Adaptation of cucumber seedlings to low temperature stress by reducing nitrate to ammonium during it’s transportation

Low temperature severely depresses the uptake, translocation from the root to the shoot, and metabolism of nitrate and ammonium in thermophilic plants such as cucumber (Cucumis sativus). Plant growth is inhibited accordingly. However, the availability of information on the effects of low temperature on nitrogen transport remains limited. Using non-invasive micro-test technology, the net nitrate (NO3−) and ammonium (NH4+) fluxes in the root hair zone and vascular bundles of the primary root, stem, petiole, midrib, lateral vein, and shoot tip of cucumber seedlings under normal temperature (NT; 26 °C) and low temperature (LT; 8 °C) treatment were analyzed. Under LT treatment, the net NO3− flux rate in the root hair zone and vascular bundles of cucumber seedlings decreased, whereas the net NH4+ flux rate in vascular bundles of the midrib, lateral vein, and shoot tip increased. Accordingly, the relative expression of CsNRT1.4a in the petiole and midrib was down-regulated, whereas the expression of CsAMT1.2a–1.2c in the midrib was up-regulated. The results of 15N isotope tracing showed that NO3−-N and NH4+-N uptake of the seedlings under LT treatment decreased significantly compared with that under NT treatment, and the concentration and proportion of both NO3−-N and NH4+-N distributed in the shoot decreased. Under LT treatment, the actual nitrate reductase activity (NRAact) in the root did not change significantly, whereas NRAact in the stem and petiole increased by 113.2 and 96.2%, respectively. The higher net NH4+ flux rate in leaves and young tissues may reflect the higher NRAact in the stem and petiole, which may result in a higher proportion of NO3− being reduced to NH4+ during the upward transportation of NO3−. The results contribute to an improved understanding of the mechanism of changes in nitrate transportation in plants in response to low-temperature stress.


Background
Cucumber (Cucumis sativus L.) is an important vegetable crop worldwide and a model plant system for studying sex determination and vascular biology [1]. It is native to the tropics and is sensitive to low temperature [2]. Cucumber is widely cultivated in greenhouses in northern China during the winter and spring seasons. Low temperature is a crucial environmental factor that limits the development and productivity of cucumber crops [3].
Nitrogen (N) is the mineral nutrient required in the highest amount by plants [4]. It is crucial for the biosynthesis of amino acids, proteins, and nucleic acids [5]. Nitrogen contributes approximately 2% of plant dry matter and exerts the greatest nutrient influence (up to 50%) on the growth and yield of plants [6,7]. The absorption and utilization of N by plants under normal temperatures have been clarified. Plant roots absorb N primarily as nitrate (NO 3 − ) and ammonium (NH 4 + ), especially as NO 3 − for terrestrial plants [8]. The NO 3 − absorbed by plants is first reduced to NH 4 + before it can be metabolized. This reduction is catalyzed by nitrate reductase (NR) and nitrite reductase (NiR) [9]. Of these enzymes, NR is considered to be the rate-limiting step in N assimilation [10]. Activity of NR and NiR can be detected in many plant organs (e.g., the root, stem, cotyledon, inflorescence stalk, flower, petiole, and leaf) [11][12][13][14].
The absorption and transportation of NO 3 − and NH 4 + in plants is mediated by nitrate transporters (NRTs) and ammonium transporters (AMTs), respectively [15]. Four families of nitrate-transporting proteins have been identified to date: nitrate transporter 1 family (NRT1), nitrate transporter 2 family (NRT2), chloride channel family (CLC), and slow anion channel-associated homologs (SLAC1/SLAH) [16]. The ammonium transporter gene family of vascular plants consists of two clades, comprising AMTs and methylammonium permeases (MEPs) [17]. The regulation of NO 3 − uptake and transport is often highly correlated with changes in expression of relevant transporter genes [18,19].
The uptake of inorganic N forms is favored by warm temperatures, especially NO 3 − uptake [20]. In many crop species, particularly those originating from tropical and subtropical regions, low temperature restricts the uptake capacity of the root and distribution of NO 3 − and NH 4 + in the shoot and, consequently, plant growth and metabolism are inhibited [21][22][23]. Furthermore, reduction in temperature decreases N translocation more strongly than uptake and, as a result, the N concentration in the root increases [24]. However, previous studies mainly focused on the changes in physiological characteristics under low temperature. Hence the molecular mechanism of these phenomena remains unclear. Limited information is available on the adaptation of NO 3 − and NH 4 + transport in vascular bundles under low-temperature stress.
The use of stable N isotopes has typically been the most important method in the study of N absorption and transportation, but only enables monitoring of N absorption and distribution within a certain period. It is difficult to monitor the dynamic transport of N. No effective technology to study N transport under low temperature has previously existed. In recent years, noninvasive micro-test technology (NMT) has provided a novel means to detect ion velocity in living plant tissues [25]. The development and application of NO 3 − and NH 4 + sensors for NMT provide convenience for intuitive detection of net NO 3 − and NH 4 + flow rates [26,27]. The purpose of the present study was to study the effects of low temperature on the absorption and transportation of NO 3 − and NH 4 + , treating the plant as a whole using NMT technology, in combination with 15 N isotope tracing and quantitative reverse transcription-PCR (qRT-PCR) technology.
We observed that low temperature reduced the net NO 3 − flux rate in the root hair zone and vascular bundles of cucumber seedlings, whereas the net NH 4 + flux rate was enhanced in vascular bundles of the midrib, lateral vein, and shoot tip. To further understand the regulation of N transportation by low temperature, the uptake and distribution of 15 N-NO 3 − and 15 N-NH 4 + , NR and NiR activities and gene expression, and relative expression of CsNRT and CsAMT were measured under normal temperature (NT) and low temperature (LT) treatments. The results will aid in understanding the adaptability of inorganic N transport in thermophilic plants to low-temperature stress.

Net NO 3
− and NH 4 + flux rates First, we observed that LT (8°C) treatment significantly depressed the net NO 3 − flux rate in the root hair zone and in the vascular bundles of other organs of cucumber seedlings. Compared with NT (26°C) treatment, the net NO 3 − influx rate in the root hair zone and the net NO 3 − efflux rate in the vascular bundles of the primary root, stem, petiole, midrib, lateral vein, and shoot tip under LT treatment decreased significantly (Fig. 1), indicating that the uptake and upward transport of NO 3 − was significantly inhibited by low temperature.
Compared with the net NO 3 − flux rate, the change in net NH 4 + flux rate under LT treatment was different. The net NH 4 + flux rate in the root hair zone and vascular bundles of the primary root, stem, and petiole of cucumber seedlings under LT treatment decreased significantly compared with those under NT treatment. In contrast, the net NH 4 + flux rate in the vascular bundles of the midrib, lateral vein, and shoot tip increased significantly (Fig. 2). Compared with the net NO 3 − flux rate, the net NH 4 + flux rate at the detection sites was markedly lower under NT treatment, but significantly higher in the lateral vein and shoot tip under LT treatment. These results indicated that the inhibition of net NO 3 − flux rate at low temperature was more severe than the effect on net NH 4 + flux rate.
Nitrogen uptake per plant, N concentration, and N distribution in different organs The effects of low temperature on the uptake and distribution of NO 3 − -N and NH 4 + -N in cucumber seedlings were further explored using an isotope tracer method. Compared with NT treatment, NO 3 − -N, NH 4 + -N, and total N uptake per plant under LT treatment decreased significantly (Table 1). These effects were consistent with the change in net NO 3 − and NH 4 + flux rates in the root hair zone under LT treatment ( Figs. 1 and 2). Under LT treatment, the ratio of NO 3 − -N to total N decreased significantly, whereas the ratio of NH 4 + -N to total N increased significantly, compared with those under NT treatment. This result indicated that, compared with NH 4 + -N uptake, low temperature inhibited NO 3 − -N uptake more severely.
Under NT treatment, the NO 3 − -N concentrations at the detection sites of cucumber seedlings were significantly higher than NH 4 + -N concentrations (Fig. 3 Exposure of cucumber seedlings to low temperature resulted in a significant increase in not only NO 3 − -N but also NH 4 + -N distribution proportion in the root (Fig. 4). Thus, LT treatment significantly reduced the distribution proportion of NO 3 − -N and NH 4 + -N in the shoot. This finding indicated that low temperature inhibited the transportation of NO 3 − and NH 4 + from  the root to the shoot, and resulted in N accumulation in the root. Under LT treatment, the distribution proportion of NO 3 − -N and NH 4 + -N in all aerial organs decreased, except for the NH 4 + -N distribution proportion in the stem.

Expression of CsNRT and CsAMT genes in the petiole and midrib
To investigate the response of CsNRT and CsAMT genes in cucumber seedlings to low temperature, the relative expression of 34 genes was quantified. Exposure to low temperature decreased expression of CsNRT1.4a in the petiole and midrib significantly, whereas the expression levels of CsAMT1.2a, CsAMT1.2b, and CsAMT1.2c were significantly enhanced in the midrib (Fig. 5). Thus, the expression of these genes may be strongly associated with NO 3 − and NH 4 + transport in the petiole and midrib. Compared with those under NT treatment, the expression levels of CsNRT1.1, CsNRT1.3, CsNRT1.7, CsNRT1.8, CsCLCa, and CsCLCe in the petiole and midrib, and that of CsNRT1.2b in the midrib were upregulated. Therefore, these genes were not indicated to play crucial roles in nitrate transport in the petiole and midrib.
The relative expression of CsNRT1.2a, CsNRT1.5a, CsNRT1.10, CsCLCc, CsCLCd, CsAMT2, and CsAMT3.3 in the petiole and midrib, and those of CsNRT1.2b, CsNRT1.4a, CsNRT1.4b, CsCLCa, CsCLCb, CsAMT1.2a, and CsAMT1.2b in the petiole were not significantly affected by LT treatment. The relative expression levels of CsNRT1.2c, CsNRT1.5b, CsNRT1.5c, CsNRT1.9, CsSLAH1-4, CsCLCf, CsCLCg, CsAMT1.1a, and CsAMT1.1b in the petiole and midrib under the NT and LT treatments were substantially lower than those of the genes shown in Fig. 5. Therefore, data on their relative expression levels are not presented. Activities of NR and NiR, and expression of CsNR and CsNiR genes The enzymes NR and NiR catalyze the nitrate-to-nitrite and nitrite-to-ammonium reduction processes, respectively, in plants [10,28]. The NRA max reflects the maximum amount of enzyme protein indirectly and NRA act indicates actual NR activity in situ [29]. After LT treatment for 5 h, NRA max in the root decreased significantly, whereas that in the stem, petiole, and midrib increased significantly, compared with that under NT Compared with that under NT treatment, NRA act in the stem and petiole increased significantly, whereas that in the midrib and blade decreased significantly under LT treatment (Fig. 6B). No significant difference in NRA act in the root was observed between the NT and LT treatments. The NiR activity in the root, stem, petiole, and blade was not significantly decreased by LT treatment, except for the midrib (Fig. 6C). The C. sativus genome contains three NR family genes (CsNR1, CsNR2, and CsNR3) according to Reda et al. [30]. Compared with that under NT treatment, expression of CsNR1 in the root, stem, petiole, and midrib was down-regulated under LT treatment, whereas no significant difference in relative expression of CsNR1 in the blade was observed between the NT and LT treatments (Fig. 7A). Under LT treatment, expression of CsNR2 in the leaf (including the petiole, midrib, and blade) was up-regulated, whereas expression of CsNR2 in the root decreased, compared with that under NT treatment (Fig.  7B). No significant difference in CsNR2 expression in the stem was observed between the LT and NT treatments. Compared with that under NT treatment, expression of CsNR3 in the root, stem, petiole, and midrib under LT treatment was up-regulated (Fig. 7C). The relative expression of CsNR1 in the root was substantially higher than that in other organs under the NT and LT treatments (Fig. 7A). The relative expression of CsNR2 in the leaf was higher than that in the root and stem (Fig. 7B). Similar to CsNR2, a high expression level for CsNR3 was observed in the midrib, and especially in the blade, under the NT and LT treatments. The results presented in Figs. 6A and 7A, B, C suggested that CsNR1 may be the dominant NR gene expressed in the root, and that CsNR3 may be the dominant NR gene expressed in the leaf. CsNR2 and CsNR3 may play a leading role together in the stem and petiole.
Compared with CsNR genes, the differences in relative expression levels of CsNiR among different organs were small (Fig. 7D). The LT treatment enhanced the expression of CsNiR in the petiole, midrib, and blade to a certain extent. The highest expression level of CsNiR was observed in the root in both NT and LT treatments, but its expression was not affected significantly by low temperature. − uptake was greater than that of NH 4 + uptake. This may be because uptake of NO 3 − is energy dependent [35]. The energy requirements for absorption and assimilation of NO 3 − are several-fold higher than those of NH 4 + [36]. With the occurrence of low-temperature stress, the energy absorbed and utilized by leaves decreased significantly [37]. Thus, under LT treatment, the uptake of NO 3 − by roots would be severely inhibited as a result of energy limitation.

Discussion
The transport of NO 3 − is induced by NO 3 − itself and promoted by photosynthesis [38]. Under low temperature the xylem sap transport in cucumber is reduced severely [39]. Laine et al. [34] reported that low temperature decreases xylem N translocation and results in N accumulation in the roots of Secale cereale and Brassica napus. The present results confirmed that low temperature not only inhibited uptake of NO 3 − -N and NH 4 + -N, but also inhibited their upward transportation. The degree of inhibition of NO 3 − -N upward transportation was almost identical to that of NH 4 + -N under low temperature. Recently, Anwar et al. [23] reported that low temperature reduces N content in roots of cucumber seedlings, but does not significantly reduce N content in the shoot. This conflict may be due to the different detection methods used. The total N contents were detected by Anwar et al. [23], whereas the 15 N concentrations were detected in the present experiment.
The net NO 3 − and NH 4 + flux rates detected by NMT showed that, compared with NT treatment, the change in net NO 3 − /NH 4 + flux rate in the stem and petiole under LT treatment differed from that in the leaf vein and shoot tip. In contrast to the change in net NO 3 − flux rate, the net NH 4 + flux rate in the midrib, lateral vein, and shoot tip was increased by LT treatment (Figs. 1 and 2). This result was inconsistent with the uptake and distribution of 15 N-NH 4 + in the leaf and shoot tip ( Table 1, Figs. 3 and 4). We selected the petiole and midrib as target tissues to study the effect of low temperature on the relative expression of nitrate and ammonium transporter genes.
Nitrate uptake by plants is regulated by transcriptional regulation [40]. Two environmental factors, temperature and nutrient concentration, significantly influence the expression of nutrient transporter genes [41]. However, few studies have examined the function of cucumber N transporters to date [42][43][44]. Little information on the regulatory pathways involved in the effect of low temperature on the expression of N transporter genes in cucumber has been reported. Among nitrate transporters, NRT1.1 (NPF6.3) is regarded to be a dual-affinity nitrate transporter that participates in nitrate absorption and transport [45,46]. The present results showed that CsNRT1.1 may not be the dominant gene involved in nitrate transportation in the petiole and midrib of cucumber. In Arabidopsis AtNRT1.8 is associated with stress-induced NO 3 − redistribution [47]. Under LT treatment, the relative expression of CsNRT1.8 was up-regulated in the petiole and midrib. This response may allow an increase in nitrate transportation to the root, thus reducing the net ion flux rate in the petiole and midrib. In Arabidopsis AtNRT1.4 and AtNRT1.7 are responsible for nitrate transport to the petiole and leaf [48,49]. We identified two homologs of AtNRT1.4 in cucumber, CsNRT1.4a and CsNRT1.4b. Compared with that under NT treatment, the expression of CsNRT1.4a in the petiole and midrib under LT treatment was down-regulated, whereas the expression of CsNRT1.4b was upregulated. The different responses in relative expression level of these genes to low temperature may reflect their different functions. CsNRT1.7 is involved in NO 3 − recycling in cucumber [42]. Under low temperature CsNRT1.7 in the petiole and midrib was up-regulated. This response may reduce NO 3 − upward transport to the leaves to some extent. AtClCa and AtCLCe are critical for nitrate transport into vacuoles in Arabidopsis [50,51]. In the present experiment, the relative expression levels of CsCLCa and CsCLCe in the petiole and midrib of cucumber seedlings were upregulated by low temperature. This response may lead to increased nitrate storage in vacuoles under low temperature. The AMT1 subfamily of Arabidopsis plays an important role in the stage of NH 4 + absorption [52]. The MEP subfamily (AtAMT2) may play a role in the transport of NH 4 + from the apoplast to the symplast [53]. In the present experiment, upregulation of CsAMT1.2a-1.2c in the midrib may have contributed to the higher net NH 4 + flux rate under low temperature.
Under low temperature a higher proportion of NO 3 − was reduced to NH 4 + during its transportation in the stem and petiole Under low temperature, although the total amount of NO 3 − -N and NH 4 + -N absorbed by cucumber seedlings and transported from the root to the shoot decreased, the net NH 4 + flux rate increased in the midrib, lateral vein, and shoot tip. To elucidate the mechanism responsible, the morphological changes in N during the transport process were studied.
Nitrate reductase is the key rate-limiting enzyme in nitrate reduction [10]. In higher plants the activity of NR is regulated at the phosphorylation and transcriptional levels [54]. Under low temperature, NRA max in the root decreased, whereas NRA act did not change significantly, which indicated that low temperature reduced the amount of enzyme protein but had no significant effect on the apparent activity of the enzyme (Fig. 6). Therefore, the amount of NR protein may be redundant in the root. The NRA act and NRA max in the stem and petiole increased significantly, which indicated that the change Fig. 8 Regulation of low temperature on net NO 3 − and NH 4 + flux rates in cucumber seedlings. The color of the arrows indicates the changes in relevant indicators: pink indicates increase, and blue indicates decrease. The thickness of the arrows indicates the degree of increase or decrease in enzyme protein content was consistent with the change in enzyme apparent activity, and NRA act in the stem and petiole was predominantly regulated by low temperature at the transcriptional level. Compared with those under NT treatment, NRA max in the midrib and blade under low temperature did not decrease, whereas NRA act decreased significantly in the midrib and blade. This response indicated that the effect of low temperature on NRA act in the midrib and blade may be predominantly through protein phosphorylation. Overall, low temperature had no effect on NRA act in the root, but significantly increased NRA act in the stem and petiole of cucumber seedlings. These changes may account for the increased proportion of NO 3 − reduced to NH 4 + during its transportation in the stem and petiole. The qRT-PCR analysis of NR gene expression and NRA max analysis indicated that CsNR1 and CsNR3 may be the dominant NR gene in the root and leaf, respectively, of cucumber seedlings (Figs. 6A, 7A, C).

Biological significance of the increase in net NH 4 + fluxes in vigorously growing tissues under low temperature
Plants transfer nutrients to young tissues and seeds under unsuitable environmental conditions [55]. This process has been an important adaptive strategy during terrestrial plant evolution. In the present experiment, although the uptake and upward transportation of NH 4 + decreased under low temperature, the net NH 4 + flux rate in the midrib, lateral vein, and shoot tip increased significantly. This response may be due to the transformation of NO 3 − during transportation. A greater proportion of NO 3 − was reduced to NH 4 + during the upward transportation of NO 3 − under low temperature. The energy consumption during the N transportation process was reduced accordingly. This strategy may be an adaptation of plant N transport to the decrease in energy supply under low-temperature stress. According to Han et al. [56], under low-temperature stress, the NO 3 − -N content and NR activities in tomato leaves significantly decrease, whereas the NH 4 + -N content significantly increases. Under drought stress, NH 4 + nutrition can limit the effect of water deficit by osmotic adjustment and thereby limit oxidative damage [57]. Therefore, assuming that NH 4 + plays a role in the prevention of stress-induced peroxidation, the increase in NH 4 + content in leaves and young tissues is not only beneficial for utilization of N nutrition, but also improves the stress tolerance of the plant.
The predominant N sources of cucumber are NO 3 − -N and NH 4 + -N. Compared with a single NO 3 − -N source or a single NH 4 + -N source, a compound N source is more conducive to N absorption and plant growth [31]. Plant preference for NO 3 − -N or NH 4 + -N is associated with species and is influenced by environmental conditions and growth stage [58][59][60]. Kant [61] considered that improvement of nitrate uptake and transport would enhance plant growth, resulting in improved crop yields. In the future, further in-depth research on nitrate and ammonium transport in the roots of cucumber seedlings and research on enhancing the N nutrition status of plants by improving the ratio of NO 3 − -N to NH 4 + -N under low temperature is required.

Conclusions
Our results provide evidence that cucumber seedlings reduce a greater proportion of NO 3 − to NH 4 + during the process of upward transport of NO 3 − under low temperature. This action may reduce the dependence of N transport on energy and enable plants to adapt to the decrease in energy supply under lowtemperature stress. Compared with NO 3 − , the absorption of NH 4 + -N and net NH 4 + flux rate in the root hair zone, vascular bundles of the primary root, and the stem is less inhibited by low temperature (Fig. 8). Under low temperature, the net NH 4 + flux rate in vascular bundles of the midrib, lateral vein, and shoot tip is increased, which is the opposite response to that of net NO 3 − flux rate. In line with these responses, under low temperature, the relative expression of CsNRT1.4a in the petiole and midrib is downregulated, whereas the expression of CsAMT1.2a-1.2c in the midrib is up-regulated. The NRA act in the stem and petiole increases significantly, which is predominantly regulated at the transcriptional level by low temperature. Given the importance of cucumber as a greenhouse vegetable crop, this study enhances understanding of the low-temperature tolerance of a thermophilic plant and contributes to improved winter cultivation techniques of tender vegetables in a greenhouse.

Plant materials and growth conditions
All experiments in this study were conducted in controlled-environment chambers (Memmert ICH L260). Seeds of cucumber (Cucumis sativus L.) 'Xintai Mici' (China Vegetable Seed Technology Co., Ltd., Beijing, China) were incubated in the dark until germination at 28°C. The seedlings were grown in a vermiculite-sand mixture (1:2, v/v) and supplied with halfstrength modified Hoagland's nutrient solution at 26°C/ 17°C (day/night) [26]. The photosynthetic photon flux density, photoperiod, and relative humidity (RH) were 350 μmol·m − 2 ·s − 1 , 12 h/12 h (light/dark), and 70-80%, respectively. When the cotyledons of the seedlings were fully expanded, the seedlings were supplied with fullstrength modified Hoagland's nutrient solution (pH 6 The seedlings were divided into two groups and exposed to either normal temperature (NT; 26°C) or low temperature (LT; 8°C) for 5 h. During treatment, the light intensity and RH were identical to the seedling growth conditions. After treatment, the seedlings were harvested for physiological and genetic analyses.

Measurement of net NO 3 − and NH 4 + fluxes
The net NO 3 − and NH 4 + fluxes were measured at the YoungerUSA Xuyue (Beijing) BioFunction Institute using a NMT system (NMT100 Series, YoungerUSA, LLC, Amherst, MA, USA; Xuyue (Beijing) Science & Technology Co., Ltd., Beijing, China) and imFluxes V2.0 software (YoungerUSA, LLC). The method followed that of Lei et al. [64] with some modifications. The root sample was excised about 2 cm from the apex. The primary root, stem, petiole, midrib, lateral vein, and shoot tip were sampled at the positions indicated in Fig. 9. A seedling was only used once. The sample was fixed with a belt. The transverse section of each sampled organ was immediately incubated in the measuring solution (1.625 mM Ca (NO 3 ) 2 , 0.25 mM NH 4 NO 3 , 0.1 mM MgSO 4 , and 0.3 mM MES; pH 6.0) to equilibrate for 15 min because a rapid and large efflux of NO 3 − and NH 4 + occurred after the samples were excised. The flux rate gradually decreased and stabilized within 15 min. The solution was then sucked out and fresh measuring solution was injected. Uptake of NO 3 − and NH 4 + was measured in the root hair zone (Figs. S1, S2). Transport of NO 3 − and NH 4 + was measured in the transverse sections After treatment for 5 h, roots of the seedlings in Experiment 2 were rinsed for 2 min in identical, unlabeled modified Hoagland's nutrient solution. The root surface was dried with absorbent paper. In addition, the root, stem, cotyledon, first petiole, first blade, second petiole, second blade, and shoot tip were sampled, respectively. All samples were defoliated at 105°C and dried to constant weight at 55°C. The fresh weight, dry weight, and water content of the samples were determined in accordance with Oliviero et al. [65]. The samples were ground to a fine powder. Total N and 15 N contents were measured by means of continuous-flow isotope-ratio mass spectroscopy using a vario PYRO cube elemental analyzer coupled to an IsoPrime 100 isotope-ratio mass spectrometer [66]. During the analysis process, 12 samples were interspersed with a laboratory sample for correction.

Measurement of NR activity
The NR activity was measured using the method described by Reda and Klobus [67] with some modifications. Plant tissues (1.0 g fresh weight) were ground in a chilled mortar with 5 mL extraction buffer. The mixture was homogenized and centrifuged at 15000 g for 10 min at 4°C. The supernatant was used to measure NR activity in the presence and/or absence of MgCl 2 in accordance with the method of Glaab and Kaiser [68]. The reaction medium was incubated for 10 min at 27°C, and then the NR activity was recorded by measuring the NO 2 − produced.

Measurement of NiR activity
The NiR activity was measured following the method described by Liu et al. [69].
Total RNA was extracted using the RNAprep pure Plant Kit (TIANGEN, Beijing, China) in accordance with the manufacturer's instructions. The concentration of RNA was quantified by spectrophotometric measurement at λ = 260 nm and RNA integrity was checked on agarose gels [70]. First-strand cDNA was synthesized using the FastQuant RT Kit (TIANGEN) following the manufacturer's instructions. The cDNA was analyzed by qRT-PCR using the Hieff qPCR SYBR Green Master Mix (11203ES03, YEASEN) on an ABI 7500 Real Time PCR System (Applied BioSystems) [71]. Transcripts of TIP41 (PP2A phosphatase activator; GW881871) were used to standardize the cDNA samples for different genes because its expression is insensitive to low temperature [72]. Specific primers were designed using Primer Premier 5 software [73] and the cucumber genome database [1]. Oligonucleotides used are listed in Additional file 1 (Table S1).

Data analysis
Two-way analysis of variance (ANOVA) followed by the least significant difference test was performed. All statistically significant differences were identified as P < 0.05. Graphpad Prism 5 was used for graphical presentation.