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Abstract Chilling stress was one of the most sever abiotic stress to restrict cucumber plant growth. The effects of different concentrations of spermidine （0.1， 0.5， 1 and 5 mM） on photosynthetic characteristics， antioxidant （catalase， ascorbate peroxidase， superoxide dismutase and peroxidase） enzyme activities， malondialdehyde （MDA） content， electrolyte leakage and H2O2 content of cucumber seedlings under chilling stress were studied. The results showed that chilling stress reduced photosynthetic capacity and the value of the maximal photochemical efficiency of PSII （Fv/Fm）， and increased electrolyte leakage and the content of MDA and H2O2. Exogenous Spd application led to a tendency of photosynthetic characteristics and antioxidant enzyme activities to first increase and then decrease with the concentration of exogenous Spd increasing. Furthermore， electrolyte leakage and the contents of MDA and H2O2 significantly decreased in plants treated with Spd. Results of this study suggest that exogenous Spd can improve cucumber tolerance to chilling stress， and the optimal concentration of Spd to alleviate chilling stress in cucumber was 0.5 mM in the present study.
Key words Chilling stress; Exogenous spermindine; Cucumis sativus; Antioxidant enzyme
Received： August 2， 2018 Accepted： October 29， 2018
Supported by Shanghai Science and Technology Commission Project， China （18ZR1433200）; National Key Technology R&D Program （2014BAD05B0505）; Shanghai Agriculture Applied Technology Development Program， China （Grant No.20170201）.
Hong WANG （1979-）， female， P. R. China， PhD， devoted to research about regulation of light environment in greenhouse.
*Corresponding author. Email： wmzh69@126.com.
Cucumber is an important vegetable crop which must be grown yearround to meet market requirement. But cucumber is always threatened by adverse environmental stresses， among which chilling is one of the most devastating factors[1]. Chilling retards plant growth and restrict productivity worldwide. Accordingly， it is important to develop appropriate strategies to tackle the chilling stress.
Chilling stress can lead to series of physiological and biochemical changing to plant. It usually leads to membrane lipid peroxidation and accumulation of proline and malondialdehyde （MDA）[2] and significant changes of other cell components[3]. Furthermore， chilling stress can result in the increasing hydrogen peroxide （H2O2） accumulation in chilled leaves[4] and inhibit the electron transport of chloroplast[5]. When plants are suffering from chilling stress， it could modulate the content of endogenous hormones to increase the adaptation to chilling stress[6]， such as abscisic acid （ABA）[7]， polyamines （PAs）[8]， and their biosynthetic or responsive genes[9-10]. Polyamines （PAs） are low molecular weight and aliphatic nitrogen organic cation and also play an important role in the regulation of plant to adverse environmental stress[11]. Putrescine （Put）， spermidine （Spd） and spermine （Spm） are the main PAs， each of which may be present in a free， soluble conjugated or insoluble bound form[12]. PAs can alleviate osmotic damage caused by membrane lipid peroxidation and combine antioxidant enzymes to permeate to the sites of oxidant stress within cells[13]. Moreover， intensive work has revealed that PAs play important roles in protecting the photosynthetic apparatus from adverse effects of environmental stresses[14].
Previous studies have indicated that Spd was involved in the chilling stress. Shen et al.[15] reported that Spd content in cucumber leaves significantly increased in coldtolerant cultivars， while Put and Spm did not change during chilling. Zhang et al.[16] also indicated that exogenous Spd can reverse the increase of MDA content and electrolyte leakage caused by chilling. But the concentrations of exogenous Spd applied are different among cultivars examined and need further study[17-18]. So the objective of this study was to understand the effects of different concentrations of exogenous Spd on photosynthesis and antioxidative system in cucumber during chilling stress.
Materials and Methods
Plant material and chilling treatments
Cucumber （Cucumis sativus L.cv. Zhongnong No.26） seeds were germinated in a growth medium filled with a mixture of peat and vermiculite （2∶1， V∶V） in trays in an indoor growth chamber. When the first true leaf fully expanded， seedlings were transplanted into plastic pots （10 cm diameter and 10 cm deep， one seedling per pot） containing the same medium. The seedlings were watered daily with halfstrength Enshi nutrient solution. Temperature and relative humidity in the growth chamber were maintained at 25 ℃/20 ℃ （day/night） and 60%-70% （day/night）， respectively. After planting for 25 d， the seedlings were subjected to different treatments： ① normal temperature （25 ℃/20 ℃）; ② 8 ℃ chilling; ③ chilling+0.1 mM Spd; ④ chilling+0.5 mM Spd; ⑤ chilling+1mM Spd; ⑥ chilling+5 mM Spd for 8 d. All treatments were repeated for three times.
Measurements of gas exchange and chlorophyll fluorescence
CO2 assimilation （Pn）， stomatal conductance （gs） and intracellular CO2 concentration （Ci） were measured simultaneously on the 4th leaf with the portable photosynthesis system （LI6400; LiCOR Lincoln NE， USA）. Data were recorded at 25 ℃， with 200 μmol/（m 2·s） photosynthetic photon flux density （PPFD） and a reference CO2 concentration of 450 μmol/mol， using ambient humidity （ 40%-60%， relative humidity）. Chlorophyll fluorescence was measured using an ImagingPAM Chlorophyll Fluorometer comprising a computeroperated PAMcontrol unit （Walz， Effeltrich， Germany）. The seedlings were deposited in a dark room and were accommodated blackly for more than 20 min. The maximal photochemical efficiency of PSII （Fv/Fm） calculated according to the methods outlined by Ven （1990）[19].
Assays of antioxidant enzyme and H2O2
For the enzyme assays， 0.3 g of leaves were homogenized with 3 ml of icecold 25 mM potassium phosphate buffer （pH 7.8） containing 0.2 mM EDTA and 2% PVP （adding 2 mM AsA when measured Ascorbate peroxidase）. The homogenates were centrifuged at 12 000 g for 20 min at 4 °C and the obtained supernatants were used as enzyme extract.
Catalase （CAT， EC 1.11.1.6） activity was measured according to Patra et al.， by measuring the decrease in absorbance at 240 nm[20]. Ascorbate peroxidase （APX， EC 1.11.1.11） was analyzed according to Nakano and Asada by estimating the rate of ascorbate oxidation at 290 nm[21]. The activity of guaiacol peroxidase （GPOD， EC 1.11.1.7） was assayed according to the method of Cakmak and Marschner[22]. Superoxide dismutase （SOD， EC 1.15.1.1） was measured using the method of Giannopolitis and Ries[23]. One unit of SOD activity was defined as the amount of enzyme required to cause a 50% inhibition of the rate of nitroblue tetrazolium （NBT） reduction at 560 nm.
The content of H2O2 in leaves was assayed by monitoring the absorbance of the titaniumperoxide complex at 415 nm， using the method of Brennan and Frenkel[24].
Determination of soluble proteins， electrolyte leakage and MDA content
To determine membrane permeability， the electrolyte leakage from leaf tissues was measured by the electrical conductivity method. The content of malondialdehyde （MDA） was measured by the thiobarbituric acid reaction method according to Hodegs et al.[25].
Statistical analysis
The differences between treatments were established using ANOVA （analysis of variance）. Means separation was performed by the Duncans multiple range test （P<0.05）.
Results and Analysis
Effects of exogenous application of spermidine on photosynthetic capacity of cucumber seedlings under chilling stress
CO2 assimilation （Pn）， stomatal conductance （gs） and intracellular CO2 concentration （Ci） were measured （Fig. 1）. Chilling stress remarkably reduced seedling photosynthesis. For example， Pn and gs were 59.43% and 77.83% lower than that grown under control treatment. Furthermore， exogenous application of Spd significantly improved the Pn and gs under chilling stress. In general， the Pn and gs first increased and then declined with the concentration of exogenous Spd increasing. For example， the Pn and gs increased by 14.27%， 53.23%， 48.46%， 31.65% and 47.81%， 92.68%， 97.23%， 18.49% in 0.1 mM Spd， 0.5 mM Spd， 1 mM Spd and 5 mM Spd treatments compared with chilling treatment， respectively. In addition， there were no significant differences in Ci among different chilling treatments expect chilling+0.5 mM Spd treatment. The maximal PSII efficiency （Fv/Fm） was significantly affected by chilling stress. As shown in Fig. 2， Fv/Fm value deceased 58.8% in chilling treatment compared with control treatment. Moreover， exogenous application of Spd significantly increase the value of Fv/Fm with the increase of Spd concentration， but the differences among 0.5， 1， and 5 mM Spd treatments were not significant.
Effects of exogenous application of spermidine on MDA content and electrolyte leakage in cucumber leaves under chilling stress
The effects of chilling treatment on MDA content and electrolyte leakage were examined. As shown in Fig. 4， chilling stress caused relatively higher MDA content and increased electrolyte leakage. To some extent， exogenous Spd alleviated the chillinginduced increases in MDA content and electrolyte leakage. For example， 0.5 mM Spd decreased the MDA content and electrolyte leakage by 30.27% and 24.19%， respectively， compared with chillingtreated treatment.
Effects of exogenous application of spermidine on antioxidant enzyme activities in cucumber leaves under chilling stress
The activities of several representative enzymes， including CAT， APX， SOD and GPOD （Fig. 3）， were measured in cucumber leaves to determine the effects of exogenous Spd application on antioxidant enzymes under chilling stress. The activity of the antioxidant enzymes in leaves increased rapidly under chilling
stress. Furthermore， the exogenous Spd increased the activities of these four enzymes， and exogenous Spd initially increased and then decreased in a dosedependent effect. During chilling stress， CAT activity was the highest in 0.5 mM Spd treatment， and similar results were found in the activities of APX， SOD and GPOD. For example， treatment with 0.5 mM Spd increased the activities of CAT， APX， SOD and GPOD by 54.23%， 91.51%， 57.51% and 65.49%， respectively， compared with the treatment of chilling stress.
Effects of exogenous application of spermidine on H2O2 content in cucumber leaves under chilling stress
As shown in Fig. 5， chilling stress resulted in greater increases in H2O2 level， compared with the controls. Whereas treatment with exogenous Spd reduced the levels of H2O2. The levels of H2O2 decreased and then increased， with the minimum values in the plants treated with 0.3 mM Spd （Fig. 5）.
Discussions
In this study， chilling stress caused reductions in Pn， gs and the value of the maximal photochemical efficiency of PSII （Fv/Fm）， and increases in electrolyte leakage and the content of MDA and H2O2 in cucumber plants. Recent work indicated that exogenous Spd can increase plant tolerance to chilling stress[15]. One of the most rapid effects of chilling was the decrease of net photosynthetic rate. Allen and Ort[26] found that the declines in photosynthesis observed at cold temperatures have been attributed to energy dissipation， decrease enzymatic activities of Calvin cycle and the stomatal closure which compromises gas exchange and CO2 fixation. Exogenous Spd application led to an increase in Pn and stomata opening （Fig.1）. The decrease of photochemical efficiency of photosystem （PS） II （Fv/Fm） in chilling treatment indicates the reduced capacity of PS II to utilize incident light[27]. Laloi et al.[28] also reported that chilling inhibited the electron transport of chloroplast. This is accordance with our result that chilling significantly decreased Fv/Fm， and exogenous Spd application can increase the value of Fv/Fm to first increase and then decrease with the concentration of exogenous Spd increasing.
MDA content and electrolyte leakage can use as important indicators for the damage extent of ROS[29]. Our results showed that induced chilling increased the MDA content and electrolyte leakage （Fig.3）. In contrast， exogenous Spd application maintained a low level of MDA and electrolyte leakage， suggesting weak lipid peroxidation， in agreement with Mostofa et al.[30]. Furthermore， chilling stress could result in changes in the activities of antioxidant enzymes， including SOD， POD， APX and CAT， which may alleviate damages caused by ROS[31-32]. Our results showed that cucumber seedlings could reduce H2O2 content by enhancing the activities of SOD， POD and CAT during chilling stress （Fig. 4 and Fig. 5）， which was in accordance with the findings of Kumar et al.[33]and Xu et al.[34]. These results were approved by the findings of Mostofa et al.[30] who suggested that Spd enhanced heat tolerance in rice seedlings by increasing the activities of antioxidant enzymes. H2O2 was emerged through SOD action and was further scavenged by CAT. Our results showed that the content of H2O2 significantly decreased in plants treated with Spd. The concentration of H2O2 was decreased and then increased with concentration of exogenous Spd increasing， and this defense response was in agreement with the increase in antioxidant enzyme activities. Results of this study suggest that exogenous Spd can increase cucumber tolerance to chilling stress， and the optimal concentration of Spd to alleviate chilling stress in cucumber was 0.5 mM in our present study.
References
[1] Xu PL， Guo， YK， Bai JG， et al. Effects of longterm chilling on ultrastructure and antioxidant activity in leaves of two cucumber cultivars under low light[J]. Physiologia Plantarum， 2008， 132： 467-478. [2] Dai AH， Nie YX， Yu B， et al. Cinnamic acid pretreatment enhances heat tolerance of cucumber leaves through modulating antioxidant enzyme activity[J]. Environmental and Experimental Botany， 2012， 79： 1-10.
[3] Lee SH， Singh AP， Chung GC， et al. Chilling root temperature causes rapid ultrastructural changes in cortical cells of cucumber （Cucumis sativus L.） root tips[J]. Journal of Experimental Botany， 2002， 53： 2225-2237.
[4] Zhou YH， Yu JQ， Mao WH， et al. Genotypic variation of Rubisco expression， photosynthetic electron flow and antioxidant metabolism in the chloroplasts of chillexposed cucumber plants[J]. Plant and Cell Physiology， 2006， 47： 192-199. 2006.
[5] Kudoh H， Sonoike K. Irreversible damage to photosystem I by chilling in the light： cause of the degradation of chlorophyll after returning to normal growth temperature[J]. Planta， 2002， 215： 541-548.
[6] Wang F， Cheng FM， Liu YI， et al. Dynamic changes of plant hormones in developing grains at rice filling stage under different temperature[J]. Acta Agronomica Sinca， 2006， 1： 25–29.
[7] Anderson MD， Prasad TK， Martin BA， et al. Differential gene expression in chillingacclimated maize seedlings and evidence for the involvement of abscisic acid in chilling tolerance[J]Plant Physiology， 1994， 105： 331-339.
[8] Cuevas JC， LopezCobollo R， Alcazar R， et al. Putrescine is involved in Arabidopsis freezing tolerance and cold acclimation by regulating ABA levels in response to low temperature[J]. Plant Physiology， 2008， 148： 1094-1105.
[9] Moschou PN， Delis ID， Paschalidis KA， et al. Transgenic tobacco plants overexpressing polyamine oxidase are not able to cope with oxidative burst generated by abiotic factors[J]. Physiologia Plantarum， 2008， 133： 140-156.
[10] Moschou PN， Paschalidis KA， Delis ID， et al. Spermidine exodus and oxidation in the apoplast induced by abiotic stress is responsible for H2O2 signatures that direct tolerance responses in tobacco[J]. Plant Cell， 2008， 20： 1708-1724.
[11] Puyang X， A M， Han L， et al. Protective effect of spermidine on salt stress induced oxidative damage in two Kentucky bluegrass （Poa pratensis L.） cultivars[J]. Ecotoxicology and environmental safety， 2015， 117： 96-106.
[12] Groppa MD， Benavides MP. Polyamines and abiotic stress： recent advances[J]. Amino Acids， 2008， 34： 35-45.
[13] Mostofa MG， Yoshida N， Fujita M. Spermidine pretreatment enhances heat tolerance in rice seedlings through modulating antioxidative and glyoxalase systems[J]. Plant Growth Regulation， 2014， 73： 31-44. [14] He L， Nada K， Tachibana S. Effects of Spd pretreatment through the roots on growth and photosynthesis of chilled cucumber plants （Cucumis sativus L.）[J]. Journal of the Japanese Society for Horticultural Science， 2002， 71： 490-498.
[15] Shen W， Nada K， Tachibana S. Involvement of polyamines in the chilling tolerance of cucumber cultivars[J]. Plant Physiology， 2000， 124： 431-439.
[16] Zhang W， Jiang B， Li W， et al. Polyamines enhance chilling tolerance of cucumber （Cucumis sativus L.） through modulating antioxidative system[J]. Scientia Horticulturae， 2009， 122： 200-208.
[17] Duan J， Li J， Guo S， et al. Exogenous spermidine affects polyamine metabolism in salinitystressed Cucumis sativus roots and enhances shortterm salinity tolerance[J]. Journal of plant physiology， 165： 1620-1635.
[18] Zrig A， Tounekti T， Vadel AM， et al. Possible involvement of polyphenols and polyamines in salt tolerance of almond rootstocks[J]. Plant Physiology and Biochemistry， 2011， 49： 1313-1322.
[19] Van KO， Snel JFH. The use of chlorophyll fuorescence nomenclature in plant stress physiology[J]. Photosynthesis research， 1990， 25： 147-150.
[20] Patra H K， Kar M， Mishra D. Catalase activity in leaves and cotyledons during plant development and senescence[J]. Biochemie und Physiologie der Pflanzen， 1978， 172： 385-390.
[21] Nakano Y， Asada K. Hydrogen peroxide is scavenged by ascorbatespecific peroxidase in spinach chloroplasts[J]. Plant and Cell Physiology， 1981， 22： 867-880.
[22] Cakmak I， Marschner H. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase， ascorbate peroxidase， and glutathione reductase in bean leaves 1[J]. Plant Physiology， 1992， 98： 1222-1227.
[23] Giannopolitis CN， Ries SK. Superoxide dismutases： I. occurrence in higher plants[J]. Plant Physiology， 1997， 59： 309-314.
[24] Brennan T， Frenkel C. Involvement of hydrogen peroxide in the regulation of senescence in Pear 1[J]. Plant Physiology， 1977， 59： 411-416.
[25] Hodges DM， Delong JM， Forney CF， et al. Improving the thiobarbituric acidreactivesubstances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds[J]. Planta， 1999， 207： 604-611.
[26] Allen DJ， Ort DR. Impacts of chilling temperatures on photosynthesis in warmclimate plants[J]. Trends in Plant Science， 2000， 6（1）： 36-42.
[27] Jung S， Steffen KL， Jae Lee H. Comparative photoinhibition of a high and a low altitude ecotype of tomato （Lycopersicon hirsutum） to chilling stress under high and low light conditions[J]. Plant Science， 1998， 134（1）： 69-77. [28] Laloi C， Apel K， Danon A. Reactive oxygen signaling： The latest news[J]. Current Opinon in Plant Biology， 2004， 7： 323-328.
[29] Dai AH， Nie YX， Yu B， et al. Cinnamic acid pretreatment enhances heat tolerance of cucumber leaves through modulating antioxidant enzyme activity[J]. Environmental and Experimental Botany， 2012， 79： 1-10.
[30] Mostofa MG， Yoshida N， Fujita M. Spermidine pretreatment enhances heat tolerance in rice seedlings through modulating antioxidative and glyoxalase systems[J]. Plant Growth Regulation， 2014， 73： 31-44.
[31] Qiu J， Wang RM， Yan JZ， et al. Seed film coating with uniconazole improves rape seedling growth in relation to physiological changes under waterlogging stress[J]. Plant Growth Regulation， 2005， 47： 75-81.
[32] Guo ZF， Ou WB， Lu SY. Differential responses of antioxidative system to chilling and drought in four rice cultivars differing in sensitivity[J]. Plant Physiology and Biochemistry， 2006， 44： 828-836.
[33] Kumar S， Kaur R， Kaur N， et al. Heatstress induced inhibition in growth and chlorosis in mungbean （Phaseolus aureus Roxb.） is partly mitigated by ascorbic acid application and is related to reduction in oxidative stress[J]. Acta Physiology Plant， 2011， 33： 2091-2101.
[34] Xu SC， Hu J， Li YP， et al. Chilling tolerance in Nicotiana tabacum induced by seed priming with putrescine[J]. Plant Growth Regulation， 2011， 63： 279-290.
Key words Chilling stress; Exogenous spermindine; Cucumis sativus; Antioxidant enzyme
Received： August 2， 2018 Accepted： October 29， 2018
Supported by Shanghai Science and Technology Commission Project， China （18ZR1433200）; National Key Technology R&D Program （2014BAD05B0505）; Shanghai Agriculture Applied Technology Development Program， China （Grant No.20170201）.
Hong WANG （1979-）， female， P. R. China， PhD， devoted to research about regulation of light environment in greenhouse.
*Corresponding author. Email： wmzh69@126.com.
Cucumber is an important vegetable crop which must be grown yearround to meet market requirement. But cucumber is always threatened by adverse environmental stresses， among which chilling is one of the most devastating factors[1]. Chilling retards plant growth and restrict productivity worldwide. Accordingly， it is important to develop appropriate strategies to tackle the chilling stress.
Chilling stress can lead to series of physiological and biochemical changing to plant. It usually leads to membrane lipid peroxidation and accumulation of proline and malondialdehyde （MDA）[2] and significant changes of other cell components[3]. Furthermore， chilling stress can result in the increasing hydrogen peroxide （H2O2） accumulation in chilled leaves[4] and inhibit the electron transport of chloroplast[5]. When plants are suffering from chilling stress， it could modulate the content of endogenous hormones to increase the adaptation to chilling stress[6]， such as abscisic acid （ABA）[7]， polyamines （PAs）[8]， and their biosynthetic or responsive genes[9-10]. Polyamines （PAs） are low molecular weight and aliphatic nitrogen organic cation and also play an important role in the regulation of plant to adverse environmental stress[11]. Putrescine （Put）， spermidine （Spd） and spermine （Spm） are the main PAs， each of which may be present in a free， soluble conjugated or insoluble bound form[12]. PAs can alleviate osmotic damage caused by membrane lipid peroxidation and combine antioxidant enzymes to permeate to the sites of oxidant stress within cells[13]. Moreover， intensive work has revealed that PAs play important roles in protecting the photosynthetic apparatus from adverse effects of environmental stresses[14].
Previous studies have indicated that Spd was involved in the chilling stress. Shen et al.[15] reported that Spd content in cucumber leaves significantly increased in coldtolerant cultivars， while Put and Spm did not change during chilling. Zhang et al.[16] also indicated that exogenous Spd can reverse the increase of MDA content and electrolyte leakage caused by chilling. But the concentrations of exogenous Spd applied are different among cultivars examined and need further study[17-18]. So the objective of this study was to understand the effects of different concentrations of exogenous Spd on photosynthesis and antioxidative system in cucumber during chilling stress.
Materials and Methods
Plant material and chilling treatments
Cucumber （Cucumis sativus L.cv. Zhongnong No.26） seeds were germinated in a growth medium filled with a mixture of peat and vermiculite （2∶1， V∶V） in trays in an indoor growth chamber. When the first true leaf fully expanded， seedlings were transplanted into plastic pots （10 cm diameter and 10 cm deep， one seedling per pot） containing the same medium. The seedlings were watered daily with halfstrength Enshi nutrient solution. Temperature and relative humidity in the growth chamber were maintained at 25 ℃/20 ℃ （day/night） and 60%-70% （day/night）， respectively. After planting for 25 d， the seedlings were subjected to different treatments： ① normal temperature （25 ℃/20 ℃）; ② 8 ℃ chilling; ③ chilling+0.1 mM Spd; ④ chilling+0.5 mM Spd; ⑤ chilling+1mM Spd; ⑥ chilling+5 mM Spd for 8 d. All treatments were repeated for three times.
Measurements of gas exchange and chlorophyll fluorescence
CO2 assimilation （Pn）， stomatal conductance （gs） and intracellular CO2 concentration （Ci） were measured simultaneously on the 4th leaf with the portable photosynthesis system （LI6400; LiCOR Lincoln NE， USA）. Data were recorded at 25 ℃， with 200 μmol/（m 2·s） photosynthetic photon flux density （PPFD） and a reference CO2 concentration of 450 μmol/mol， using ambient humidity （ 40%-60%， relative humidity）. Chlorophyll fluorescence was measured using an ImagingPAM Chlorophyll Fluorometer comprising a computeroperated PAMcontrol unit （Walz， Effeltrich， Germany）. The seedlings were deposited in a dark room and were accommodated blackly for more than 20 min. The maximal photochemical efficiency of PSII （Fv/Fm） calculated according to the methods outlined by Ven （1990）[19].
Assays of antioxidant enzyme and H2O2
For the enzyme assays， 0.3 g of leaves were homogenized with 3 ml of icecold 25 mM potassium phosphate buffer （pH 7.8） containing 0.2 mM EDTA and 2% PVP （adding 2 mM AsA when measured Ascorbate peroxidase）. The homogenates were centrifuged at 12 000 g for 20 min at 4 °C and the obtained supernatants were used as enzyme extract.
Catalase （CAT， EC 1.11.1.6） activity was measured according to Patra et al.， by measuring the decrease in absorbance at 240 nm[20]. Ascorbate peroxidase （APX， EC 1.11.1.11） was analyzed according to Nakano and Asada by estimating the rate of ascorbate oxidation at 290 nm[21]. The activity of guaiacol peroxidase （GPOD， EC 1.11.1.7） was assayed according to the method of Cakmak and Marschner[22]. Superoxide dismutase （SOD， EC 1.15.1.1） was measured using the method of Giannopolitis and Ries[23]. One unit of SOD activity was defined as the amount of enzyme required to cause a 50% inhibition of the rate of nitroblue tetrazolium （NBT） reduction at 560 nm.
The content of H2O2 in leaves was assayed by monitoring the absorbance of the titaniumperoxide complex at 415 nm， using the method of Brennan and Frenkel[24].
Determination of soluble proteins， electrolyte leakage and MDA content
To determine membrane permeability， the electrolyte leakage from leaf tissues was measured by the electrical conductivity method. The content of malondialdehyde （MDA） was measured by the thiobarbituric acid reaction method according to Hodegs et al.[25].
Statistical analysis
The differences between treatments were established using ANOVA （analysis of variance）. Means separation was performed by the Duncans multiple range test （P<0.05）.
Results and Analysis
Effects of exogenous application of spermidine on photosynthetic capacity of cucumber seedlings under chilling stress
CO2 assimilation （Pn）， stomatal conductance （gs） and intracellular CO2 concentration （Ci） were measured （Fig. 1）. Chilling stress remarkably reduced seedling photosynthesis. For example， Pn and gs were 59.43% and 77.83% lower than that grown under control treatment. Furthermore， exogenous application of Spd significantly improved the Pn and gs under chilling stress. In general， the Pn and gs first increased and then declined with the concentration of exogenous Spd increasing. For example， the Pn and gs increased by 14.27%， 53.23%， 48.46%， 31.65% and 47.81%， 92.68%， 97.23%， 18.49% in 0.1 mM Spd， 0.5 mM Spd， 1 mM Spd and 5 mM Spd treatments compared with chilling treatment， respectively. In addition， there were no significant differences in Ci among different chilling treatments expect chilling+0.5 mM Spd treatment. The maximal PSII efficiency （Fv/Fm） was significantly affected by chilling stress. As shown in Fig. 2， Fv/Fm value deceased 58.8% in chilling treatment compared with control treatment. Moreover， exogenous application of Spd significantly increase the value of Fv/Fm with the increase of Spd concentration， but the differences among 0.5， 1， and 5 mM Spd treatments were not significant.
Effects of exogenous application of spermidine on MDA content and electrolyte leakage in cucumber leaves under chilling stress
The effects of chilling treatment on MDA content and electrolyte leakage were examined. As shown in Fig. 4， chilling stress caused relatively higher MDA content and increased electrolyte leakage. To some extent， exogenous Spd alleviated the chillinginduced increases in MDA content and electrolyte leakage. For example， 0.5 mM Spd decreased the MDA content and electrolyte leakage by 30.27% and 24.19%， respectively， compared with chillingtreated treatment.
Effects of exogenous application of spermidine on antioxidant enzyme activities in cucumber leaves under chilling stress
The activities of several representative enzymes， including CAT， APX， SOD and GPOD （Fig. 3）， were measured in cucumber leaves to determine the effects of exogenous Spd application on antioxidant enzymes under chilling stress. The activity of the antioxidant enzymes in leaves increased rapidly under chilling
stress. Furthermore， the exogenous Spd increased the activities of these four enzymes， and exogenous Spd initially increased and then decreased in a dosedependent effect. During chilling stress， CAT activity was the highest in 0.5 mM Spd treatment， and similar results were found in the activities of APX， SOD and GPOD. For example， treatment with 0.5 mM Spd increased the activities of CAT， APX， SOD and GPOD by 54.23%， 91.51%， 57.51% and 65.49%， respectively， compared with the treatment of chilling stress.
Effects of exogenous application of spermidine on H2O2 content in cucumber leaves under chilling stress
As shown in Fig. 5， chilling stress resulted in greater increases in H2O2 level， compared with the controls. Whereas treatment with exogenous Spd reduced the levels of H2O2. The levels of H2O2 decreased and then increased， with the minimum values in the plants treated with 0.3 mM Spd （Fig. 5）.
Discussions
In this study， chilling stress caused reductions in Pn， gs and the value of the maximal photochemical efficiency of PSII （Fv/Fm）， and increases in electrolyte leakage and the content of MDA and H2O2 in cucumber plants. Recent work indicated that exogenous Spd can increase plant tolerance to chilling stress[15]. One of the most rapid effects of chilling was the decrease of net photosynthetic rate. Allen and Ort[26] found that the declines in photosynthesis observed at cold temperatures have been attributed to energy dissipation， decrease enzymatic activities of Calvin cycle and the stomatal closure which compromises gas exchange and CO2 fixation. Exogenous Spd application led to an increase in Pn and stomata opening （Fig.1）. The decrease of photochemical efficiency of photosystem （PS） II （Fv/Fm） in chilling treatment indicates the reduced capacity of PS II to utilize incident light[27]. Laloi et al.[28] also reported that chilling inhibited the electron transport of chloroplast. This is accordance with our result that chilling significantly decreased Fv/Fm， and exogenous Spd application can increase the value of Fv/Fm to first increase and then decrease with the concentration of exogenous Spd increasing.
MDA content and electrolyte leakage can use as important indicators for the damage extent of ROS[29]. Our results showed that induced chilling increased the MDA content and electrolyte leakage （Fig.3）. In contrast， exogenous Spd application maintained a low level of MDA and electrolyte leakage， suggesting weak lipid peroxidation， in agreement with Mostofa et al.[30]. Furthermore， chilling stress could result in changes in the activities of antioxidant enzymes， including SOD， POD， APX and CAT， which may alleviate damages caused by ROS[31-32]. Our results showed that cucumber seedlings could reduce H2O2 content by enhancing the activities of SOD， POD and CAT during chilling stress （Fig. 4 and Fig. 5）， which was in accordance with the findings of Kumar et al.[33]and Xu et al.[34]. These results were approved by the findings of Mostofa et al.[30] who suggested that Spd enhanced heat tolerance in rice seedlings by increasing the activities of antioxidant enzymes. H2O2 was emerged through SOD action and was further scavenged by CAT. Our results showed that the content of H2O2 significantly decreased in plants treated with Spd. The concentration of H2O2 was decreased and then increased with concentration of exogenous Spd increasing， and this defense response was in agreement with the increase in antioxidant enzyme activities. Results of this study suggest that exogenous Spd can increase cucumber tolerance to chilling stress， and the optimal concentration of Spd to alleviate chilling stress in cucumber was 0.5 mM in our present study.
References
[1] Xu PL， Guo， YK， Bai JG， et al. Effects of longterm chilling on ultrastructure and antioxidant activity in leaves of two cucumber cultivars under low light[J]. Physiologia Plantarum， 2008， 132： 467-478. [2] Dai AH， Nie YX， Yu B， et al. Cinnamic acid pretreatment enhances heat tolerance of cucumber leaves through modulating antioxidant enzyme activity[J]. Environmental and Experimental Botany， 2012， 79： 1-10.
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