24-Epibrassinolide promotes NO3− and NH4+ ion flux rate and NRT1 gene expression in cucumber under suboptimal root zone temperature
Suboptimal root zone temperature (RZT) causes a remarkable reduction in growth of horticultural crops during winter cultivation under greenhouse production. However, limited information is available on the effects of suboptimal RZT on nitrogen (N) metabolism in cucumber seedlings. The aim of this study is to investigate the effects of 24-Epibrassinolide (EBR) on nitrate and ammonium flux rate, N metabolism, and transcript levels of NRT1 family genes under suboptimal RZT in cucumber seedlings.
Suboptimal RZT (LT) negatively affected on cucumber growth and proportionately decreased EBR contents, bleeding rate, root activity, enzyme activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT), nitrate (NO3−) influx rate, ammonium (NH4+) efflux rate, and transcript levels of nitrate transporter (NRT1) encoding genes. However, exogenous EBR reduced the harmful effects of suboptimal RZT and increased endogenous EBR contents, bleeding rate, root activity, enzyme activities of NR, NiR, GS, and GOGAT, NH4+ and NO3− flux rates and contents, and N accumulation. EBR-treated seedlings also upregulated the transcript levels of nitrate transporters CsNRT1.1, CsNRT1.2A, CsNRT1.2B, CsNRT1.2C, CsNRT1.3, CsNRT1.4A, CsNRT1.5B, CsNRT1.5C, CsNRT1.9, and CsNRT1.10, and downregulated CsNRT1.5A and CsNRT1.8. LT treatment upregulated the expression level of CsNRT1.5A, while exogenous BZR application downregulated the expression level of NRT1 genes.
These results indicate that exogenous application of EBR alleviated the harmful effects of suboptimal RZT through changes in N metabolism, NH4+ and NO3− flux rates, and NRT1 gene expression, leading to improved cucumber seedlings growth. Our study provides the first evidence of the role of EBR in the response to suboptimal RZT in cucumber, and can be used to improve vegetable production.
Keywords24-Epibrassinolide Root zone temperature NO3− and NH4+ flux rates NRT1 genes
Ammonium Transporter 1
Analysis of Variance
BRI1-EMS SUPPERSSOR 1
BRASSINAZOLE RESISTANT 1
Cold Responsive Genes
Heat Shock Proteins
Least Significant Difference
Non-invasive Micro-test Technology
Nitrate Transporter 1
Root Zone Temperature
Environmental factors influence plant growth and developments, and temperature is particularly important. Air temperature is unstable, while root zone temperature (RZT) is considered both stable and more important for study . Temperature and light intensity are key factors affecting plant growth and development , which are also influenced by humidity and nutrient availability [3, 4]. Among these factors temperature is very important, that effects overall plant developmental process in short time [3, 7]. The previous studies reported that low temperature stress leads to over production of reactive oxygen species (ROS) and reduce antioxidant enzyme activates, reduction in chlorophylls and photosynthetic capacity, hormonal imbalance, ion uptake and accumulation, thus caused a significant reduction in plant growth and yield [2, 4, 6, 8]. Physiological and molecular studies have shown that plant growth is affected by RZT . RZT influences physiological and biological processes, thereby affecting nutrient uptake and availability [5, 6]. Plant nutrient absorption rate is dependent on RZT , and can alter ion balance and nitrogen metabolism ; a small increase in RZT could induce large changes in plant growth and development .
During winter cultivation, air temperature is unstable, while soil temperature changes slowly and is maintained around 10–15 °C . RZT plays a critical role in plant root physiology, morphology, growth, nutrient and water uptake, and translocation from root to leaf. Even horticultural crops exposed to suboptimal RZT may experience heavy losses of early productivity [3, 5, 6, 7]. Earlier studies reported that RZT severely affected nitrogen metabolism in cucumber and reduced growth and yield . These studies demonstrated the importance of RZT on plant growth and development. The potential mechanisms of growth inhibition at ambient RZT may involve water and nutrient uptake rates but are largely unknown.
Nitrogen (N) is an essential macronutrient and its availability in soil affects plant growth and development, as well as all metabolic processes . N is a major constituent of proteins and nucleotides, as well as of chlorophyll, numerous metabolites, and cellular components . Nutrient availability and uptake affect plant growth and development . Ammonium (NH4+) and nitrate (NO3−) are the principal soil N sources for plants . Plant fine roots absorb NO3− and assimilate NH4+ into organic N via the GOGAT enzyme, once inside root cells, nitrate (NO3−) can be reduced to ammonium (NH4+) by nitrate and nitrite reductase and then assimilated into organic nitrogen through the glutamine synthase (GS)-GOGAT cycle [13, 14, 15, 16]. Plants take up nitrate and transport it across the specialized plasma membrane made of root epidermal and cortical cells through a complex transport system [17, 18]. The mechanisms by which nitrate influx and efflux occur have been characterized at both the physiological and molecular levels [13, 19]. Plant cells consist of two nitrate uptake systems; one is a low-affinity system, either constitutive low-affinity system (cLATS) or inducible low affinity transport system (iLATS), which are encoded by NRT1 genes; the other is a high affinity transport system, either constitutive high affinity transport system (cHATS) or inducible high affinity transport system (iHATS), which are encoded by NRT2 genes [12, 20]. In Arabidopsis, eleven NRT1 and seven NRT2 gene homologues have been identified, but a limited number are considered responsible for nitrate uptake from soil [13, 21]. In iHATS, NRT2.1 and 2.2 and NRT1.1 in iLATS appear to play a major role in NO3− influx [11, 17]. LATS and HATS are involved in root xylem loading and unloading of nitrate (AtNRT1.5 and AtNRT1.8) and transport into the leaf [12, 17].
Brassinosteroids (EBRs) are growth-promoting steroid phytohormones in plants [22, 23]. EBRs play vital roles in a wide range of developmental processes in plants from germination to fruit development [24, 25]. Exogenous application of EBRs regulates a variety of physiological, biochemical, and molecular processes which enhance plant tolerance to a variety of abiotic stresses, such as low temperatures, heavy metals, and drought [25, 26]. 24-epibrassinolide (EBR) is the most active synthetic analog of the EBR family and can improve tolerance of low temperatures in pepper, tomato, eggplant, cucumber, and ryegrass [22, 27, 28, 29, 30]. The mechanism of EBR activity in plant responses to abiotic stress has been reported [25, 31, 32, 33]. EBR promotes plant tolerance to heat, cold, drought, and salinity by correlating with higher expression of stress marker genes, including heat shock proteins (HSPs) and cold responsive genes (COR) [25, 29, 34]. EBR Plant exposed to low/cold stress caused negative effects on chlorophyll, photosynthesis, nutrients accumulation and antioxidant enzyme activity, thus leads to reduced plant growth and yield. The pervious study reported that chilling stress downregulate thousands of genes in involved in many developmental process, including chlorophyll and photosynthesis, antioxidant enzymes, hormones and transcriptional factors, while exogenous EBR application reduce the negative effect of chilling/cold stress on pepper seedling . Cold stress caused a significant reduction in antioxidant enzyme activities and increase ROS (reactive oxygen species) accumulation, thus leads to reduce chlorophylls and photosynthetic capacity, while exogenous EBR application reduce the harmful effects and improve growth . These findings are suggested that, EBR enhances biosynthesis of chlorophyll and photosynthetic machinery and activates stress tolerance enzymes, thus reduce the harmful effects of abiotic stresses [35, 36, 37]. A previous study reported that EBR regulated nitrogen uptake and metabolism in Arabidopsis via the EBR signaling pathway [23, 38]. Additionally, EBR receptor BRI1 (BRASSINOSTEROID INSENSITIVE 1) mutant bri1–5 induced expression of AMT1 (ammonium transporter 1) and GS and GOGAT encoded genes, showing that EBR signaling transcription factors BES1 (BRI1-EMS SUPPERSSOR 1) and BZR1 (BRASSINAZOLE RESISTANT 1) are involved in pathways of EBR-mediated nitrogen metabolism and uptake [23, 39, 40, 41]. A recent study reported that EBR enhanced low temperature and weak light stress tolerance in tomato, by improving nitrogen metabolism, stimulating nitrate and ammonium accumulation, and accelerating nitrogen conversion into free amino acids . These amino acids are involved in biosynthesis of chlorophylls, proteins, primary and secondary metabolites, and enzyme biosynthesis . These findings suggest an active role for EBR in stress and in nitrogen uptake and metabolism to reduce the harmful effect of stress. However, little is known about the role of EBR in nitrate and ammonium ion influx and in regulation of nitrogen metabolism under suboptimal RZT. This study will comprehensively determine the role of EBR in nitrogen uptake, metabolism, and accumulation under suboptimal RZT in cucumber seedlings.
Cucumber, which is widely grown in greenhouses in northern parts of China during summer and winter seasons, is intolerant to suboptimal RZT, leading to large yield losses [2, 9]. Therefore, suboptimal RZT is a major limiting factor for winter cultivation of cucumber in greenhouses [9, 42, 43]. In this study, we investigated the effect of EBR on cucumber seedling physiology and growth under suboptimal RZT. We hypothesized that exogenous EBR applied to leaves may enhance cucumber seedling growth by increasing enzyme activities and expression of genes involved in nitrogen metabolism as well as regulating nutrient uptake (ion influx rate). The key objectives of this study were to: (1) investigate the effect of RZT on plant physiology; (2) examine whether exogenous EBR application can effectively enhance nitrogen metabolism and uptake rate (ion influx rate); and (3) examine whether exogenous EBR regulates NRT1 expression in cucumber. The results could improve understanding of the role of EBR in nitrogen metabolism, uptake, and response to RZT, which is useful for greenhouse vegetable production.
Effect of EBR on cucumber seedlings growth under suboptimal RZT
Changes in cucumber seedling growth after seven days under suboptimal RZT with EBR and BZR application
Plant Height (cm)
Hypocotyl Diameter (mm)
Leaf Area (mm2)
Total FW (g)
6.13 ± 0.41 a
3.84 ± 0.07 a
678.33 ± 37.78a
3.97 ± 0.15 a
3.88 ± 0.36 a
4.50 ± 0.35 b
2.71 ± 0.08 b
483.55 ± 50.11 b
2.46 ± 0.29 b
2.36 ± 0.41 b
6.56 ± 0.34 a
3.74 ± 0.08 a
719.01 ± 33.86 a
3.98 ± 0.27 a
3.57 ± 0.31 a
4.39 ± 0.42 b
2.96 ± 0.17 b
440.27 ± 32.83 b
2.29 ± 0.12 b
2.23 ± 0.38 b
Effect of exogenous EBR application on endogenous accumulation of EBR
Effect of EBR on root activity and bleeding rate
Suboptimal RZT also negatively affected bleeding rate (Fig. 2 B. In the LT treatment, bleeding rate was reduced by 46.73% compared to that of the NT treatment, while exogenous EBR application significantly increased bleeding rate by 47.31, and 55.65%, compared to that of the LT and BZR treatments (Fig. 2 B). The bleeding rate did not differ detectably between the NT and EBR treatments. These findings suggest that suboptimal RZT negatively affected root activity and bleeding rate and caused a significant reduction in growth.
Effect of EBR on NR, NiR, GS and GOGAT enzyme activities
Effect of EBR on NH4 + and NO3 − fluxes rate
The results indicated that, NO3− influx rate in cucumber roots were also adversely affected by suboptimal RZT, as shown in Fig. 4. The average influx rates indicate that exogenous EBR application significantly increased NO3− influx rate by 72.11 and 86.02%, when compared with LT and BZR treatments, respectively (Fig. 4). NO3− flux rate decreased significantly (by 54.85%) in the LT compared to the NT treatment. Additionally, EBR increased the nitrate influx rate by 15.75% compared to that of the NT treatment, but this difference was not significant (Fig. 4). The influx rate of nitrate did not differ significantly between LT and BZR (Fig. 4).
Similarly, the NH4+ efflux rate was significantly higher in the EBR treatment, but significantly lower in the BZR and LT treatments (Fig. 5). Exogenous EBR application increased average NH4+ efflux rate by 15.75, 71.01, and 76.44%, as compared to the NT, LT, and BZR treatments, respectively (Fig. 5). The NT treatment showed a 71.53% increase in average NH4+ flux rate compared to the LT treatment (Fig. 5). The difference between the NT and EBR treatments was not significant from 0 to 2.5 min (Fig. 5) but became significant over time (Fig. 5). The differences between the LT and BZR treatments were not significant (Fig. 5). These results suggest that suboptimal RZT caused negative effects on cucumber roots and led to a reduction in the flux rate of NO3− and NH4+ which decreased cucumber seedling growth rate. Additionally, exogenous EBR application reduce the detrimental effects of suboptimal RZT through increasing NH4+ and NO3− flux rates.
The effect of EBR on N accumulation under suboptimal RZT
Effect of EBR on NO3 − and NH4 + contents
Nitrate transporter (NRT1) gene expression
Transcription levels of CsNRT1.1, CsNRT1.2A, CsNRT1.2B, CsNRT1.2C, CsNRT1.3, CsNRT1.4A, CsNRT1.5B, CsNRT1.5C, CsNRT1.9, and CsNRT1.10 significantly increased in EBR treated seedlings under suboptimal RZT. Additionally, EBR downregulated the expression of CsNRT1.5A and CsNRT1.8, while CsNRT1.4B showed the same trend in the NT, EBR, and BZR treatments, but was downregulated by the LT treatment. Among the LATS gene family, CsNRT1.1, CsNRT1.2A, CsNRT1.2B, CsNRT1.2C, and CsNRT1.5B showed higher expression than did other members of this family when treated with exogenous EBR under suboptimal RZT. These findings indicated that EBR activated the expression of NRT1 genes and led to increased N metabolism, thus improving cucumber seedling growth under suboptimal RZT.
EBR is a growth-promoting steroid hormone which plays an active role in a wide range of developmental processes, including abiotic stress tolerance [25, 34, 35]. EBR increases tolerance to abiotic stresses including chilling , heat , drought , and salinity . The previous study shows that EBR regulate thousands of genes in pepper to reduce the harmful effects of chilling stress . Nitrogen is an essential, that’s promotes plant growth and development, as well as alleviate the inhibitory effects of abiotic stresses, but their metabolism is sensitive suboptimal RZT . The previous studies suggested that nitrogen metabolism plays a fundamental role in biosynthesis of chlorophyll and photosynthetic capacity . It has been widely observed that abiotic stress induced reduction in chlorophyll and photosynthetic capacity accompanied by the decrease in the nitrogen metabolic enzyme activities, like NR, NiR, GS, GOGAT [2, 6, 12]. Exogenous EBR application alleviates the harmful effects of low temperature and weak light stress through enhancing the nitrogen metabolism and photochemical efficiency in tomato seedling . Suboptimal RZT causes a significant reduction in plant growth and growth-related parameters [1, 2]. Previous studies reported that exogenous EBR application increased low temperature stress tolerance, regulated levels of endogenous plant hormones (including EBR contents), and regulated expression of an EBR biosynthesis gene (CsDWF) [34, 47, 48]. In the present study, endogenous EBR contents increased in EBR treated seedlings (Fig. 1) and resulted in significant increases in cucumber seedling growth under suboptimal RZT (Table 1). These findings are in line with those of a previous study, who reported that exogenous EBR increased cucumber and pepper seedlings growth under low temperature stress [29, 48].
Correlation (Pearson) analysis between root activity, bleeding and influx rate
NO3− flux rate
NH4+ flux rate
NO3− flux rate
NH4+ flux rate
Nitrogen is an important constituent of basic nitrogen-containing compounds such as amino acids, proteins, chlorophylls, and nucleotides, which play important roles in plant growth and development [12, 17, 57, 51]. Therefore, understanding the physiological and molecular mechanisms of nitrogen metabolism and responses to suboptimal RZT is important for agronomic approaches to enhance nitrogen use efficiency in crops and reduce losses [13, 20, 58]. Most plants absorb inorganic nitrogen from soil as ammonium (NH4+) and nitrate (NO3−), which serve as N source . NH4+ can be assimilated to glutamine by the GS and GOGAT enzymes [59, 60]. Plant roots absorb NO3− which is then converted into NH4+ by NR and NiR enzymes for synthesis of amino acids, proteins, and nucleotides , thus N accumulation in roots and shoots is important for plant growth and development . A previous study revealed that RZT significantly reduced N accumulation in leaf and root tissues of cucumber, and suggested that N uptake depends on the temperature root zone . Our results indicated that N contents (total N, nitrate, and ammonium contents) under suboptimal RZT were much lower than those of EBR treated seedlings, are suggested that EBR can reduce the harmful effects of suboptimal RZT, as presented in Fig. 6. Under suboptimal RZT, root activity and bleeding rate (Fig. 2) were significantly lower than in the EBR treatments, which indicated that EBR alleviates the harmful effects induced by suboptimal RZT and may explain why EBR increased N accumulation (Fig. 6). These findings are suggested that, suboptimal RZT reduce N accumulation, thus leads to reduce cucumber seedlings growth (Table 1). The results build upon those of previous studies in which RZT negatively affected plant growth through reduced nutrient accumulation [2, 6, 61].
Enzyme activities are very sensitive and reduce very quickly under abiotic stresses [42, 49, 52, 62]. Previous studies indicated that exogenous EBR application positively regulated the activities of enzymes involved in nitrogen metabolism (NR, NiR, GS, and GOGAT) [28, 63]. We investigated the activity of these enzymes involved in N metabolism, as presented in Fig. 3. Our results indicated that the activities of these enzymes (NR, NiR, GS and GOGAT) under suboptimal RZT were much lower than in the EBR treatment. We proposed that suboptimal RZT might have cause a reduction in enzyme activities (Fig. 3), thus leading to a significant reduction in nitrate and ammonium contents (Fig. 7). Our results suggested that the NR, NiR, GS and GOGAT enzymes activities, and assimilation of nitrate and ammonium were promoted after EBR application in cucumber, as exposed to stress. These findings are supported by an earlier study which reported that EBR enhanced the activity and expression levels of GS and GOGAT enzymes and genes in Arabidopsis and concluded that BZR1 and BES1 transcription factors might be involved in different pathways of BR-mediated nitrogen metabolism and uptake [14, 23, 52]. Therefore, exogenous EBR application regulated N metabolism under suboptimal RZT, thus leading to improved growth, as presented in Table 1.
Suboptimal RZT significantly reduced root activity and bleeding rate, both of which may affect nutrient and water uptakes in cucumber seedlings (Fig. 2). N acquisition in plants is primarily regulated by plant hormones [28, 64], which may activate nitrogen signaling pathway to promotes the flux rate of NH4+ and NO3− ion in roots . Previous studies reported that ion flux is sensitive to external stimuli (abiotic stresses), which can cause a significant reduction in ion uptake/flux rate [1, 2]. Our previous results suggested, that suboptimal RZT severely reduced the enzyme activities involved in N metabolism (Fig. 3). Therefore, we speculated that EBR plays a role in NH4+ and NO3− flux rates in cucumber roots under suboptimal RZT. As predicted, NH4+ efflux and NO3− influx rate in cucumber roots under suboptimal RZT were significantly lower than in the EBR treatments (Figs. 4 & 5). Additionally, we compared NH4+ efflux and NO3− influx rates under the EBR treatment with those of the NT and BZR treatments to make clear the role of EBR. Our findings suggested that EBR increased NH4+ and NO3− flux rates and reduced harmful effects, thus leads to significant increment in nitrogen accumulation (Fig. 6). The earlier studies suggested that ammonium and nitrate flux rates are affected by abiotic stresses [8, 46, 66]. EBR is a steroid hormone and induces plant tolerance to a variety of stresses [51, 64, 67, 68]. These findings are suggested that, EBR reduces the negative effect of suboptimal RZT, through increasing NH4+ and NO3− flux rates under suboptimal RZT [28, 69, 70]. Additionally, NRT1s protein family plays an important role in nitrate absorption from soil and translocation to various plant tissues, and these proteins were significantly upregulated by exogenous EBR application, as presented in Fig. 8. Activation of the EBR signal transduction pathway may lead to upregulation of the AMT1, NRT1, and GS/GOGAT genes in Arabidopsis [23, 71, 72]. The positive correlation was reported between ion flux rate, root activity and bleeding rate (Table 2), are suggesting that EBR minimized the detrimental effects induced by suboptimal RZT, and could explain mechanism of NH4+ and NO3− influx rates, that’s significantly higher in EBR treated seedlings. These findings provide evidence that EBR enhanced NH4+ and NO3− acquisition capacity, which may have significantly increased nitrogen metabolism and cucumber seedling growth (Table 1).
Plants absorb nitrate and ammonium from soil through various transporters: NO3− is absorbed by NRT1 protein family members and incorporated into amino acids through the GS and GOGAT enzymes [2, 15, 73]. The NRT1s gene family is responsible for the overall mechanism of nitrate absorption and translocation in plants [12, 74]. However, suboptimal RZT is unfavorable for N acquisition and metabolism, which significantly reduces horticultural production [2, 7, 54]. We investigated the effect of EBR using transcript levels of NRT1 genes, which play specific roles in nitrate absorption and translocation in various plant tissue. Previous studies reported a positive correlation between the flux rates of nitrate and ammonium with the transcript levels of NRT1 genes . The results of this study indicated that exogenous EBR application significantly induced the expression of CsNRT1 genes, which were downregulated by suboptimal RZT and exogenous BZR (Fig. 8). These findings suggest that exogenous EBR activated the expression levels of NRT1 genes, potentially contributing to the observed increase in nitrate (Fig. 4) and ammonium flux rates (Fig. 5), enzyme activities (Fig. 3), and N accumulation in leaf and root tissues of EBR treated seedlings (Fig. 6). The CsNRT1s gene upregulated after exogenous EBR application, are suggested that BZR1 and BES1 transcription factors might be involved directly in NRT1s regulation [14, 15, 23]. A previous study reported that CsNRT1 genes showed variable expression patterns across plant tissues and suggested that NRT1s proteins are primarily responsible for nitrate absorption and translocation. Among these, CsNRT1.1, CsNRT1.3, CsNRT1.4B, CsNRT1.5A and CsNRT1.8 regulate nitrate, whereas other members of this family (CsNRT1.9, CsNRT1.2 s, CsNRT1.4A, CsNRT1.5B, and CsNRT1.5C) also appear to play distinct physiological roles in plants [12, 16]. These variable roles help to explain why NTR1 genes showed different expression patterns, as presented in Fig. 8. The EBR signaling pathway is known to mediate AMT1 encoded genes and induce N-metabolism and uptake in Arabidopsis . In our study, similar temporal levels of NRT1 genes were observed, indicating that suboptimal RZT down-regulated NRT1 genes and significantly reduced growth [2, 7]. In a recent study, EBR affected N metabolism by increasing nitrate and ammonium contents and enzyme activities (NR, NiR, GS, and GOGAT), which increased the tolerance of tomato to low light and temperature [23, 28, 49]. These findings are concluding that, exogenous EBR alleviates the adverse effect of suboptimal RZT by modulating nitrogen metabolism, thus leading to improved cucumber seedling growth.
Plant material and growth conditions
Cucumber (Cucumis sativus L. Cv. Zhongnong 26) seeds were obtained from; The Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China. The detail method and treatments are same as previously described by Anwar et al. 
Measurement of growth
Plant height and root length were determined using a ruler, while fresh weight was determined using a digital balance . The seedling index was used to calculate as (stem thickness / plant height + root FW / shoot FW) * FW of whole plant.
Determination of root activity and bleeding rate
The root activity and bleeding rate were investigated six days after exposure to suboptimal RZT. Eight seedlings per treatment were cut below the cotyledons and the incisions were quickly covered with absorbent cotton to collect bleeding sap for two hours. The bleeding rate was calculated from the weight increments of absorbent cotton after two hours. The root activity was determined using TTC (C19H15CIN4) .
Enzyme activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) were determined using assay kits (COMINBIO) with a UV-1800 spectrophotometer following the manufacturer’s instructions .
Determination of nitrate and ammonium contents
Nitrate (NO3−) and ammonium (NH4+) contents in the leaf were determined after seven days of exposure to suboptimal RZT, using the methods described by . The OD was read at 410 nm and 625 nm, and contents were calculated using a standard curve .
Measurement of NO3 − and NH4 + flux rate at the root surface with NMT
Net NH4+ flux of cucumber seedlings roots was measured by using Non-invasive Micro-test Technology (NMT Physiolyzer®, Younger USA LLC, Amherst, MA 01002, USA) in Xuyue (Beijing) Sci. & Tech. Co., Ltd., Beijing, China .
The cucumber roots were fixed to the bottom of petri dish using resin blocks and filter paper strips, the root tip was exposed, then incubated in the testing solution (2.625 mM Ca (NO3)2, 0.1 mM MgSO4, 0.25 mM NH4NO3, 0.3 mM MES, pH 6.0) for 20 min. After that, roots were transferred to a petri dish containing 5 ml of fresh testing solution. Then placed the root sample on the detection platform, and the NH4+ flux microsensor (NH4+ liquid ion exchanger: XY-SJ-NH4; NH4+ flux microsensor: XY-CGQ-01; Xuyue (Beijing) Sci. &Tech. Co., Ltd., Beijing, China.) was positioned close to the root tip (root hair zone) of cucumber seeding. The tip of NH4+ flux microsensor was about 5 μm form the root surface without touched the root. 10 min for each sample and 6 replicates per group. Use imFluxes software (imfluxes.com, Xuyue (Beijing) Sci. & Tech. Co., Ltd., Beijing, China) to obtained NH4+ flux data and process them. NO3− flux detection steps are exactly the same as NH4+ .
Estimation of total nitrogen contents
Endogenous EBR contents determination
EBR contents were determined using an enzyme-linked immunosorbent assay technology (ELISA) at the College of Agronomy and Biotechnology, China Agricultural University, Beijing, China .
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated using RNAprep Pure Kit (TANGEN) and Fast Quant RT Kit (TANGEN) was used to synthesized first strand cDNA, as described by Anwar et al. . Additionally, primers were designed by using Primer Premier 5 software (Additional file 1: Table S1).
Statistix 8.1 software (www.statistix.com) was used to analyze the difference between treatments. The figures were drown by using Graphpad Prism 5 (www.graphpad.com), as described by Anwar et al. .
AA and XY, LY conceived and designed the study. AA perform the whole experiment and wrote the manuscript. YL and CH review the manuscript. All authors read and approved the final manuscript.
This work was supported by the National Key Research and Development Program of China (2016YFD0201006) to Yansu Li, the Earmarked fund for Modern Agro-industry Technology Research System (CARS-25-C-01) to Xianchang Yu, the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS) to Xianchang Yu, and the Key Laboratory of Horticultural Crop Biology and Germplasm Innovation, Ministry of Agriculture, China. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Ethics approval and consent to participate
Consent for publication
The authors have declared that they have no competing interests.
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