Introduction

Salt stress is one of the major environmental factor limiting crop growth and yield in the worldwide (Parida and Das 2005). Salt affected plants show symptoms of small leaves, leaf necrosis (Maliro et al. 2008). The salt-induced decrease in growth may involve several physiological dysfunctions caused by the ionic toxicity and alterations in the ionic balance and water status in the rhizosphere (Feng et al. 2007; Geissler et al. 2009). It has been well documented that salinity often causes a decrease in photosynthesis of plants (Sudhir and Murthy 2004; Shu et al. 2013). Salt stress induced the inhibition of photosynthesis through stomatal limitation (Li et al. 2007) and non-stomatal limitations including stomatal closure, photosynthetic pigment loss, reduction of Rubisco activity, inhibition of carbon assimilation, and degradation of membrane proteins in the photosynthetic apparatus (Mittal et al. 2012; Gil et al. 2013).

Polyamines (PAs) are ubiquitous low-molecular-weight aliphatic amines that are involved in a wide array of fundamental processes in plants, such as replication and gene expression, plant growth and development, senescence and response to abiotic and biotic stresses (Kusano et al. 2007; Takahashi et al. 2010). In higher plants, the most commonly PAs are the diamine putrescine (Put), triamine spermidine (Spd), and tetraamine spermine (Spm), presenting in the free, soluble conjugated and insoluble bound forms (Shu et al. 2012). Among the three major forms of PAs, Spd is closely associated with stress tolerance in plants (Kasukabe et al. 2004). Foliar application of Spd alleviated salinity-induced damages to maize (Jiang et al. 2000) and cucumber plants (Duan et al. 2008). Chattopadhayay et al. (2002) found that salinity-induced injury of rice was greatly mitigated by adding Spd to salinized nutrient solution. Exogenous Spd plays vital roles in preventing the electrolyte and/or amino acid leakage or recovering the plasma membrane damage in three varieties of indica rice cultivars in response to salinity (Roychoudhury et al. 2011).

It has been suggested that higher plants sprayed with Spd can alleviate salinity-induced decrease in photosynthetic efficiency to some extent, but this effect strongly depends on Spd concentrations and stress levels. The potential role of PAs in maintaining the photochemical efficiency of stressed-plants has become a research focus. Duan et al. (2008) showed that exogenous application of Spd increased net photosynthetic rate and actual photochemical efficiency of PSII in salt-stressed cucumber seedlings. Beauchemin et al. (2007) reported that high concentration of Spd was shown to elicit strong inhibitory effects on PSII sub-membrane fractions of spinach. Moreover, exogenous Spd involved in the regulation of plant stomatal opening, and reduced negative feedback inhibition to photosynthesis caused by carbohydrate accumulation in leaves (Chen et al. 2011).

However, little information is currently available in literature regarding the possible mechanisms of exogenous Spd alleviating salt stress on inhibition of carbon assimilation in leaves. In the present study, cucumber, an important horticultural crop, was selected as the plant material because it is highly sensitive to salinity (Duan et al. 2008). Therefore, we examined the ability of cucumber plants to achieve salinity tolerance by applying exogenous Spd. The purpose of this study was to analyze the role of exogenous Spd on photosynthetic carbon assimilation of cucumber seedlings under salt stress.

Materials and methods

Plant materials and treatments

Cucumber plants (Cucumis sativus L. cv. Jinchun No. 2, a salinity-sensitive cultivar) were cultured in a temperature-controlled greenhouse under natural light conditions at Nanjing Agricultural University. The seeds were placed in Petri dishes that were lined with two layers of filter paper moistened with sterile water, and the seeds were germinated in a growth chamber at 29 ± 1°C. The germinated seeds were then sown in washed quartz sand. Seedlings of similar size with two fully expanded true leaves were transplanted to troughs (the length, width and depth are 95, 45 and 16 cm, respectively) containing 65 L of half-strength Hoagland solution (pH 6.5 ± 0.1, electric conductivity 2.0–2.2 dS m−1). The air temperatures in the greenhouse were kept at 28 ± 1 °C/19 ± 1 °C (day/night), 14/10 h (day/night) photoperiod was imposed with a maximum photosynthetic photon flux density (PPFD) of about 1,200 μmol m−2 s−1 and relative air humidity fluctuated between 60 and 75 %. The nutrient solution was maintained between 20 and 25 °C and it was aerated with an air pump at intervals of 30 min to keep dissolved oxygen at 8.0 ± 0.2 mg L−1.

After 2 days pre-culture, seedlings received four different nutrient solution treatments as follows: (1) Cont, half-strength Hoagland solution; (2) Spd, half-strength Hoagland solution with 0.1 mM Spd (Sigma Chemical Co.); (3) NaCl, half-strength Hoagland solution with 50 mM NaCl; and (4) NaCl + Spd, half-strength Hoagland solution with 50 mM NaCl and 0.1 mM Spd. The respective analyses were carried out in triplicate using 36 plants per treatment. The nutrient solution was renewed every 2 days. After 9 days of salt treatment, the third fully expended leaf from the top of seedlings was used for analysis the growth parameters, photosynthesis, and chlorophyll fluorescence. Samples used for chemical analyses were kept frozen at −80 °C until analysis.

Measurement of growth parameters

The following parameters were measured using a scanner (Epson Eepression 1680) and image analysis software (WinRHIZO, Regent Instruments, Canada): root surface area, leaf area of the third fully expanded leaves. For determination of fresh weight, shoots and roots were separated and weighed after being washed with sterile distilled water. The dry weight was obtained after drying at 75 °C in an oven for 72 h.

Measurement of photosynthesis and chlorophyll fluorescence parameters

Net photosynthetic rates (P N ) was measured using a portable photosynthesis system LI-6400 (Li-COR lnc., Lincoln, NE, USA) that maintained external CO2 concentration at 380 ± 10 μmol mol−1 and light intensity at 1,000 μmol photons m−2 s−1. The relative contribution of stomatal limitation value (Ls) to photosynthesis, which is the proportional decrease in light-saturated (A) attributed to stomatal component, was calculated according to Farquhar and Sharkey (1982): Ls = 1 − Ci/Ca, where, Ca is the ambient CO2 concentration.

Chlorophyll fluorescence was measured with a portable fluorometer (PAM-2100, Walz, Effeltrich, Germany) on dark-adapted leaves just before the onset of the light period as described by Lu et al. (2003). Minimal fluorescence (Fo) was determined by illuminating the leaf with a dim red light modulated at 0.6 kHz. Maximal fluorescence of a dark-adapted leaf (Fm) was obtained during a subsequent saturating light pulse (0.8 s; 8,000 μmol m−2 s−1). The following parameters were calculated according to the following formulas as described by Kooten and Snel (1990). The maximal photochemical efficiency of PSII [Fv/Fm = (Fm − Fo)/Fm], non-photochemical quenching coefficient [qN = (Fm − Fm′)/(Fm − Fo′)], photochemical quenching coefficient [qP = (Fm′ − Fs)/(Fm′ − Fo′)] and actual photochemical efficiency of PSII [ΦPSII = (Fm′ − Fs)/Fm′].

Internal-CO2–response curves of photosynthesis (A/Ci)

The CO2 response curves were measured by stepping down CO2 from 350 to 250, 200, 150, 100 and 50 μmol mol−1, and then returned to 350 μmol mol−1 for 4 min re-acclimation until the initial state was attained. The CO2 was then stepped up to saturation from 350 to 500, 700, 900 and 1,200 μmol mol−1. The measurements were taken at constant PPFD of 1,000 μmol m−2 s−1, ambient relative humidity (50 %), and leaf temperature of 25 °C, using the LI-COR auto program ‘A/Ci-curve’. The maximum rate of Rubisco activity (Vcmax), light saturated rate of electron transport (Jmax), carboxylation efficiency (CE) and the triose-phosphate utilization rate (TPU) were derived from A/Ci curves with the help of PHOTOSYN software version 1.1.2 (Dundee Scientific Ltd., Dundee, UK) that employs the Farquhar biochemical model as modified by Dubois et al. (2007).

Analysis of Rubisco and aldolase activities

To analyze Rubisco activity, a method described by Wang et al. (2009) with a slight modification. Leaf samples (0.2 g of fresh weight) were used to assay Rubisco activity. The extraction buffer for Rubisco activity measurement consisted 33 mM Tris–HCl (pH 7.5), 0.67 mM Na2-EDTA, 33 mM MgCl2 and 10 mM NaHCO3. The initial activity was measured in 0.15 mL of a reaction mixture consisting of 100 mM Tris–HCl (pH 8.0), 2 mM Na2-EDTA, 20 mM MgCl2, 10 mM dithiothreitol (DTT), 10 mM ATP, 0.4 mM NADH, 140 mM NaHCO3, 1 U of 3-phosphoglyceric phosphokinase, 2.5 U of glyceraldehyde 3-phosphate dehydrogenase, 0.06 mM ribulose 1,5-bisphosphate (RuBP) and 25 μL of enzyme solution. The enzyme activity was estimated by following the decrease in A340 for 3 min. Aldolase activity was assayed according to Mustroph and Albrecht (2003). The reaction was started by adding 200 µL of 5 mM d-fructose 1,6-biphosphate, and the aldolase activity was estimated by following the decrease in A340 for 1 min.

Total soluble protein content was measured by using Bradford method (Bradford 1976).

Measurement of Rubisco and aldolase contents

Protein was extracted from leaves using a mixture consisting of 0.5 M Tris–HCl (pH 6.8), 20 % (v/v) glycerol, 2 % (w/v) sodium dodecyl sulfate (SDS), 5 % (v/v) β-mercaptoethanol and 0.01 % (w/v) bromophenol blue. The quantified protein was then denatured at 95 °C in a water bathing for 3–5 min and it was then stored at –20 °C until analysis.

SDS–polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (1970) using a 5 % stacking gel and a 12.5 % separating gel. The SDS-PAGE was performed to separate stored protein as described above. After electrophoresis, protein bands were visualized with Coomassie Blue R250, and the stained gels were scanned with an automatic gel image analyzer (model number JS2380, Shanghai Peiqing Technology Company, China) to estimate the protein levels.

The expression levels of Rubisco and aldolase was assayed by western Blot analysis. Proteins (15 μg from each sample) separated by SDS-PAGE were transferred to a 0.45 μm PVDF membrane and detected with antibodies raised against Rubisco or aldolase. The membrane was blocked with 5 % nonfat dry milk for 2 h and was then washed with TBST three times. The membrane was then probed with either a rabbit anti-Rubisco (1:3,000 dilution) or a rabbit anti-aldolase antibody (1:2,000 dilution) in TBST supplemented with 5 % nonfat dry milk. After an overnight incubation at 4 °C, the membrane was washed with TBST and incubated at room temperature for 1 h with a goat anti-rabbit IgG HRP-conjugate (1:1,000 dilution) in TBST supplemented with 5 % nonfat dry milk. The membrane was then washed with TBST three times and developed with DAB and H2O2.

RT-PCR analysis

Total RNA was extracted from leaves as described in the TRI reagent protocol (Takara Bio Inc). For all samples, the total RNA (l μg) was converted to cDNA using a Superscript first-strand synthesis system for RT-PCR according to the manufacturer’s instructions (Takara Bio Inc). Primers were designed from the peptide sequences obtained after mass analysis. PCR conditions were optimized for each primer set. PCR was carried out after denaturing cDNA at 94 °C for 5 min. The PCR consisting of 30 cycles of the following temperature: 94 °C for 30 s, annealing temperature as shown in Table 1 for 30 s, and extension at 72 °C for 35 s. The final PCR extension step was at 72 °C for 7 min. This amplified cDNA fragments were isolated using agarose gel electrophoresis.

Table 1 Design of primers for targeted genes
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Statistical analysis

Measurements were performed in triplicate except for growth analyses, which were performed with ten plants. All data were statistically analyzed with SAS software (SAS institute, Cary, NC) using Duncan’s multiple range test at the P < 0.05 level of significance.

Results

Growth of cucumber seedlings

As shown in Table 2, salt stress treatment for 9 days significantly reduced fresh weight, dry weight, shoot height, leaf area and root surface area of cucumber plants as compared to the control. However, the subsequent supply of 0.1 mM Spd to nutrient solution alleviated the inhibition of plant growth induced by salinity. Under the control conditions, Spd exerted no significant effects on growth parameters.

Table 2 Effects of exogenous Spd on growth and relative water content of cucumber seedlings under salt stress for 9 days
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Gas exchange parameters

As shown in Fig. 1a–f, compared to the control, salt stress caused a significant decrease in net photosynthesis rates (P N ), stomatal conductance (Gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr) of cucumber leaves by 58.56, 68.17, 33.52 and 51.91 %, respectively. Exogenous Spd alleviated the salt stress-induced the decrease in P N , Gs and Tr, but no significant effects on Ci, WUE and Ls. Spd application to the control plants did not affect these gas exchange parameters of cucumber plants.

Fig. 1
figure 1

Effects of Spd on gas exchange parameters of cucumber seedlings under salt stress. The respective parameters were measured at the day 9 after the start of 50 mM NaCl and/or 0.1 mM Spd treatments was added to the Hoagland’s solution. Vertical bars represent the mean ± SD of results from three independent experiments. Different letters indicate significant differences at P < 0.05, according to Duncan’s multiple range tests. P N , net photosynthesis rates (a); Gs, stomatal conductance (b); Ci, intercellular CO2 concentration (c); Tr, transpiration rate (d); WUE, water use efficiency (e); Ls, stomatal limitation value (f). Cont, Spd, NaCl and NaCl + Spd were as described in “Materials and methods”. Spd, 0.1 mM; NaCl, 50 mM

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Chlorophyll fluorescence

As shown in Table 3, the maximal photochemical efficiency of PSII (Fv/Fm) showed no significant change in salt-stressed plants. As compared to the control, salt stress significantly decreased the photochemical quenching coefficient (qP) and actual photochemical efficiency of PSII (ΦPSII), but markedly increased the non-photochemical quenching coefficient (qN) in cucumber leaves. Application of exogenous Spd significantly increased the values of qP and ΦPSII, and decreased qN value of plants with NaCl. There were no significant differences in chlorophyll fluorescence parameters between the control plants and those supplied with Spd.

Table 3 Effects of Spd on chlorophyll fluorescences of cucumber seedlings under salt stress for nine days
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Internal-CO2–response curves (P N /Ci)

The response of net photosynthetic rate to different intercellular CO2 concentration was examined (Fig. 2). The net photosynthetic rate with incremental increases in intercellular CO2 concentrations showed saturation and a decrease in P N at higher CO2 concentrations. Salt stress significantly caused the inhibition in CO2–response curves (P N /Ci) of cucumber seedlings as compared with the control. However, exogenous Spd alleviated salt-induced inhibition of the CO2–response curves. Calculated values of Vcmax, Jmax, TPU and CE obtained from CO2–response curves. As compared to the control, these values of Vcmax, Jmax, TPU and CE in salt-stressed cucumber leaves were decreased by 54.6, 65.9, 78.4 and 68.9 %, respectively (Table 4). Application of exogenous Spd significantly increased the values of Vcmax, Jmax, TPU and CE of plants with NaCl. There were no significant differences in these parameters between the control plants and those applied Spd to salinized nutrient solution.

Fig. 2
figure 2

Effect of exogenous Spd on photosynthetic response to CO2 (P N –Ci) of cucumber seedlings under salt stress. The respective parameters were measured at the day 9 after the start of 50 mM NaCl and/or 0.1 mM Spd treatments was added to the Hoagland’s solution. P N , Ci, Cont, Spd, NaCl and NaCl + Spd were as described in “Materials and methods”. Spd, 0.1 mM; NaCl, 50 mM

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Table 4 Effects of exogenous Spd on Vcmax, Jmax, TPU and CE of photosynthetic response to CO2 of cucumber seedlings under salt stress for 9 days
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Contents and activities of Rubisco, aldolase

Rubisco activity of cucumber leaves increased after three days of salinity stress, but decreased at 6 and 9 days (Fig. 3a). Salinity changed Rubisco activity to 162.16, 74.13 and 58.62 % at three, six and nine days compared with the control. Exogenous Spd applied to salinized nutrient solution significantly elevated Rubisco activity. Salt also decreased aldolase activity to 67.51, 78.74 and 74.37 % compared with the control at three, six and nine days, respectively (Fig. 3b). These effects of salinity on aldolase activity were improved by exogenous Spd, but remained lower than values grown in normal condition. Under non-saline conditions, exogenous Spd exerted no effects on Rubisco and aldolase activities in cucumber seedlings.

Fig. 3
figure 3

Effects of Spd on the activities of Rubisco (a) and aldolase (b) in leaves of cucumber seedlings under salt stress. Vertical bars represent the mean ± SD of results from three independent experiments. Different letters indicate significant differences at P < 0.05, according to Duncan’s multiple range tests. Cont, Spd, NaCl and NaCl + Spd were as described in “Materials and methods”. Spd, 0.1 mM; NaCl, 50 mM

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Western blot analysis was used to measure the relative Rubisco and aldolase contents. Compared with the control, the relative Rubisco content was decreased by 102.16, 95.37 and 89.30 % at three, six and nine days in cucumber leaves under salt stress (Fig. 4a, b). Exogenous Spd applied to the salinized nutrient solution significantly increased the Rubisco content. Salt decreased aldolase activity by 78.43 % when compared to the control after three days (Fig. 4c). The salinity effects on aldolase content was improved by exogenous Spd, but the aldolase level remained lower than values found in the normal conditions. However, under non-saline conditions, exogenous Spd had no significant effects on Rubisco and aldolase activities in salt-stressed cucumber seedlings.

Fig. 4
figure 4

Effects of Spd on the Rubisco and aldolase contents in leaves of cucumber seedlings under salt stress. The expression levels of Rubisco and aldolase (optical density) estimated by western blot analysis (a). The Rubisco (b) and aldolase (c) contents. Different letters indicate significant differences at P < 0.05, according to Duncan’s multiple range tests. Cont, Spd, NaCl and NaCl + Spd were as described in “Materials and methods”. Spd, 0.1 mM; NaCl, 50 mM

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Key gene expression of carbohydrate metabolism

RT-PCR was used to analyze the relative transcript levels of ten genes involved in the Calvin cycle in cucumber leaves after exposure to salt stress for three days, cucumber actin gene was used as the reference gene. As shown in Fig. 5a, transcriptional levels of these genes significantly varied with the addition of salt and Spd. Under salt stress conditions, the cucumber plants had increased transcriptional levels of genes including ribulose-1,5-bisphosphate (RCA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribrokinase (PRK) and decreased transcriptional levels for ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RbcL), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS), fructose-1,6-bisphosphate aldolase (ALD), triose-3-phosphate isomerase (TPI), fructose-1,6-bisphosphate phosphatase (FBPase) and 3-phosphoglyceric acid kinase (PGK), but the transcriptional levels of sedoheptulose-1,7-bisphosphate phosphatase (SBPase) was not affected by salt stress. Exogenous Spd regulated salt stress-mediated accumulation of transcripts. The features of mRNA accumulation were classified into four groups as follows: (1) salt stress increased the expression but exogenous Spd reduced the expression (e.g., GAPDH and PRK); (2) salt stress decreased the expression but exogenous Spd increased the expression (e.g., RbcL, RbcS, FBPase, TPI, ALD and PGK); (3) salt stress increased the expression and exogenous Spd further increased the expression (e.g., RCA); (4) salt stress or Spd had little effect (e.g., SBPase). Accumulation of mRNA transcripts for RbcL, RbcS, TPI and PRK had a greater response to salt stress than others, and RbcL, PRK, ALD, RCA had a greater response to exogenous Spd (Fig. 5b).

Fig. 5
figure 5

Effect of exogenous Spd on key gene expression of carbohydrate metabolism in leaves of cucumber seedlings under salt stress after 3 days treatments. Transcript abundance (a). The relative abundance ratio of genes (b). A single concentration of cDNA was also used for amplification with ACTIN (AF171095, actin) primers. ACTIN was used as the internal standard to determine the extent of cDNA amplification. Cont, Spd, NaCl and NaCl + Spd were as described in “Materials and methods”. Spd, 0.1 mM; NaCl, 50 mM. Abbreviations for gene names have been defined in the footnote of Table 1

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Discussion

The experiments of the present contribution clearly showed that cucumber seedlings exhibit its large increase in growth when application of exogenous Spd supplied to the nutrient solution with 50 mM NaCl (Table 2).

Salinity often caused unfavorable effects on the growth and photosynthesis of plants (Duan et al. 2008). In the present study, we observed that salt stress decreased P N , Gs, Ci and Tr in cucumber seedlings, but increased stomatal limitation value (Ls) (Fig. 1a–d, f). Therefore, we hypothesized that one of the possible mechanism responsible for low P N values may be stomatal limitation, which is associated with a decrease in the CO2 concentration in the cellular spaces of the leaf (Ci) (Praxedes et al. 2006). In addition, although the value of the photochemical quenching coefficient (qP) and the actual photochemical efficiency of PSII (ΦPSII) were significantly reduced in salt-stressed cucumber leaves, Fv/Fm was still unchanged with a stable level at approximately 0.8, suggesting that PSII reaction centers were not photo-inhibited by salt stress treatment for nine days (Table 3). The result is consistent with the findings of Lu et al. (2003). Application of exogenous Spd significantly increased the P N value and caused a higher biomass accumulation in salt-stressed cucumber plants, as compared to plants with salt. The increase P N may be due to the ability of exogenous Spd to increase stomatal conductance, thus producing more CO2 in the pore space of the cell and improving the efficiency of CO2 assimilation.

Many stressful environments, such as salinity could induce limitation of CO2 fixation and further decrease photosynthetic capacity (Barhoumi et al. 2007). Some studies have shown that one of the most important biochemical limitations involved in salt-related down-regulation of photosynthesis was limitation in the content or activity of Rubisco (Lu et al. 2009). He et al. (2014) reported that decrease of Rubisco content and activity in salt-sensitive soybean which caused low carboxylation efficiency. In this study, salt stress significantly increased the initial Rubisco activity and Rubisco content after 3 days of treatment, but significantly decreased the activity and content of Rubisco at six and nine days. We also observed that salt stress caused a significant decrease in the maximum carboxylase activity of Rubisco (Vcmax), the maximal velocity of RuBP regeneration (Jmax) and carboxylation efficiency (CE). These results are in agreement with those of Yamane et al. (2012) showed that reduction in P N was attributed to inhibition of Rubisco under the long-term salinity. However, exogenous Spd alleviated the reduction of salt stress on the above mentioned parameters which indicate that application of Spd strongly regulated photosynthetic carbon assimilation. The earlier reports also showed that foliar exogenous application of 1 mM Spd markedly reversed cinnamic acid-induced the inhibition of Rubisco activity in cowpea leaves (Huang and Bie 2010). It may be explained that Spd exhibited positive charges which interact with Rubisco under transglutaminase form of protein-glutamyl-polyamines-glutamyl-protein (Besford et al. 1993).

Aldolase is a key enzyme in plants, which is involved not only in glycolysis and gluconeogenesis in the cytoplasm, but also in regulating CO2 fixation and product generation through the Calvin cycle in chloroplasts (Christine 2003). In the Calvin cycle, aldolase as an enzyme of the glycolytic pathway, which reversibly catalyzes the conversion of glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) to fructose 1,6-bisphosphate (Fru-1,6-P2), and erythrose 4-phosphate (Ery4P) and DHAP to sedoheptulose 1,7-bisphosphate (Haake et al. 1998). The increased expression of FBPase activity in salt-stressed rice was previously reported (Abbasi and Komatsu 2004), and it has been suggested that expression of the protein may play a role in the acclimation of rice seedlings to the anaerobic conditions created by oxidative stress generated in a saline environment. Enhanced expression of aldolase would increase the flow of carbon through the Calvin cycle and lead to an increase in sucrose and amino acid production. These traits would also lead to osmolite production and contribute to stress tolerance. In the present study, levels of aldolase activity and content were reduced in the leaves during salt stress. This is similar with those of Yang et al. (2008) who reported that the activity of photosynthesis-related enzymes including aldolase was inhibited by salt stress in tobacco. However, exogenous Spd significantly increased activity and content of aldolase in the salt-stressed plants, and our results suggested that Spd accelerated carbon assimilation pathway through the Calvin cycle, leading to an increased in photosynthetic efficiency of plants may contribute to salt tolerance.

Plants can sense stress signals and pass them to organelles to initiate the adaptive response, this adaptation is partly completed by regulating gene expression (mRNA abundance). RT-PCR was used to analyze the relative transcript levels of ten genes involved in the Calvin cycle in plants after exposure to salt stress treatment for 3 days. Rubisco activity is regulated by light, stromal pH and Mg2+. It must be carbamylated at Lys-201 of the large subunit, and the carbamate binds Mg2+ to fully activate the enzyme for both carboxylation and oxygenation (Lorimer 1981). In our study, salt stress decreased the gene expression of RbcL and RbcS at three days after the treatment, but the decrease was alleviated by Spd (Fig. 5b). However, Rubisco activity and content were enhanced by salt stress (Fig. 4a–b). One possible explanation for this discrepancy may be that salt stress induced the inhibition of photosynthesis mainly occurred at the transcriptional level of Rubisco. PGK is the first enzyme in the glycolytic pathway and produces ATP through a phosphoryl transfer from 1,3-bisphosphoglycerate to ADP resulting in 3-phosphoglycerate (Marston et al. 2010). Fructose 1,6-bisphosphatase (FBPase) dephosphorylates fructose 1,6-bisphosphate to produce fructose 6-phosphate and inorganic phosphate in higher plants (Lee and Hahn 2002). Kelly and Latzko (1976) reported that FBPase has an important regulatory role in running the Calvin cycle and transporting photosynthetic products. TPI and aldolase continuously catalyze the readily reversible conversion of glyceraldehyde 3-phosphate (G3P) and 3-phosphoglycerate (PGA), which is followed by the conversion of GAP and DHAP to Fru-1,6-P2 (Kang et al. 2005). The turnover rate in the Calvin cycle is largely determined by the steps with the substrates RuBP, PGA and F6P (Fridlyand and Scheibe 1999), Therefore, the carbon acquisition in Cyanobacteria may be somewhat limited under certain conditions by the activities of aldolase and TPI activities available for RuBP production. Uematsu et al. (2012) found that increased plastid aldolase stimulated RuBP regeneration and promoted CO2 fixation of transgenic tobacco plants. Moreover, phosphorus is essential for plant photosynthesis, and TPI is important for the phosphorus element. If the rate of TPI use is slower than the production of TPI, the rate of phosphorus release will be decreased, restricting photosynthesis and phosphorus regeneration (Sharkey et al. 1986). In the present study, we observed the down-regulation of FBPase, ALD and TPI in salt-stressed leaves, which implied that salt stress might inhibit the transport of photosynthetic products and use of phosphorus at the transcriptional level. Moreover, salt treatment may lead to feedback inhibition of photosynthesis and the limitation of phosphorus regeneration. The decreased expression of ALD and PGK may indicate that salt stress reduces the transformation process of carbon assimilation. However, exogenous Spd alleviated the down-regulation of these genes to some extent, especially for RbcL and ALD. Moreover, the up-regulation of GAPDH, PRK and RCA may be explained by an adaptation to salt stress.

In conclusion, the present study demonstrates that exogenous Spd could enhance salinity tolerance of cucumber seedlings. The increased tolerance may be involved in two possible mechanisms. One mechanism is by decreasing stomatal limitation, and the other mechanism might alleviate the disorder on carbon assimilation through regulating gene expression of key enzymes in the Calvin cycle, especially in the carboxylation phase.