DOI: 10.1007/s10535-014-0395-8 BIOLOGIA PLANTARUM 58 (2): 311-318, 2014

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Exogenous 24-epibrassinolide ameliorates high temperature-induced inhibition of growth and photosynthesis in Cucumis melo Y.P. ZHANG1,2, J. HE3, S.J. YANG2, and Y.Y. CHEN1,2* Horticultural Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, P.R. China1

Shanghai Key Laboratory of Protected Horticultural Technology, Shanghai 201106, P.R. China2 College of Biosciences and Biotechnology, Yangzhou University, Yangzhou 225009, P.R. China3 Abstract This study was carried out to better understand the role of 24-epibrassinolide (EBR) in thermotolerance of melon (Cucumis melo L.). The melon seedlings were pretreated with various concentrations of EBR (0, 0.05, 0.1, 0.5, 1.0, and 1.5 mg dm-3) as foliar spray and then exposed to a high temperature (HT) stress. Exogenous EBR (0.5 - 1.5 mg dm-3) alleviated HT-caused growth suppression. In parallel, 1.0 mg dm-3 EBR attenuated the decrease in chlorophyll content, net photosynthetic rate, stomatal conductance, maximum quantum efficiency of photosystem (PS) II, quantum yield of PS II, and photochemical quenching of chlorophyll a fluorescence in HT-stressed plants, and inhibited transpiration rate and non-photochemical quenching. Furthermore, exogenous EBR also significantly reduced the content of malondialdehyde (MDA) and increased the content of soluble proteins and free proline, and activities of antioxidant enzymes including superoxide dismutase, guaiacol peroxidase, catalase, and ascorbate peroxidase under the HT stress. The results show that protective effects of EBR against the HT stress in the melon seedlings were most likely mediated through the improvement of photosynthesis and the stimulation of antioxidant capacity.

Additional key words: ascorbate peroxidase, brassinosteroids, catalase, chlorophyll, malondialdehyde, melon, net photosynthetic rate, peroxidase, photosystem II, proline, stomatal conductance, superoxide dismutase, transpiration rate. Introduction High temperature (HT) stress, one of the environmental factors negatively affecting crop production worldwide, might increase in future due to global warming and climatic change (Wahid et al. 2007, Ainsworth and Ort 2010). High temperature leads to severe retardation in growth and development mostly due to injury of photosynthetic apparatus (Wise et al. 2004). HT also cause oxidative stress by overproduction of reactive oxygen species (ROS), which can damage lipids, proteins, and nucleic acids (Mittler 2002, Wahid et al. 2007, Zhang et al. 2012). In plants, the removal of ROS are governed by antioxidative enzymes like superoxide

dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and quaiacol peroxidase (POD), and by a non-enzymatic system, such as ascorbate and glutathione (Mittler 2002). Therefore, HT tolerance is often correlated well with high photosynthetic efficiency and effective antioxidant capacity (Wahid et al. 2007, Asthir et al. 2012). Brassinosteroids (BRs) are essential regulators of plant growth and development at very low concentrations (Sasse 2003, Bajguz and Hayat 2009, Hayat et al. 2011). Furthermore, BRs have also been targeted in many plant species to improve their tolerance to different

Submitted 21 January 2013, last revision 20 August 2013, accepted 2 September 2013. Abbrevations: APX - ascorbate peroxidase; BRs - brassinosteroids; CAT - catalase; Chl - chlorophyll; ci - intercellular CO2 concentration; E - transpiration rate; EBR - 24-epibrassinolide; F0 - minimal fluorescence; Fv/Fm - variable to maximum fluorescence ratio in dark adapted leaves (maximum quantum yield of PS II photochemistry); Fv´/Fm´ - variable to maximum fluorescence ratio in steady-state conditions (efficiency of excitation energy capture by open PS II centers); gs - stomatal conductance; MDA - malondialdehyde; NBT - nitroblue tetrazolium; NPQ - non-photochemical quenching; ΦPSII - effective quantum yield of PS II photochemistry; POD - peroxidase; PS - photosystem; qP - photochemical quenching; ROS - reactive oxygen species; SOD - superoxide dismutase. Acknowledgements: We are grateful for funding supported by the Shanghai Prosper Agriculture by Science and Technology Plan, China (Grant No. 2009-2-1) and the Open Fund of Shanghai Key Lab of Protected Horticultural Technology. * Author for correspondence; fax: (+86) 21 52630133, e-mail: youyuanchen@126.com

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environmental stresses, such as chilling stress (Liu et al. 2009), water stress (Li et al. 2012), salinity (Ding et al. 2012), heavy metals (Fariduddin et al. 2009), or phenanthrene (Ahammed et al. 2013). BRs under stress conditions may stimulate several processes, such as nucleic acid and protein syntheses (Kagale et al. 2007), enzyme activities (Ogweno et al. 2008), photosynthetic efficiency (Hayat et al. 2010, Hasan et al. 2011), and increase fruit set (Ali et al. 2008). The involvement of BRs in thermotolerance has drawn much attention over the past two decades (Dhaubhadel et al. 2002, Singh and Shono 2005, Kagale et al. 2007, Ogweno et al. 2008, Hayat et al. 2010, Janeczko et al. 2011, Mazorra et al. 2011). However, the mechanisms of BRs-induced

thermotolerance are still far from being complete. Melon (Cucumis melo L.) is an economically important and widely cultivated greenhouse crop, which is greatly affected by HT stress (Mavi and Demir 2007). Melon seedlings face HT in summer-autumn in China, resulting in premature aging and a decline in yield and quality. It is actually important to improve thermo-tolerance of melon. Therefore, the objective of the present study aimed to investigate the effects of 24-epibrassinolide (EBR) at different concentrations on the growth, photosynthetic gas exchange, photosystem II photochemistry, and the activities of antioxidant enzymes in melon plants under a HT stress in order to elucidate the mechanisms of BRs-induced thermotolerance.

Materials and methods Seeds of melon (Cucumis melo L.cv. Baiyuxiang) from the Horticultural Research Institute, Shanghai Academy of Agricultural Sciences, China were sterilized with 1 % (m/v) sodium hypochlorite for 10 min and rinsed thoroughly with sterile distilled water. Then they were sown on two layers of filter paper moistened with distilled water in Petri dishes and germinated at 28 C. The germinated seeds were planted in plastic plates in a growth chamber at day/night temperatures of 28/18 C, a photosynthetic photon flux density (PPFD) of 400 μmol m-2 s-1, and a relative humidity of 70 - 80 %. All plants were watered daily with a half-strength Hoagland solution. When seedlings were at a 3 - 4 leaf stage, the EBR and HT treatments were started. The EBR (Sigma, St. Louis, USA) was dissolved in a small amount of absolute ethanol and then brought into distilled water. The seedlings were treated with 0 (distilled water), 0.05, 0.1, 0.5, 1.0, 1.5 mg dm-3 EBR. Each plant was daily sprayed with 50 cm3 of the EBR solution for 4 d (a total of 200 cm3 plant-1). Then the seedlings were divided into two groups, a control (30/20 C) and a high temperature stress (42/32 C). After two days, the second leaves were used for various determinations and at least three replications were carried out. Shoots were harvested, rinsed three times in distilled water, blotted on filter paper and then weighed. The stem diameter (the diameter of the cotyledon node along the opening direction of the cotyledon) was measured by a vernier caliper. The leaf area was determined as maximum leaf length × maximum leaf width × 0.66 (a correction factor). Amounts of chlorophyll (Chl) a, Chl b, and Chl a + b were assayed according to Lichtenthaler (1987) in acetone extract and the absorbance was read at 470, 663, and 645 nm on a spectrophotometer DU730 (Beckman, Coulter, USA). Net photosynthetic rate (PN), stomatal conductance (gs), transpiration rate (E), and intercellular CO2 concentration (ci) were measured on the third fully expanded leaf using a portable photosynthesis system

(LI-6400, LI-COR, Lincoln, NE, USA). During the measurements, photosynthetic photon flux density was set to 800 µmol m-2 s-1, relative humidity to about 85 %, leaf temperature to 25 C, and ambient CO2 concentration to 400 μmol mol-1. The measurement of photosynthesis was done once for each leaf and for five different leaves per treatment. Chl a fluorescence was measured using a portable pulse-modulated fluorometer (PAM-2100, Walz, Effeltrich, Germany). Before each measurement, leaves were dark-adapted for at least 30 min. The minimal fluorescence (F0) was determined at weak modulated radiation. A 0.8 s saturating pulse of 8 000 μmol m-2 s-1 was used to determine the maximal fluorescence (Fm). Then the actinic radiation of 600 μmol m-2 s-1 was applied and after the leaf reached steady-state photosynthesis, the steady-state fluorescence (Fs) was recorded and a second 0.8 s saturating pulse of 8 000 μmol m-2 s-1 was applied to determine the maximal fluorescence (Fm´) in the light adapted state. The actinic radiation was turned off and after 3 s of far-red radiation, the minimal fluorescence in the light-adapted state (Fo´) was determined. The maximumal quantum efficiency of photosystem (PS) II (Fv/Fm), the quantum yield of PS II (ФPSII), the efficiency of excitation energy capture by open PS II centers (Fv’/Fm’), non-photochemical quenching (NPQ), and photochemical quenching (qP) were calculated as Fv/Fm, (Fm´- Fs)/Fm´, Fv´/Fm´, Fm/Fm´- 1, and (Fm’ - Fs)/(Fm’ - F0’), respectively (Yu et al. 2004). Lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA) produced by the thiobarbituric acid reaction as described by Jiang and Zhang (2001). Protein content was determined according to the method of Bradford (1976) with bovine serum albumin as standard. Proline was measured according to the method of Bates et al. (1973) after its extraction at room temperature with a 3 % (m/v) 5-sulphosalicylic acid solution.

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For the determination of enzyme activities, leaf segments (0.5 g) were homogenized in 5 cm3 of 50 mM potassium phosphate buffer, pH 7.0, containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 2 % (m/v) soluble polyvinylpyrrolidone (PVP), with the addition of 1 mM ascorbate in the case of APX assay. The homogenate was centrifuged at 15 000 g and 4 °C for 30 min and the supernatant was immediately frozen with liquid N2 and stored at -70 °C for the following assays. SOD (EC 1.15.1.1) activity was assayed by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium (NBT) according to the method of

Beauchamp and Fridovich (1971). CAT (EC 1.11.1.6) activity was determined spectrophotometrically by following the decrease of absorbance of H2O2 at 240 nm as described by Aebi (1984) with a slight modification. APX (EC 1.11.1.11) activity was determined according to Nakano and Asada (1981) by monitoring the rate of ascorbate oxidation at 290 nm. POD (EC 1.11.1.7) activity was measured using guaiacol as substrate (Nickel and Cunningham 1969). All data were subjected to analysis of variance and means were compared by the Duncan’s multiple range test at 5 % level of probability.

Results The melon seedlings exposed to the HT stress exhibited a significant reduction in morphological attributes like stem diameter, leaf area, and shoot fresh mass (Fig. 1A,B,C). However, exogenous EBR significantly alleviated the decrease in melon growth under the HT stress in a con-centration dependent manner. The 1.0 and 1.5 mg dm-3 EBR almost nullified the inhibition of the shoot fresh mass caused by the HT stress. No significant effect on growth attributes was observed in the seedlings treated

with EBR alone. To examine whether EBR-regulated growth was associated with changes in photosynthesis under the HT stress, the amount of chlorophyll (Fig. 1D,E,F) was firstly detected. The content of Chl a, Chl b, and Chl a+b significantly decreased under the HT treatment, which was counteracted by exogenous EBR, especially 1.0 mg dm-3 EBR. Besides, the HT-stressed plants displayed a significant decrease in PN and gs (Fig. 2A,B)

Fig. 1. The effects of different concentrations of 24-epibrassinolid (EBR) on the stem diameter (A), leaf area (B), shoot fresh mass (C), and content of chlorophyll a (D), chlorophyll b (E) and chlorophyll a+b (F) per fresh mass unit of melon seedlings under high temperature stress. The melon plants were continuously sprayed with different concentration of EBR for 4 d and were then exposed to high temperature for 2 d. Data are means ± SD of three replicates. The means marked with different letters indicate significant difference between treatments at P < 0.05 according to the Duncan’s multiple range test.

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and an increase in E (Fig. 2D) in comparison with the control, which was partially restored by the foliar application of EBR. In particular, 1.0 and 1.5 mg dm-3

EBR could return the values of PN and gs to the control levels. However, the HT stress and EBR pretreatment had little effect on ci (Fig. 2C).

Fig. 2. The effects of different concentration of 24-epibrassinolid (EBR) on the net photosynthetic rate (PN) (A), stomatal conductance (gs) (B), intercellular CO2 concentration (Ci) (C), and transpiration rate (E) (D) of melon seedlings grown under high temperature stress. Data are means ± SD of five replicates. The means marked with different letters indicate significant differencesbetween treatments at P < 0.05 according to the Duncan’s multiple range test.

Fig. 3. The effects of different concentrations of 24-epibrassinolid (EBR) on the minimum fluorescence (F0) (A), maximum quantum yield of PSII (Fv/Fm) (B), efficiency of excitation energy capture by open PS II centres (Fv’/Fm’) (C), quantum yield of PS II (ФPSII) (D), photochemical quenching coefficient (qP) (E), and nonphotochemical quenching coefficient (NPQ) (F) of melon seedlings grown under high temperature stress. Data are means ± SD of five replicates. The means marked with different letters indicate significant differences between treatments at P < 0.05 according to the Duncan’s multiple range test.

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All chlorophyll fluorescence parameters significantly changed in the HT-stressed plants (Fig. 3). The pretreatment with EBR significantly increased Fv/Fm, Fv’/Fm’, ФPSII and qP, but remarkably reduced Fo and NPQ under the HT stress in a concentration dependent manner. In the presence of 1.0 mg dm-3 EBR, the values of Fv/Fm and Fv’/Fm’ recovered to control levels. 1.0 mg dm-3 EBR alone significantly increased all parameters except for F0 and Fv/Fm. To examine whether EBR alleviated a HT-induced oxidative stress, MDA was measured (Fig. 4A). The HT stress resulted in a significant increase in the MDA content (up to 156 % in comparison with the control). Nevertheless, exogenous EBR markedly blocked the

oxidative stress and at 1.0 mg dm-3 EBR the MDA content under HT was only 71.0 % of the control. The HT stress significantly increased antioxidant enzymes activities (Fig. 5). In the presence of 1.0 mg dm-

3 EBR under the HT stress, the activities of SOD, POD, CAT, and APX were 13.3, 59.2, 95.5, and 33.7 %, respectively, as compared to the HT stress alone. Furthermore, the protein content was significantly decreased by the HT stress and was restored to the control level by the EBR application at 0.05 - 1.5 mg dm-

3 (Fig. 4B). On the contrary, the accumulation of proline in the HT-treated leaves was accelerated (Fig. 4C). The maximum content of proline was recorded in the plants supplied with 1.0 mg dm-3 EBR under the HT stress.

Discussion In the present experiments, a HT stress-induced inhibition in melon growth was markedly alleviated by the foliar application of EBR at appropriate concentrations (Fig. 1). These results are consistent with previous studies on tomato (Singh and Shono 2005, Ogweno et al. 2008, Mazorra et al. 2011), Vigna radiata (Hayat et al. 2010),

Fig. 4. The effects of different concentrations of EBR on the content of MDA (A), proline (B), and soluble protein (C) of melon seedlings grown under high temperature stress. Data aremeans ± SD of three replicates. The means marked withdifferent letters indicate significant differences between treatments at P < 0.05 according to the Duncan’s multiple rangetest.

and barley (Janeczko et al. 2011). Since BRs promote a transverse orientation of microtubules (Sasse 2003), an EBR-induced growth improvement might be due to the acceleration of cell elongation. The other possibilities are the effects of EBR on photosynthetic capacity or ROS metabolism which are discussed in the following sections. In this study, PN of the EBR-treated plants was higher than PN of the HT-stressed plants without EBR (Fig. 2). The decline of PN under the HT stress may be attributed to stomatal or non-stomatal limitations. When a non-stomatal limitation of PN occurs, ci increases or remains constant in parallel with decreased gs (Farquhar and Sharkey 1982). Our results show that an EBR-induced increase in PN under HT was accompanied with an increase in gs, but not in ci, implying that EBR also affected non-stomatal limitation to photosynthesis (photosynthetic pigments, Rubisco content and activity, and use of assimilation products; Dubey 2005). The foliar application of EBR significantly increased the Chl content of the melon seedlings (Fig. 1D,E,F), which is consistent with some earlier studies (Ali et al. 2007, Hasan et al. 2008, Hayat et al. 2010). The most likely reason for increased Chl content might be the increased Chl biosynthesis (Hayat et al. 2011) which lead to the improvement of the light-capturing efficiency resulting finally in a higher photosynthetic rate. PS II is believed to play a key role in the response of leaf photosynthesis to environmental perturbation (Dubey 2005). Fv/Fm is frequently used as an indicator of the photoinhibition or of stress damage to the PS II (Calatayud and Barreno 2004). Fv/Fm was significantly inhibited in the melon leaves under the HT stress (Fig. 3B) which is in agreement with the studies of Ogweno et al. (2008) and Hayat et al. (2010). However, the 1.0 mg dm-3 EBR-pretreated melon leaves under the HT stress showed a less decrease in Fv/Fm, suggesting that the EBR-induced improvement of PS II efficiency is associated with a decrease in the inhibition of electron

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flow at oxidizing site of PS II. Correspondingly, Fv´/Fm´, qP, and ΦPSII (Fig. 3) exhibited similar changes, indicating that the improved quantum yield of PS II electron transport by EBR is attributed to the increase in the photochemical quenching and efficiency of energy capture by open PS II reaction centres. A smaller decrease in NPQ in the EBR-pretreated seedlings

suggests that the EBR application results in a smaller thermal dissipation of excitation energy in the PS II antennae (Gilmore 1997). Yu et al. (2004) and Ogweno et al. (2008) reported that EBR increases the carboxylation rate of Rubisco and the maximum quantum yield of PS II, thereby increasing the capacity of CO2

assimilation in the Calvin-Benson cycle.

Fig. 5. The effects of different concentrations of EBR on the activities of SOD (A), POD (B), CAT (C), and APX (D) of melonseedlings grown under the high temperature stress. Data are means ± SD of three replicates. The means marked with different lettersindicate significant differences between treatments at P < 0.05 according to the Duncan’s multiple range test. One unit of SOD activity was defined as the amount of enzyme that gave 50 % inhibition of the reduction of NBT monitored at 560 nmfor 1 min. One unit of CAT and APX activities was defined as an absorbance change of 0.01 units per min. One unit of POD defined as the increase of 0.01 in absorbance at 470 nm per min. Chloroplasts are not only the site of photosynthesis, but also the main sites for generating ROS in both unstressed and stressed plants. ROS scavenging systems play an important role in protecting cells from photoxidative damage (Mittler 2002). High temperature could result in reduced carboxylation efficiency and increased oxidative stress leading to a down-regulation of PS II activity (Ogweno et al. 2008, Hayat et al. 2010). In the present study, The HT stress caused an increase in the MDA content (Fig. 4A) suggesting that the HT stress caused an oxidative damage in the melon plants. Simultaneously, the HT stress increased the activities of SOD, POD, CAT, and APX (Fig. 5) and the EBR pretreatment further increased the activities of these four enzymes. EBR also reduced the MDA content in the leaves of the melon plants subjected to the HT stress. Our results are in coherence with the previous reports made by Ogweno et al. (2008) and Hayat et al. (2010), who observed the effects of brassinolides in HT-stressed tomato and Vigna radiata, respectively. Recently, BRs-regulated antioxidant systems under various biotic/abiotic stresses have been reviewed (Bajguz and Hayat 2009). The enhanced activities of antioxidative enzymes by BRs

seem to be the result of de novo synthesis and/or an activation of the enzymes mediated through transcription and/or translation of specific genes (Ali et al. 2008). It has been demonstrated that the expressions of CAT and cAPX are elevated by EBR in cucumber (Xia et al. 2009). ATPA2 and ATP24a genes, which encode peroxidases, are regulated by brassinosteroids in Arabidopsis (Goda et al. 2002). Mazorra et al. (2011) observed that thermotolerance in tomato is independent of endogenous BR content, but heat stress-mediated oxidative stress depends on BR. The mechanisms of BR-mediated resistance to oxidative stress require further studies. Proline accumulating under stress performs a clear role as a compatible osmolyte. However, the effects of BRs on proline metabolism have been poorly addressed and obtained results are often conflicting. Ábrahám et al. (2003) showed that the induction of proline biosynthesis by abscisic acid and a salt stress is inhibited by BRs in Arabidopsis. Li et al. (2012) also showed that an EBR pretreatment results in a smaller increase in the proline content in drought stressed Chorispora bungeana. However, EBR-increased the free proline content in rice and Raphanus sativus under different stresses (Farooq

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et al. 2009, Ramakrishna and Rao 2012). In this study, 0.1 - 1.5 mg dm-3 BRs could further increase the content of proline in the HT-treated plants (Fig. 4B) which can be supported by the findings of Hayat et al. (2010) that 28-homobrasisnolide (HBL) can enhance proline content in Vigna radiata under a HT stress. EBR-induced accumulation of proline under stress may be associated with a reduction in proline utilization due to the minimum protein formation, proline degradation, and enhancement in proline formation due to the hydrolysis of proteins (Shahid et al. 2011) and with the stimulation of the 1-pyrroline-5-carboxylate synthetase (Ramakrishna and Rao 2012). Increased growth and soluble protein content following the application of BRs under different conditions have been reported (Saygideger and Deniz

2008, Ramakrishna and Rao 2012). In the present investigation, EBR significantly increased the protein content as compared to the HT stress alone (Fig. 4C) which might be attributed to the activation of transcription and translational processes of specific stress tolerance genes (Kagale et al. 2007). In conclusion, a dose-dependent effects of EBR on the melon seedlings under the HT stress were observed, and the optimal concentration (1.0 mg dm-3 EBR) had protective effect on growth which was closely associated with the EBR-induced increase in photosynthetic efficiency and the activities of the ROS scavenging system. Furthermore, future research should be focused on the elucidation of EBR-induced thermotolerance on cellular and molecular levels.

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