS+U+C represent normal water condition with distilled water spray, drought stress with distilled water spray, drought stress with urea spray, drought stress with spray of urea and urease inhibitor, respectively. Different letters following the values indicate significant differences among the treatment values at P < 0.05. Data represent the means ± SE of three replicates
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was recorded in DS+U+C treated plants (Fig. 3c). Over-all, the maximum value of all three enzymes was found in plants when sprayed with both urea and NBPT at day 3.
Reactive oxygen species (ROS) generation
Drought caused an increase in ROS generation in maize (Fig. 4). The contents of H2O2 and O⋅−
2 were low under
normal condition. However, their contents were provoked when plants were exposed to DS condition. Exogenously applied treatments remarkably decreased the generation of ROS, especially for urea and urease inhibitor applied. It exhibited obvious decreases in H2O2 content by 18.0, 23.8, 19.6, and 20.3% (Fig. 4a) and a decrease in O⋅−
2 content was
Fig. 3 The activities of SOD, POD and CAT enzymes of maize seed-lings fed with urea under drought stress. NDS+W, DS+W, DS+U and DS+U+C represent normal water condition with distilled water spray, drought stress with distilled water spray, drought stress with urea spray, drought stress with spray of urea and urease inhibitor, respectively. Different letters following the values indicate significant differences among the treatment values at P < 0.05. Data represent the means ± SE of three replicates
Fig. 4 H2O2 content and O⋅−
2content of maize seedlings fed with urea
under drought stress. NDS+W, DS+W, DS+U and DS+U+C repre-sent normal water condition with distilled water spray, drought stress with distilled water spray, drought stress with urea spray, drought stress with spray of urea and urease inhibitor, respectively. Different letters following the values indicate significant differences among the treatment values at P < 0.05. Data represent the means ± SE of three replicates
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by 5.4, 8.7, 24.2, and 20.9% at 1, 3, 5, and 7 days (Fig. 4b) under DS+U+C compared with those in DS+W condi-tion, respectively. Moreover, the ROS-scavenging effects induced by the above exogenous spraying treatments were more predominate in the later period of drought stress.
MDA content
Drought stress induced greater accumulation of MDA com-pared with that under normal condition. The foliar-applied with urea, especially for both urea and NBPT sprayed, decreased the MDA content to a great extent, particularly at the later stages of drought stress. Their decrement was in 9.2, 13.8, 19.8, and 25.5% at days 1, 3, 5, and 7 under DS+U+C condition, compared to the control, respectively (Fig. 5).
Soluble protein content
Soluble protein content showed a considerable increase under DS. Application of urea and NBPT steeply increased soluble protein content, especially for urea-sprayed only (Fig. 6). There was no significant difference between DS+U and DS+U+C condition, except for day 3 under DS+U condition. Soluble protein content showed the same trend with increasing first and then decreasing under all conditions. Sprayed with urea and urease inhibitor treat-ment resulted in lower values than those obtained with urea application only in most cases. The highest value of soluble protein content was observed at day 3 in single
urea-sprayed plants, which increased by 12.0% compared with that under DS+W condition.
Soluble sugar content
Soluble sugar content was gradually increased with the pro-longation of treatments under DS condition. Application of urea caused a slight increase under DS condition. However, foliar-sprayed with urea and urease inhibitor resulted in a greater increase in soluble sugar content by 14.1, 18.1, 7.0, and 14.2% at different days under DS+U+C compared with that in DS+W, respectively (Fig. 7).
Discussion
Drought stress seriously affects plant growth due to consid-erable stress-induced changes in many physio-biochemical processes. Maintenance of water status in plant tissues is one of the most important adaptive responses to drought. In view of Blum (2009), relative water content (RWC) reflected leaf water status and cellular metabolic level under stress conditions. It had been reported that drought declined RWC in several plants, including wheat (Triticum aestivum L.) (Arjenaki et al. 2012), pistachio (Pistacia vera L.) (Esmaeilpour et al. 2014), sunflower (Helianthus ann-uus L.) (Ghobadi et al. 2013), radish (Raphanus sativus L.) (Akram et al. 2015), cucumber (Cucumis sativus L.) (Naz et al. 2016) and cauliflower (Brassica oleracea L.) (Latif
Fig. 5 MDA content of maize seedlings fed with urea under drought stress. NDS+W, DS+W, DS+U and DS+U+C represent normal water condition with distilled water spray, drought stress with dis-tilled water spray, drought stress with urea spray, drought stress with spray of urea and urease inhibitor, respectively. Different letters fol-lowing the values indicate significant differences among the treatment values at P < 0.05. Data represent the means ± SE of three replicates
Fig. 6 The content of soluble sugar of maize seedlings fed with urea under drought stress. NDS+W, DS+W, DS+U and DS+U+C repre-sent normal water condition with distilled water spray, drought stress with distilled water spray, drought stress with urea spray, drought stress with spray of urea and urease inhibitor, respectively. Different letters following the values indicate significant differences among the treatment values at P < 0.05. Data represent the means ± SE of three replicates
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et al. 2016). In the current study, the RWC content at day 7 reduced by 18.0% compared with that at day 1. Exog-enously applied urea effectively improved RWC and alle-viated stress-induced damage in plant, particularly under DS+U+C condition. Similarly, urea and urease inhibitor also played an important role in enhancing biomass pro-duction under DS. Urea-induced enhancement in growth was well documented for many plant species (Shivay and Prasad 2012; Wu et al. 2008). Application of urea not only increased nitrogen nutrition but stimulated the accumu-lation of nitric oxide (NO) as signal molecule to regulate drought tolerance (Menchyk 2012; Stiegler et al. 2011; Zhang et al. 2009). The urease inhibitor could keep urea molecules intact in plants because it restricted the urea decomposition. Urea then acted as a signaling molecule to regulate physiological responses to stress, such as osmotic adjustment (Dawar 2010). For example, addition of NBPT to foliar-supplied urea improved nitrogen uptake by 22% in cotton under low salinity condition (Kawakami et al. 2013). In our study, enhanced water status induced by urea and NBPT may assist plants in maintaining their normal physi-ological functions including nitrogen metabolism under drought stress condition (Cruchaga et al. 2011; Kawakami et al. 2013; Kaya and Higgs 2003).
Photosynthesis is one of the most important physiologi-cal processes in all green plants, which directly links with plant biomass production (Demura and Ye 2010; Zhu et al. 2008). However, it is very sensitive to drought stress. Syn-thesis and accumulation of photosynthetic pigments might
decrease due to drought stress thereby suppressing pho-tosynthesis (Lichtenthaler and Wellburn 1983; Rajasekar et al. 2015; Sudhir and Murthy 2004). In the present study, chlorophyll content of maize plants decreased under water stress. Moreover, urea application significantly increased the chlorophyll content and a further improvement in this attribution was observed under DS+U+C treatment. It is evident that nitrogen is a basic element of chlorophyll structure (Wettstein et al. 1995) and exogenous supply of urea seemed to supply a sufficient amount of nitrogen to promote the synthesis and accumulation of chlorophyll in plants (Ghobadi et al. 2013; Kumawat et al. 2015; Zhao et al. 2008). From the results of chlorophyll content, it was obvious that there was no significant effect on chlorophyll content in DS+U, while the treatment DS+U+C had a sig-nificant effect on improvement of chlorophyll content.
Carotenoids serve as an accessory pigment for harvest-ing light used by the photosynthetic apparatus to protect plants against photoinhibition damage (Croce and Amer-ongen 2014; Lichtenthaler and Wellburn 1983). They could greatly accumulate under short-term drought, but their syn-thesis was impaired or degradation, which was promoted under prolonged drought stress condition (Ashraf and Har-ris 2013). In our study, the changing pattern of carotenoid content was similar with the chlorophyll content recorded here. For example, it decreased considerably under drought, but a slight increase was noted under DS+U and DS+U+C condition. Urea was believed to provide more appropri-ate substrates to synthesize chlorophyll and carotenoids (Kumawat and Mahla 2015). Previous studies showed that urea application in optimum concentration could enhance photosynthetic pigment contents (Houles et al. 2007), which exhibited a positive relationship between nitrogen content and photosynthetic processes like carboxylation and electron transport rate (Ripullone et al. 2003). Urea application could stimulate photosynthesis, enhance leaf dry matter production and promote plant growth in most plants (Kumawat and Mahla 2015; Maleva et al. 2013).
Generally, drought stress may trigger some metabolic adaptive processes in plants to suppress over-accumula-tion of reactive oxygen species (ROS), including H2O2 and O⋅−
2, which would cause serious damage to cellular
structures and metabolic processes under stress conditions (Sharma et al. 2012). The over-production of ROS could be adequately controlled by antioxidant enzymes includ-ing SOD, POD and CAT (Das and Roychoudhury 2014). Higher activities of these antioxidant enzymes could effi-ciently help plants resist various adverse environmental stresses (Meloni et al. 2003; Sharma et al. 2012; Talbi et al. 2015). In this study, we also found higher activities of SOD, CAT and POD enzymes under drought stress condition, and foliar-sprayed with urea and urease inhibi-tor had a beneficial effect on improving the activities of
Fig. 7 The content of soluble protein of maize seedlings fed with urea under drought stress. NDS+W, DS+W, DS+U and DS+U+C represent normal water condition with distilled water spray, drought stress with distilled water spray, drought stress with urea spray, drought stress with spray of urea and urease inhibitor, respectively. Different letters following the values indicate significant differ-ences among the treatment values at P < 0.05. Data represent the means ± SE of three replicates
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antioxidase enzymes, especially under DS+U+C condi-tion, which suggested that exogenous application with urea and urease inhibitor showed a better ability to scavenge stress-induced ROS. These results are in agreement with the research of Yuan et al. (2013), which demonstrated that urea could significantly increase antioxidase activities. It was proposed that the antioxidant protection induced by urea could be related well to osmotic adjustment in plants under drought stress (Zhang et al. 2012a, b). Moreover, plant water status played an essential role in activating and modulating of antioxidant defense systems under drought condition (Del Amor and Cuadra-Crespo 2011; Hasanuz-zaman et al. 2012). In this study, SOD and CAT activities in urea-sprayed plants were significantly higher than those in water-sprayed plants under drought stress. As for POD, it exhibited a slight but not significant increase under DS+U and DS+U+C condition compared with that under DS+W. The enhancements in antioxidase activities might activate more efficient antioxidant systems for ROS elimination and oxidative damage mitigation to resist to drought stress (Das and Roychoudhury 2014; Talbi et al. 2015). Kaya et al. (2015a, b) reported that exogenous nitric oxide reduced MDA and H2O2 contents, stimulated the activities of SOD, CAT, and POD enzymes, and enhanced chlorophyll con-tent under salinity stress. The stimulation in antioxidant enzymes activities could be attributed to NBPT which could interfere with urea nutrition in maize plants thereby limiting influx as well as the subsequent assimilation pathway (Zanin et al. 2015). In our study, higher levels of H2O2 and O⋅−
2 were observed under DS condition. Similar
results were reported in rice (Oryza sativa L.) (Cunha et al. 2016), wheat (Sairam et al. 2002), alfalfa (Medicago sativa L.) (Wang et al. 2009), and chickpea (Cicer arietinum L.) (Kukreja et al. 2005) under various environmental stress conditions. Urea was reported to effectively increase nitro-gen and nitric oxide content in plants (Zhang et al. 2012). Nitrogen metabolism needs NADP(H) which is produced in chloroplasts or mitochondria to provide energy, however, they were also the main organelles to generate of ROS. It showed a close relationship between nitrogen metabolism and ROS generated in plants (Naylor 2012). Moreover, nitric oxide served as an endogenous signal under drought stress and it can also reduce the generation of ROS in plants (Pasqualini et al. 2015). In this study, a significant decrease in H2O2 and O⋅−
2 contents were observed under
DS+U and DS+U+C condition, more prominent being in DS+U+C. MDA had been considered as an indicator of membrane lipid peroxidation (Møller et al. 2007) and considerable concentration of MDA was increased under drought condition (Mohammadkhani and Heidari 2007; Yang and Miao 2010). Less membrane stability showed ROS-induced lipid peroxidation (Møller et al. 2007). In an earlier study, the MDA content in maize plants was
increased in droughty-plants compared with that in the con-trol plants (Mohammadkhani and Heidari 2007). Similarly, the MDA content substantially increased under DS condi-tion in this study, and a further enhancement was observed under DS+U and DS+U+C condition, especially under DS+U+C treatment. Our results were similar to those stud-ies reported earlier which showed a decreases in MDA con-tent caused by urea application (Maleva et al. 2015).
Plants accumulate considerable organic solutes in cyto-plasm in response to drought stress, such as proline, total soluble sugar, soluble protein and so on (Blum 2016). These compatible solutes protected the functioning of nor-mal metabolic processes under drought stress, which could mediate adaptive changes in response to external osmo-lality and maintain osmotic balance as well as continuous water influx to the plants (Chan et al. 2013). Osmolytes also increased the stability of macromolecules without interfer-ing with their functional activities (Yancey et al. 1982). They also scavenged free radicals under severe drought stress (Padmavathi and Rao 2013). In our study, dramatic increases in contents of total soluble sugar and soluble pro-tein were observed in maize seedlings when spraying exog-enous chemicals. The increasing in sugar content induced by urea supplied may be attributed to increase nitrogen accumulation, which may in turn excite metabolic activ-ity and improve osmotic adjustment ability (Zhang et al. 2012a, b). However, urea and NBPT application led to a marked increase in soluble sugar content, compared with that under DS+W treatment. The NBPT can significantly suppress urease activity thereby promoting urea accumula-tion in the leaves of spinach (Spinacia oleracea L.) and pea (Pisum sativum L.) (Cruchaga et al. 2011). After spraying urea, the increase in substrate concentration could promote soluble protein synthesis in plants under water limited con-dition (Tarighaleslami et al. 2012). Moreover, urea can also increase nitric oxide content (Kaya et al. 2015a, b). It was widely known that exogenous nitric oxide could increase the soluble protein content in stressed-plants (Kaya et al. 2015a, b). Similarly, application of urea and urease inhibi-tor significantly increased the soluble protein content. This suggested that the urease inhibitor could inhibit efficiently urea decomposition to regulate soluble protein synthesis and accumulation (Cruchaga et al. 2011; Rajale and Prasad 1974).
Conclusion
Foliar-applied urea with or without urease inhibitor enhanced drought tolerance of maize seedlings. Urea-sprayed significantly increased leaf relative water content, dry weight and photosynthetic pigment content, raised the activities of SOD, CAT and POD, decreased accumulation
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of H2O2, O⋅−
2 and MDA, and increased the contents of solu-
ble sugar and soluble protein under drought stress. A fur-ther change was observed in these variables by spraying the mixture of urea and urease inhibitor as compared to that with urea-sprayed only. All these urea-induced changes ultimately increased the drought tolerance of maize seed-lings. Therefore, this biological strategy could be encour-aged for wide application in maize production.
Acknowledgements This research was supported by the Founda-tion of State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau (A314021402-1514), China Postdoctoral Science Foundation (QN2009069) and Sci-tech Development Foundation of NWSUAF (A2990215264). WG, PFZ and LT contributed equally to this work.
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