Increased Ammonium Enhances Suboptimal-Temperature Tolerance in Cucumber Seedlings

Nitrate nitrogen (NO3−-N) is widely used in the cultivation of the cucumber (Cucumis sativus L.). In fact, in mixed nitrogen forms, partially substituting NO3−-N with NH4+-N can promote the absorption and utilization of nitrogen. However, is this still the case when the cucumber seedling is vulnerable to the suboptimal-temperature stress? It remains unclear as to how the uptake and metabolism of ammonium affect the suboptimal-temperature tolerance in cucumber seedlings. In this study, cucumber seedlings were grown under suboptimal temperatures at five ammonium ratios (0NH4+, 25%NH4+, 50%NH4+, 75%NH4+, 100%NH4+) for 14 days. Firstly, increasing ammonium to 50% promoted the growth and root activity and increased protein and proline contents but decreased MDA content in cucumber seedlings. This indicated that increasing ammonium to 50% enhanced the suboptimal-temperature tolerance of cucumber seedlings. Furthermore, increasing ammonium to 50% up-regulated the expression of the nitrogen uptake-transport genes CsNRT1.3, CsNRT1.5 and CsAMT1.1, which promoted the uptake and transport of nitrogen, as well as the up-regulation of the expression of the glutamate cycle genes CsGOGAT-1-2, CsGOGAT-2-1, CsGOGAT-2-2, CsGS-2 and CsGS-3, which promoted the metabolism of nitrogen. Meanwhile, increased ammonium up-regulated the expression of the PM H+-ATP genes CSHA2 and CSHA3 in roots, which maintained nitrogen transport and membranes at a suboptimal temperature. In addition, 13 of 16 genes detected in the study were preferentially expressed in the roots in the increasing ammonium treatments under suboptimal temperatures, which, thus, promoted nitrogen assimilation in roots to the enhance the suboptimal-temperature tolerance of cucumber seedlings.


Introduction
The growth of cucumber needs a large amount of nitrogen (N) fertilizer. Nitrate nitrogen (NO 3 − -N) can promote the vegetative growth of the cucumber grown under suitable temperatures [1]. Cucumbers cultivated in the spring have a good market price, but the seedling stage is vulnerable to the low temperature of the spring. Low temperature stress can be divided into chilling stress and suboptimal-temperature stress, especially as suboptimal temperatures occur more frequently for cucumber planting in south China, where low temperatures can affect the plants' N utilization efficiency and lead to reduced yield and quality [2,3]. Increasing the ratio of ammonium nitrogen (NH 4 + -N) can stimulate N uptake in the crop and is an important way to increase N utilization efficiency [4,5]. Wang et al. [6] found that 25% NH 4 + -N could significantly increase morphological parameters, including the weight of root dry matter and root surface area in lettuce, while 50% NH 4 + -N did not significantly inhibit root growth in lettuce. Temperature is the main factor that affects the selective uptake of NO 3 − -N and NH 4 + -N in plants [7]. Zhang et al. (2017) [8] found that low temperatures were able to inhibit NO 3 − -N uptake, while a mixture of NO 3 − -N and NH 4 + -N could significantly promote N uptake under low-temperature conditions in cotton seedlings. NH 4 + -N and NO 3 − -N uptake are mediated, respectively, via membrane-localized ammonium transporters (AMT) and nitrate transporters (NRT) in plant roots, and the energy needed for this process is provided through ATP. Narumol et al. [9] showed that compared to NH 4 + -N uptake and utilization, the process of NO 3 − -N uptake and reduction required more energy. Under low-temperature conditions, the energy supply in plants becomes insufficient and NO 3 − -N uptake and reduction may be inhibited, thereby causing N deficiency that further affects plant growth. NH 4 + is absorbed by the roots and then transported to other parts of the plant via AMT on the cell membrane [10]. In tomato, the AMT1.1 gene is first expressed in root hairs [11], also confirming that this gene plays a role in NH 4 + uptake. NO 3 − is absorbed through two NO 3 − -N transport systems, NRT1 and NRT2 [12]. The rate of NO 3 − uptake in plants correlates well with the expression level of NRT [13]. Roots are important organs for nutrient uptake [14]. Roots can also alter their structures to adapt to the changes in the growth environment [15]. Previous studies on increasing NH 4 + nutrition showed that within a suitable range, a higher NH 4 + concentration could promote root growth and development [16,17] (Song et al., 2005;Wang et al., 2006). However, there are few reports on the effect of N on the nutrient uptake of roots under suboptimal-temperature stress. After N is absorbed by the plant, it is used to synthesize amino acids, which need the regulation of a series of N metabolism genes. By searching and analysing genes encoding key enzymes in nitrate reduction and glutamate/glutamine cycle, Ma et al. [18] identified 18 genes that were involved in NO 3 − -N and NH 4 + -N assimilation. Their findings showed that the CsNR-2 gene, CsNR-3 gene, CsNiR gene, CsGOGAT-1-1 gene, CsGS-1 gene and CsGDH-2 gene played important roles in N metabolism in cucumbers. Moreover, some substances are related to stress metabolism, including proline, proteins and sugars, and metabolites are related to osmoregulation in adverse situations. They are able to stabilize the structures of organelles and protect tissues from the damage caused by insufficient temperatures [19].
In summary, the uptake and utilization of nitrogen are closely related to temperature and that increasing the ratio of NH 4 + to N could affect nitrogen uptake and utilization at optimal temperatures. However, whether increasing ammonium is beneficial for N uptake and utilization at suboptimal temperatures in cucumber seedlings was not reported. Moreover, the mechanism of how the uptake and metabolism of ammonium affects suboptimaltemperature tolerance in cucumber seedlings remains unclear. Thus, we carried out an experiment increasing NH 4 + in cucumbers under suboptimal-temperature conditions by partially substituting NO 3 − -N with NH 4 + -N, and the levels of morphology, physiology, biochemistry and gene expression were analysed. Our goal was to provide a theoretical reference for appropriate N utilization in cucumbers under suboptimal-temperature conditions during the winter-spring seasons. We studied the effect of five different compositions of NH 4 + -N and NO 3 − -N on cucumber dry weight and N utilization efficiency when grown under optimal temperatures (25 • C day/18 • C night). The A25 treatment resulted in the highest cucumber biomass (dry weight) and N utilization efficiency among five ammonium treatments, followed by A0, under optimal temperatures. A100 resulted in the lowest biomass and N utilization efficiency ( Figure 1). Under optimal temperatures, cucumber biomass and N utilization efficiency were the highest under the A25 treatment followed by the A0 treatment. Figure 1A,B shows that as the ratio of NH 4 + to NO 3 − increased, the cucumber biomass (dry weight) and N utilization efficiency exhibited similar changes. The N utilization efficiency in the A25 lowed by A0, under optimal temperatures. A100 resulted in the lowest biomass and utilization efficiency (Figure 1). Under optimal temperatures, cucumber biomass and utilization efficiency were the highest under the A25 treatment followed by the treatment. Figure 1A,B shows that as the ratio of NH4 + to NO3 − increased, the cucum biomass (dry weight) and N utilization efficiency exhibited similar changes. The N lization efficiency in the A25 treatment was higher than that in the A0 treatment; as NH4 + /NO3 − ratio increased beyond that level, N utilization efficiency significantly creased. The biomass of cucumber in 100% NO3 − -N was higher than in 100% NH4 when holding the total N amount constant, substituting 25% of NH4 + -N for NO3 − -N sulted in further growth promotion.

The Growth of Cucumber Plants under Suboptimal-Temperature Stress
While keeping the total N supply constant, using A0 (100% NO3 − -N) as the con (Figure 2), NH4 + was increased to 25%, 50%, 75% or 100% under suboptimal temp tures (18 °C day/11 °C night). The dry weights and N utilization efficiency of cucum seedlings under A50 were significantly higher than those undergoing other treatme and those under A100 and A0 were significantly lower than those in A25, A50 and A ( Figure 2). This showed that increasing NH4 + to 50% under suboptimal-temperat stress led to the highest biomass of cucumber plants. This result was different from results found under optimal temperatures, where the NH4 + to N ratio of 25% promo cucumber growth the most.

The Growth of Cucumber Plants under Suboptimal-Temperature Stress
While keeping the total N supply constant, using A0 (100% NO 3 − -N) as the control (Figure 2), NH 4 + was increased to 25%, 50%, 75% or 100% under suboptimal temperatures (18 • C day/11 • C night). The dry weights and N utilization efficiency of cucumber seedlings under A50 were significantly higher than those undergoing other treatments, and those under A100 and A0 were significantly lower than those in A25, A50 and A75 ( Figure 2). This showed that increasing NH 4 + to 50% under suboptimal-temperature stress led to the highest biomass of cucumber plants. This result was different from the results found under optimal temperatures, where the NH 4 + to N ratio of 25% promoted cucumber growth the most. lowed by A0, under optimal temperatures. A100 resulted in the lowest biomass an utilization efficiency (Figure 1). Under optimal temperatures, cucumber biomass an utilization efficiency were the highest under the A25 treatment followed by the treatment. Figure 1A,B shows that as the ratio of NH4 + to NO3 − increased, the cucum biomass (dry weight) and N utilization efficiency exhibited similar changes. The N lization efficiency in the A25 treatment was higher than that in the A0 treatment; as NH4 + /NO3 − ratio increased beyond that level, N utilization efficiency significantly creased. The biomass of cucumber in 100% NO3 − -N was higher than in 100% NH4 when holding the total N amount constant, substituting 25% of NH4 + -N for NO3 − -N sulted in further growth promotion.

The Growth of Cucumber Plants under Suboptimal-Temperature Stress
While keeping the total N supply constant, using A0 (100% NO3 − -N) as the con (Figure 2), NH4 + was increased to 25%, 50%, 75% or 100% under suboptimal temp tures (18 °C day/11 °C night). The dry weights and N utilization efficiency of cucum seedlings under A50 were significantly higher than those undergoing other treatme and those under A100 and A0 were significantly lower than those in A25, A50 and ( Figure 2). This showed that increasing NH4 + to 50% under suboptimal-tempera stress led to the highest biomass of cucumber plants. This result was different from results found under optimal temperatures, where the NH4 + to N ratio of 25% promo cucumber growth the most.   The results showed that under suboptimal-temperature stress, root volume ( Figure 3E), number of root branches ( Figure 3F), total root length ( Figure 3G) and root morphology ( Figure 4) exhibited the same pattern: 0% NH 4 + -N < 25% NH 4 + -N < 50% NH 4 + -N. This indicated that in the 0-50% range, increasing NH 4 + promoted cucumber root growth more, while in the 50-100% range, a higher ratio of NH 4 + to N resulted in lower root volume, number of root branches and total root length. The results showed that under suboptimal-temperature stress, root volume ( Figure  3E), number of root branches ( Figure 3F), total root length ( Figure 3G) and root morphology ( Figure 4) exhibited the same pattern: 0% NH4 + -N < 25% NH4 + -N < 50% NH4 + -N. This indicated that in the 0-50% range, increasing NH4 + promoted cucumber root growth more, while in the 50-100% range, a higher ratio of NH4 + to N resulted in lower root volume, number of root branches and total root length.   represent the standard error, and different letters above the bars indicate significant differences at p < 0.05, according to Tukey's multiple comparison test.

The Root Architecture of Cucumber Plants
The results showed that under suboptimal-temperature stress, root volume ( Figure  3E), number of root branches ( Figure 3F), total root length ( Figure 3G) and root morphology ( Figure 4) exhibited the same pattern: 0% NH4 + -N < 25% NH4 + -N < 50% NH4 + -N. This indicated that in the 0-50% range, increasing NH4 + promoted cucumber root growth more, while in the 50-100% range, a higher ratio of NH4 + to N resulted in lower root volume, number of root branches and total root length.   Root activity measured using the TTC method was closely correlated with the number of growth points in roots and their physiological activities. Furthermore, a higher triphenyl tetrazolium formazan value of the TTC-reduced substance indicated a higher physiological  Figure 3H showed that the cucumber root activity increased when the ratio of NH 4 + was increased from 0% to 100% under suboptimal-temperature stress. Root activity was the highest in the A100 treatment, while that in A0 treatment was the lowest.

The Root/Shoot Ratio in Cucumber Seedlings
Under suboptimal-temperature stress, the root/shoot ratio in cucumber seedlings gradually decreased when the ratio of NH 4 + to N in the NO 3 − /NH 4 + composition increased. However, increased NH 4 + -N did not decrease the root dry weight (Root (DW)) ( Table 1).

The Effect of Increased Ammonium on Adversity Metabolites under Suboptimal-Temperature Stress
The protein content reached the highest level under A50, followed by A75, whose level was lowest under A0 ( Figure 5I). The proline continuously increased when NH 4 + -N increased from 0% to 100% ( Figure 5J). The MDA content was the lowest under A50 and the highest under A0, which was opposite to the changes in protein content ( Figure 5I,K). The results show that protein content first increased, then decreased, while MDA content first decreased, then increased when the ratio of NH 4 + to N increased. Root activity measured using the TTC method was closely correlated with the number of growth points in roots and their physiological activities. Furthermore, a higher triphenyl tetrazolium formazan value of the TTC-reduced substance indicated a higher physiological activity of the root. Figure 3H showed that the cucumber root activity increased when the ratio of NH4 + was increased from 0% to 100% under suboptimal-temperature stress. Root activity was the highest in the A100 treatment, while that in A0 treatment was the lowest.

The Root/Shoot Ratio in Cucumber Seedlings
Under suboptimal-temperature stress, the root/shoot ratio in cucumber seedlings gradually decreased when the ratio of NH4 + to N in the NO3 − /NH4 + composition increased. However, increased NH4 + -N did not decrease the root dry weight (Root (DW)) ( Table 1).

The Effect of Increased Ammonium on Adversity Metabolites under Suboptimal-Temperature Stress
The protein content reached the highest level under A50, followed by A75, whose level was lowest under A0 ( Figure 5I). The proline continuously increased when NH4 + -N increased from 0% to 100% ( Figure 5J). The MDA content was the lowest under A50 and the highest under A0, which was opposite to the changes in protein content ( Figure 5I,K). The results show that protein content first increased, then decreased, while MDA content first decreased, then increased when the ratio of NH4 + to N increased. We used qRT-PCR to measure the relative expression levels of genes ( Figure 6). The  We used qRT-PCR to measure the relative expression levels of genes ( Figure 6). The nitrate-uptake and -transport gene, CsNRT1.2, had its highest expression level in the leaves under A50, while CsNRT1.3 and CsNRT1.5 had their highest expression levels in the roots under A50. CsNRT1.2, CsNRT1.3, CsNRT1.4, CsNRT1.5 and CsNRT1.8 exhibited significantly higher expression in the roots than in the leaves. Regarding ammoniumuptake and -transport genes in roots and leaves, the expression of the NH 4 + -N-transport gene CsAMT1.1 increased as the ratio of ammonium increased from 0% to 50%, while its expression decreased as the ratio of ammonium increased beyond that. Its expression level was highest under A50. Moreover, under all increased NH 4 + treatments, the CsAMT1.1 gene was expressed more highly in the roots than the leaves. Plasma membrane H + -ATPase genes in roots and leaves, namely the ATPase genes CsHA2 and CsHA3, were expressed more highly as the ratio of NH 4 + to N increased from 0% to 100%, but they displayed some differences to each other. In all treatments with increased NH 4 + , CsHA2 was higher in the roots than in the leaves, while CsHA3 was significantly lower in the roots than in the leaves.

The Expression of Nitrogen-Metabolic Glutamine Cycle Genes
The results showed that in the roots, CsGS-2 had its highest expression level under A50. CsGS-3 expression in the roots increased as the ratio of NH 4 + to N increased from 0% to 100%. In the leaves, CsGS-3 was expressed more highly, as the ratio of NH 4 + to N increased from 0% to 50%, reaching its highest level in A50. Moreover, the expression level of CsGS-3 in the roots was higher than in the leaves in all treatments with increased NH 4 + ( Figure 7). We used qRT-PCR to measure the relative expression levels of genes ( Figure 6). The nitrate-uptake and -transport gene, CsNRT1.2, had its highest expression level in the leaves under A50, while CsNRT1.3 and CsNRT1.5 had their highest expression levels in the roots under A50. CsNRT1.2, CsNRT1.3, CsNRT1.4, CsNRT1.5 and CsNRT1.8 exhibited significantly higher expression in the roots than in the leaves. Regarding ammonium-uptake and -transport genes in roots and leaves, the expression of the NH4 + -N-transport gene CsAMT1.1 increased as the ratio of ammonium increased from 0% to 50%, while its expression decreased as the ratio of ammonium increased beyond that. Its expression level was highest under A50. Moreover, under all increased NH4 + treatments, the CsAMT1.1 gene was expressed more highly in the roots than the leaves. Plasma membrane H + -ATPase genes in roots and leaves, namely the ATPase genes CsHA2 and CsHA3, were expressed more highly as the ratio of NH4 + to N increased from 0% to 100%, but they displayed some differences to each other. In all treatments with increased NH4 + , CsHA2 was higher in the roots than in the leaves, while CsHA3 was significantly lower in the roots than in the leaves.

The Expression of Nitrogen-Metabolic Glutamine Cycle Genes
The results showed that in the roots, CsGS-2 had its highest expression level under A50. CsGS-3 expression in the roots increased as the ratio of NH4 + to N increased from 0% to 100%. In the leaves, CsGS-3 was expressed more highly, as the ratio of NH4 + to N increased from 0% to 50%, reaching its highest level in A50. Moreover, the expression level of CsGS-3 in the roots was higher than in the leaves in all treatments with increased NH4 + (Figure 7). Figure 6. Effect of increasing ammonium on N-uptake and -transport gene expression in cucumber seedings under suboptimal-temperature stress. Bars represent the standard error, and different letters above the bars indicate significant differences at p < 0.05, according to Tukey's multiple comparison test. Figure 6. Effect of increasing ammonium on N-uptake and -transport gene expression in cucumber seedings under suboptimal-temperature stress. Bars represent the standard error, and different letters above the bars indicate significant differences at p < 0.05, according to Tukey's multiple comparison test.

The Expression of Nitrogen-Metabolic Glutamine Cycle Genes
The results showed that in the roots, CsGS-2 had its highest expression level under A50. CsGS-3 expression in the roots increased as the ratio of NH4 + to N increased from 0% to 100%. In the leaves, CsGS-3 was expressed more highly, as the ratio of NH4 + to N increased from 0% to 50%, reaching its highest level in A50. Moreover, the expression level of CsGS-3 in the roots was higher than in the leaves in all treatments with increased NH4 + (Figure 7). The expression of CsGOGAT-1-2, CsGOGAT-2-1 and CsGOGAT-2-2 showed similar patterns in the roots of cucumber seedlings: when NH4 + increased by 0-50%, they all increased, reaching their highest levels under A50. In the leaves, CsGOGAT-1-1, CsGOGAT-1-2, CsGOGAT-2-1 and CsGOGAT-2-2 all exhibited their highest expression levels when the NH4 + to N ratio was 50%. However, the expression levels of these four genes in the leaves were significantly lower than those in the roots under all treatments with increased NH4 + (Figure 7).

The Responses of Seedling Growth and Root Morphology to Increased Ammonium under Suboptimal-Temperature Stress
Although vegetable cultivation mainly uses NO3 − -N as the N source, increasing NH4 + nutrition appropriately can not only increase vegetable yield and reduce the content of nitrate salt but also increase N utilization efficiency [20,21]. Our study found that cucumbers required a higher ratio of NH4 + -N under suboptimal-temperature stress compared to optimal temperature conditions. Zhang et al. [8] also found that NO3 − -N uptake in cotton seedlings was significantly inhibited by suboptimal temperatures, but this inhibitory effect was alleviated by partially substituting NO3 − -N with NH4 + -N. Similar results were shown in a study on N in sweet melon seedlings by Gao et al. [22]. Regarding the reason why plants require a higher ratio of NH4 + under suboptimal-temperature conditions, Di et al. [23] proposed that both NO3 − uptake-transport and NO3 − reduction require energy, a too high NO3 − to N ratio could lead to the overconsumption of energy, and plants tend to favour the uptake and assimilation of NH4 + -N with less energy consumption under suboptimal-temperature stress.
This study found that under suboptimal temperatures, with the ratio of NH4 + to N increasing from 0% to 100%, cucumber root parameters first increased and then decreased, indicating that increasing NH4 + to 50% maximized root growth (Figure 3). However, the root/shoot ratio of cucumber seedlings gradually decreased (Table 1). Then, we further analysed root activity, which was increased when the ratio of NH4 + in- The expression of CsGOGAT-1-2, CsGOGAT-2-1 and CsGOGAT-2-2 showed similar patterns in the roots of cucumber seedlings: when NH 4 + increased by 0-50%, they all increased, reaching their highest levels under A50. In the leaves, CsGOGAT-1-1, CsGOGAT-1-2, CsGOGAT-2-1 and CsGOGAT-2-2 all exhibited their highest expression levels when the NH 4 + to N ratio was 50%. However, the expression levels of these four genes in the leaves were significantly lower than those in the roots under all treatments with increased NH 4 + ( Figure 7).

The Responses of Seedling Growth and Root Morphology to Increased Ammonium under Suboptimal-Temperature Stress
Although vegetable cultivation mainly uses NO 3 − -N as the N source, increasing NH 4 + nutrition appropriately can not only increase vegetable yield and reduce the content of nitrate salt but also increase N utilization efficiency [20,21]. Our study found that cucumbers required a higher ratio of NH 4 + -N under suboptimal-temperature stress compared to optimal temperature conditions. Zhang et al. [8] also found that NO 3 − -N uptake in cotton seedlings was significantly inhibited by suboptimal temperatures, but this inhibitory effect was alleviated by partially substituting NO 3 − -N with NH 4 + -N. Similar results were shown in a study on N in sweet melon seedlings by Gao et al. [22]. Regarding the reason why plants require a higher ratio of NH 4 + under suboptimal-temperature conditions, Di et al. [23] proposed that both NO 3 − uptake-transport and NO 3 − reduction require energy, a too high NO 3 − to N ratio could lead to the overconsumption of energy, and plants tend to favour the uptake and assimilation of NH 4 + -N with less energy consumption under suboptimal-temperature stress.
This study found that under suboptimal temperatures, with the ratio of NH 4 + to N increasing from 0% to 100%, cucumber root parameters first increased and then decreased, indicating that increasing NH 4 + to 50% maximized root growth ( Figure 3). However, the root/shoot ratio of cucumber seedlings gradually decreased (Table 1). Then, we further analysed root activity, which was increased when the ratio of NH 4 + increased under subop-timal temperatures. Root activity and N uptake ability are significantly correlated [24,25]. In this study, although the treatments with increased NH 4 + decreased the root/shoot ratio, the root dry weight and root volume did not significantly decrease (Table 1). Therefore, nutrient uptake via roots was strengthened and plant growth was promoted.

The Responses of Adversity Metabolites to Increased Ammonium under Suboptimal-Temperature Stress
The results show that the pattern of MDA content changes with the ratio of NH 4 + was exactly opposite to that of protein content when the ratio of NH 4 + -N increased ( Figure 6). These results indicated that MDA accumulation induced by suboptimal-temperature stress led to protein damage and the inhibition of the protein synthesis. MDA is the main product of membrane lipid peroxidation, and it also has inhibitory effects on protein synthesis [26]. The level of protein in plants increases under different stresses, which helps to maintain a relatively low osmotic potential in the cell [27]. Moreover, we found that the protein and proline contents in the cucumber leaves increased as NH 4 + increased from 0% to 50% under suboptimal-temperature stress, which could strengthen the plant's water retention ability and alleviate cellular damage caused by suboptimal temperatures. Thus, an increase in intracellular proline is able to maintain cell osmotic pressure and prevent excessive water loss, alleviating cell damage from low temperatures [28].

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Nitrogen uptake-transport genes In this study, CsNRT1.3, CsNRT1.5 and CsAMT1.1 in roots had their highest expression levels in the treatment of 50% NH 4 + , corresponding to the leaf protein content and seedling biomass. Additionally, the expression of seven nitrogen uptake-transport genes was higher in roots than those in leaves ( Figure 6). This indicated that during this stage, when NH 4 + was high, the main action site of these genes was the root. Tang et al. [29] and Forde et al. [30] reported the specific gene expression patterns of NO 3 − -N-uptake and -transport proteins in different tissues of crops. In addition, we also found that the expression of CsNRT1.3 and CsNRT1.5 in the roots was higher than under other conditions when NH 4 + increased to 50%, indicating that the NH 4 + to N ratio of 50% promoted the transport of NO 3 − -N from roots to shoots. Migocka et al. [13] found that CsNRT1.3 and CsNRT1.5 transported NO 3 − -N from roots to shoots. The AMT gene family contains AMT1 and AMT2. The AMT1 gene plays an important role in NH 4 + uptake in the roots from the soil [31]. In Arabidopsis, the ammonium uptake ability can be altered by the changes in the expression of the AtAMT1.1, AtAMTl.2 and AtAMTl.3 genes [32]. In this study, we found that the NH 4 + -N transporter gene CsAMT1.1 in the roots and leaves of cucumber seedlings was first upregulated and then downregulated with the increase in the NH 4 + to N ratio. These results indicate that the CsAMT1.1 gene was induced by NH 4 + -N, but a higher NH 4 + to N ratio was not necessarily better: the 50% NH 4 + to N ratio induced the upregulation of CsAMT1.1, thereby promoting NH 4 + -N uptake and transport in cucumber seedlings.
Our study found that the root PM H + -ATPase genes CsHA2 and CsHA3 were upregulated as the NH 4 + -N ratio increased from 0% to 100%, indicating that increasing NH 4 + elevated the expression of the plasma membrane H + -ATP genes. Miguel et al. [33] reported that H + -ATP was very important in the secondary transmembrane transport of nutrients, especially plant cells involved in environmental stress. It is possible that NH 4 + -N induced the depolarization of the cell membrane, which then activated the plasma membrane H + -ATPase and induced the expression of the CsHA2 and CsHA3 genes [34,35].

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Nitrogen metabolism genes The expression of GS and GOGAT genes reflect the level of N metabolism to a certain extent [36]. Our study found that in the roots, CsGS-2 expression was at its highest under the 50% NH 4 + treatment. The expression of CsGS3 in roots was increased when the ratio of NH 4 + to N increased. These data showed that the expression levels of two genes involved in the same process exhibited different response to the ratio of NH 4 + , but treatments increasing NH 4 + to a suitable level could upregulate the expression of both genes. In addition, our study showed that upon suboptimal-temperature stress, the expression of CsGOGAT-1-2, CsGOGAT-2-1 and CsGOGAT-2-2 in the leaves and roots all reached their highest levels when the NH 4 + to N ratio was 50% (Figure 7). The CsGOGAT gene family exhibited good consistency in response to increased NH 4 + , indicating that increasing NH 4 + to 50% most significantly induced their expression. Summarizing the above analysis of N metabolism genes, after the exogenous N was absorbed and transported, via the glutamine-glutamate cycle regulated by the CsGS-2, CsGS-3, CsGOGAT-1-2, CsGOGAT-2-1 and CsGOGAT-2-2 genes, amino acids and other N compounds necessary for plants were synthesized.
Moreover, the expression levels of six genes in the roots were significantly higher than those in the leaves in all treatments with increased NH 4 + . We supposed that a large amount of NH 4 + was rapidly assimilated upon entering the plant roots in order to be transported into the leaves for plant growth. As a result, they played an important role in promoting nitrogen assimilation to enhance the suboptimal-temperature tolerance of the cucumber seedlings.
Through the above research and discussion, we proposed schematic diagram of increasing ammonium to reduce the growth inhibition of cucumber seedlings under suboptimaltemperature stress (Figure 8). The expression of GS and GOGAT genes reflect the level of N metabolism to a certain extent [36]. Our study found that in the roots, CsGS-2 expression was at its highest under the 50% NH4 + treatment. The expression of CsGS3 in roots was increased when the ratio of NH4 + to N increased. These data showed that the expression levels of two genes involved in the same process exhibited different response to the ratio of NH4 + , but treatments increasing NH4 + to a suitable level could upregulate the expression of both genes. In addition, our study showed that upon suboptimal-temperature stress, the expression of CsGOGAT-1-2, CsGOGAT-2-1 and CsGOGAT-2-2 in the leaves and roots all reached their highest levels when the NH4 + to N ratio was 50% (Figure 7). The CsGOGAT gene family exhibited good consistency in response to increased NH4 + , indicating that increasing NH4 + to 50% most significantly induced their expression. Summarizing the above analysis of N metabolism genes, after the exogenous N was absorbed and transported, via the glutamine-glutamate cycle regulated by the CsGS-2, CsGS-3, CsGOGAT-1-2, CsGOGAT-2-1 and CsGOGAT-2-2 genes, amino acids and other N compounds necessary for plants were synthesized.
Moreover, the expression levels of six genes in the roots were significantly higher than those in the leaves in all treatments with increased NH4 + . We supposed that a large amount of NH4 + was rapidly assimilated upon entering the plant roots in order to be transported into the leaves for plant growth. As a result, they played an important role in promoting nitrogen assimilation to enhance the suboptimal-temperature tolerance of the cucumber seedlings.
Through the above research and discussion, we proposed schematic diagram of increasing ammonium to reduce the growth inhibition of cucumber seedlings under suboptimal-temperature stress ( Figure 8).

Materials and Plant Growth Conditions
This experiment was carried out in the greenhouse and growth chambers at the Guizhou Academy of Agricultural Sciences, China (26°35′ N, 106°42′ E), in 2020. The experimental cucumber variety was 'Zhongnong No. 26′, selectively bred at the Institute of

Materials and Plant Growth Conditions
This experiment was carried out in the greenhouse and growth chambers at the Guizhou Academy of Agricultural Sciences, China (26 • 35 N, 106 • 42 E), in 2020. The experimental cucumber variety was 'Zhongnong No. 26 , selectively bred at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Cucumber seeds were soaked in warm water, sterilized, wrapped in cotton gauze and germinated in the dark at 28 • C for 24 h. Germinated seeds were then sown in medium dishes (54 holes) filled with vermiculite. The cucumber seedlings were transferred to a liquid medium box filled with 1/2 standard Hongland nutrients (EC = 2.4-2.6 ms/cm) when two leaves appeared. The treatments were begun at the four-leaf stage of the cucumber seedlings, which were then cultivated for 14 days.

Experimental Treatments
(1) Temperature treatment: Suboptimal-temperature stress for cucumber was 18 • C (day)/11 • C (night). Optimal temperature was 28 • C (day)/21 • C (night). (2) Nitrogen source treatment: Treatment with different N sources was carried out using different liquid media for cucumber cultivation. The molar quantity of N remained the same in all liquid media (12 mmol/L), while the N forms were different. The liquid medium base was a new formulation developed based on the Hongland formulation [37] by adjusting the N forms.
Full NO 3 − -N (100% NO 3 − ) was used as the control, and different ratios of NH 4 + -N substitution were the treatments ( Table 2). In the table, A represents NH 4 + -N (ammonium), and the percentages (%) represent the ratios of NH 4 + to N and NO 3 − to N out of the total N. Five treatments with three biological replicates were applied to a total of 90 cucumber seedlings in the experiment; thus, six cucumber seedlings were collected per treatment per replicate.

Measurements of Plant Biomass
Cucumber plants were harvested 14 days after the suboptimal-temperature stress. After harvest, plants were washed with distilled water and separated into two parts, namely shoots and roots. Dry weight was measured after drying the plants in the oven at 105 • C for 20 min, followed by drying at 80 • C for 48 h.

Measurement of Root Parameters
After harvest, roots were placed in water in the organic glass dish of the root scanner. The main root and lateral roots were spread out according to the growth position of the cucumber roots; the roots were then scanned using a root scanner (Epson G101, Seiko Epson, Naganoken, Japan). The digital software Win RHIZO 2009 was used for analysis and to obtain root parameters, including root length, root surface area, root volume and number of root tips. The number of root tips referred to the total number of root tips from all levels in the root system, while the root length referred to the total length of the main root plus all root branches.

Measurement of Root Activity
Samples for the measurement of root activity were selected from white, newly developed root tips. A total of 0.2 g of root tips was weighed, and the triphenyltetrazolium chloride (TTC) method was used [38]. Root activity measured by the TTC method was represented by dehydrogenase activity.

Analysis of N Utilization Efficiency
Nitrogen content in plants (g·plant −1 DW) was calculated as (leaf dry weight × N content per unit leaf weight) + (stem dry weight × N content per unit stem weight) + (root dry weight × N content per unit root weight).
The amount of N application in plants equalled the N content added to the liquid medium.
N utilization efficiency = N content in plants/amount of N applied [5].

Biochemical Analysis
The third leaf counted from the top at 14 days was taken from each cucumber plant, immediately frozen in liquid N and stored at −80 • C for biochemical analyses. Protein content was measured using kits using the BCA method. Proline content was measured using the acid ninhydrin colorimetric assay [39]. Malondialdehyde (MDA) content was measured using the thiobarbituric acid method [28].

Total RNA Isolation and cDNA Synthesis
The total RNA of the leaves from cucumber plants was extracted using a commercial RNA extraction kit (Tiangen, Beijing, China), according to the manufacturer's instructions. cDNA was synthesized using the Prime Script RT Reagent Kit (TaKaRa, Dalian, China), according to the manufacturer's instructions. Finally, cDNA was used for quantitative real-time PCR (qRT-PCR) analysis.

Gene Expression Analysis Using qRT-PCR
To detect the gene expression levels of N-related genes in cucumbers, we utilized 9 reported genes of nitrogen uptake-transport in cucumbers and referenced the gene sequences in their experiment, including nitrate-uptake and -transport genes [13], ammonium-uptake and -transport genes [40] and plasma membrane H + -ATPase genes [31]. Meanwhile, we identified 8 genes involved in nitrogen metabolism in the cucumber, then clarified their sequences in previous experiments, including in CsGS genes and CsGOGAT genes [18] ( Table 3). The cucumber actin gene was used as an internal control to normalize the data (accession no. XM_004147305.3) [40]. Primers were designed using Primer Premier 5 software (Primer Co., Waterloo, ON, Canada), according to the acquired gene sequences. The primer sequences used for qRT-PCR are shown in Table 4.

Data Analysis
Tukey's randomized block analysis of variance (ANOVA) was used for the analysis of different N forms with the significance cut-off of p < 0.05. Each treatment was analysed three times. Pearson's correlation analysis was conducted using SPSS 22.0 software (version 22.0, International Business Machines Corporation, Armonk, New York, NY, USA).

Conclusions
This study conducted experiments to investigate the effect of exogenous N forms on N uptake and metabolism under suboptimal temperatures in cucumber seedlings. The results were as follows: first of all, the most suitable ratio of NH 4 + to N for the growth of cucumber seedlings under suboptimal-temperature stress was 50%, higher than the ratio (25%) under optimal temperatures. Cucumber seedlings in the 50% NH 4 + treatment had the highest root parameters and the corresponding biomass and protein and proline contents but the lowest MDA content. These results indicated that increasing NH 4 + nutrition to 50% elevated the tolerance of cucumber seedlings to suboptimal temperatures. Furthermore, under suboptimal-temperature stress, the level of upregulated genes involved in N uptake and metabolism was the highest when the NH 4 + ratio was 50%, which revealed that increased NH 4 + regulated the expression of these genes, contributing to suboptimal-temperature tolerance. These genes included the nitrogen-uptake genes CSNRT1.3, CSNRT1.5 and CSAMT1.1 and glutamate cycle genes CsGOGAT-1-2, CsGOGAT-2-1, CsGOGAT-2-2, CsGS-2 and CsGS-3. The expression of two plasma membrane H + -ATPase genes, CsHA2 and CsHA3, increased as the NH 4 + to N ratio increased from 0% to 100%. Additionally, 13 of 16 genes detected in the study had significantly higher expression levels in the roots than in the leaves. The significant response of these genes in the roots could have led to rapid N assimilation.