1 Introduction

Nitrogen (N) plays a vital role in the development of Brassica napus L., which is the third most widely planted oil-bearing crops. Nitrate (NO3−) was the primary source of N for most dryland crops; there was a huge demand of nitrate in the Brassica napus L. planting because of its low-N efficiency (Girondé et al. 2015). It decreased the economic benefits and intensified the risk of agricultural non-point source pollution (Girondé et al. 2015; Ju et al. 2009; Wu et al. 2019a, b). Hence, it is of great significance to increase the uptake of NO3− by Brassica napus L. in the low N environment.

It was demostrated that auxin impacted NO3− uptake by regulating the root architecture. In the low N environment, the basipetal auxin transport out of lateral roots was enhanced, resulting in decreased auxin content in the lateral root initial (Krouk et al. 2010), leading to a longer main root length and a decreased root branching. It was beneficial for plants to capture NO3− in deeper soil and abated the competition for NO3− among roots, which improved the N uptake efficiency (Krouk et al. 2010; Kazan 2013). Auxin was biosynthesized in meristematic tissues, mainly through the indole-3-pyruvic acid (IPA) pathway. That is, tryptophan is transformed into IPA under the action of tryptophan aminotransferase (TAAs), and then further synthesized into indole-3-acetic acid (IAA) under the catalysis of riboflavin monoaddition enzymes (YUCs) (Zhao 2012). The IAA content decreased by about 40% in taa1 mutant, while by about 50% in taa1 tar2 mutant (Stepanova et al. 2008; Tao et al. 2008). The IPA content increased by 1.5 times in yuc1 yuc2 yuc4 yuc6 mutant, but decreased by 33% in the plants overexpressing yuc6 gene (Mashiguchi et al. 2011). Polar transport of auxin is mediated by auxin influx (AUX1/LAX) and efflux carriers (PINs and MDR/PGPs) (Liu et al. 2010). In the aux1 mutant, the number of lateral roots was decreased by 50% because of the inhibited auxin polar transport (Liu et al. 2010; Marchant et al. 2002). Similarly, the auxin polar transport from shoot to root was significantly restrained in pin1 mutant (Okada et al. 1991). Besides, the auxin polar transport could be inhibited by some chemicals, such as N-1-naphthylphthalamic acid (NPA), as well as 2,3,5 triiodobenzoic acid (TIBA). Several studies showed that a decrease in nitrate supply tends to promote auxin translocation from shoots to roots and vice-versa (Asim et al. 2020; Forde and Lorenzo 2001; Sun et al. 2017, 2018). It implied that auxin plays an important role in plants response to low N stress. Most previous studies were focused on plant growth and root architecture in response to NO3− regulated by auxin, and the important mechanisms of the signaling overlap between NO3− and auxin concentrated on model plants, such as arabidopsis, rice, and maize (Asim et al. 2020; Sun et al. 2018). Nevertheless, a higher NO3− uptake rate correlated with a higher auxin content in the root of Brassica napus L. under low N stress was found in our study. It implied that auxin may has a directly regulation on the NO3− uptake of Brassica napus L. under low N stress. In order to find out the reasons for the increases of auxin in the root of Brassica napus L. and the mechanisms of auxin regulation on the stimulation of NO3− uptake rate under low N stress, the biosynthesized of IAA in the root and shoot of Brassica napus L., as well as auxin polar transport, were determined under low N stress compared with normal N supply condition. The contribution of in situ synthesis of auxin in root and auxin polar transport to the change of auxin content in the root of Brassica napus L. under low N stress was calculated. Moreover, the responds of NO3− transporter (BnNRT1.1) and PM-ATPase, which drived NO3− into cells, to the increased auxin content in the root of Brassica napus L. under low N stress were explored in this study.

2 Materials and Methods

2.1 Plant Culture and Sampling

The cultivated variety of Brassica napus L. used in this study was Xiangyou 15. Plants were grown in a greenhouse under natural light at day/night temperatures of 28/18 °C. After germination, the seedlings of uniform size and vigor were transplanted into normal N solution for 10 days. There were two kinds of nutrient solution in this study, normal N solution (NO3−: 15 mmol/L) and low N stress solution (NO3−: 1 mmol/L). The normal N solution contains KNO3 5 mmol/L, Ca(NO3)2•4H2O 5 mmol/L, Fe-EDTA 1 mmol/L, KH2PO4 1 mmol/L, MgSO4 5 mmol/L, B 0.5 mg/L, Mn 0.5 mg/L, Zn 0.05 mg/L, Cu 0.02 mg/L, Mo 0.01 mg/L. The low N stress solution modified the normal N solution to cut the NO3− concentration down to 1 mM by reduced KNO3 and Ca(NO3)2•4H2O, and the reduced K+ and Ca2+ were supplied by K2SO4 and CaCl2. Ten days later, four treatments were applied as follows: (1) 15 M, plants were grown in the normal N solution; (2) 1 M, plants were grown in the low N stress solution; (3) 15 M + IAA, plants were grown in the normal N supply solution contain IAA (10−8 mol/L); and (4) 1 M + NPA, plants with 5 μmol/L NPA applied to the rootstocks were grown in the low N stress solution. Each treatment consisted of six replicates arranged in a completely randomized design.

After 7 days of treatment, plant samples were collected to assay the net NO3− flux and total N accumulation. Roots and shoots were separated to measure the IAA content, expression of IAA synthesis related genes (TAA1, TAR2, YUC2, YUC4, YUC6) and biomass, respectively. The IAA polar transport related genes (AUX1, PIN1) in the shoots, BnNRT1.1 relative expression and PM H+-ATPase activities in the roots were determined as well.

2.2 Measurement of Net NO3 − Flux and Total N Accumulation

Net NO3− flux were measured using non-invasive micro-test technology (NMT) at the Younger USA (Xuyue Beijing) NMT Service Center which were described in Wu et al. (2019a, b). Total N content was measured using the Kjeldahl method (Bao 2000), and total N accumulation was caculated by total N content × biomass.

2.3 Determination of IAA Content, Relative Expression of Genes and PM H.+-ATPase Activities

The methods for extraction and purification of IAA were modified according to He et al. (2005) and Kojima et al. (2009). About 50 mg fresh weight of samples were extracted and homogenized in 50 µl of 80% methanol (containing 0.2 µg ml−1 butylated hydroxytoluence) and stored at – 20 ℃ for 48 h. After centrifugation at 20,000 g for 15 min, sediments were re-suspended in 1 ml 80% methanol at 20 ℃ for 16 h. The combined extracts were purified by passing them through C18-Sep-Pak cartridges (Waters, Milford, USA). Afterwards, samples were evaporated under vacuum to remove the organic solvent, and dissolved in 2.0 ml of TBS buffer. IAA content was determined by ELISA using monoclonal antibodies (Phytodetek, Agdia, Elkhart, IN, USA) following the protocol provided by the manufacturer.

$$\mathrm{Polar}\;\mathrm{transport}\;\mathrm{contribution}\;\mathrm{of}\;\mathrm{root}\;\mathrm{IAA}\;\mathrm{increment}\;\mathrm{in}\;1\mathrm M\;\mathrm{compared}\;\mathrm{to}\;15\mathrm M\;(\%)\;=\;(\mathrm{root}\;\mathrm{IAA}\;\mathrm{content}\;\mathrm{in}\;1\mathrm M\;-\;\mathrm{root}\;\mathrm{IAA}\;\mathrm{content}\;\;\mathrm{in}\;1\mathrm M\;+\;\mathrm{NPA})/(\mathrm{root}\;\mathrm{IAA}\;\mathrm{content}\;\mathrm{in}\;1\mathrm M-\mathrm{root}\;\mathrm{IAA}\;\mathrm{content}\;\mathrm{in}\;15\mathrm M)\;\times\;100\%.$$
$$\mathrm{Contribution}\;\mathrm{of}\;in\mathit\;situ\;\mathrm{synthesis}\;\mathrm{of}\;\mathrm{auxin}\;\mathrm{in}\;\mathrm{root}\;(\%)\;=\;1\;-\;\mathrm{polar}\;\mathrm{transport}\;\mathrm{contribution}.$$

Total RNA was extracted from frozen material and using Trizol reagent (Takara). Total RNA samples were treated with the RNase-free DNase Kit (Takara) to remove DNA contamination. After being reverse transcripted into cDNA, the qPCR reactions were performed using a SYBR Green system. A 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) was used to carry out the PCR procedure. PM H+-ATPase activity was determined according to the Zhu et al. (2009) method.

2.4 Statistical Analysis

Data variance was analyzed using the GLM procedure of IBM SPSS Statistics 20.0. Differences were compared using the Duncan test. In all analyses, p < 0.05 was taken to indicate statistical significance. Figures were constructed in GraphPad Prism 5.0.

3 Results

3.1 Response of NO3 − Uptake Rate to the Low N Stress and Exogenous IAA as well as NPA Treatment

The NO3− influx of 1 M was significantly higher than that of 15 M by 246.62% (Fig. 1a); however, the root biomass of 1 M was significantly decreased by 31.72% compared with 15 M (Fig. 1b), which results in a 35.66% lower total N accumulation in 1 M compared with 15 M (Fig. 1c).

Fig. 1
figure 1

Effects of low nitrogen stress on NO3− influx (a), root biomass (b) and total N accumulation (c) in Brassica napus L. 15 M, normal N treatment (NO3−: 15 mmol/L); 1 M, low N stress treatment (NO3.−: 1 mmol/L). Dissimilar letters were significantly different at p < 0.05 according to the Duncan test. Bars on the columns stands for ± standard error (SE (n = 6))

Full size image

Exogenous IAA was significantly increased the NO3− uptake rate, the NO3− influx of 15 M + IAA was 145.23% higher than that of 15 M (p < 0.05) (Fig. 2). The NO3− uptake rate was significantly inhibited by NPA treatment; the NO3− influx of 1 M + NPA was 45.14% lower than that of 1 M.

Fig. 2
figure 2

NO3− influx in root under different treatments. 15 M, normal N treatment (NO3−: 15 mmol/L); 1 M, low N stress treatment (NO3−: 1 mmol/L); 15 M + IAA, the normal N supply solution contain auxin (IAA, 10−.8 mol/L); 1 M + NPA, plants grown in low N stress solution with 5 μmol/L N-1-naphthylphthalamic acid (NPA, an inhibitor of polar auxin transport) applied to the rootstocks. Dissimilar letters were significantly different at p < 0.05 according to the Duncan test. Bars on the columns stands for ± standard error (SE (n = 6))

Full size image

3.2 IAA Content of Different Treatments

In the root, the IAA content of 1 M was significantly higher than that of 15 M by 23.11% (Fig. 3a), the IAA increment in 1 M compared with 15 M originated from root synthesis shared with 29.20%, and polar transport from shoot shared with 70.80% (Fig. 3b). The IAA content of 15 M + IAA was 11.91% higher than that of 15 M, and the difference reached to a significant level. The IAA content of 1 M + NPA was significantly lower than that of 1 M by 13.29% (Fig. 3a).

Fig. 3
figure 3

IAA contents of different treatments (a) and contribution of root IAA increment in 1 M compared with 15 M (b). 15 M, normal N treatment (NO3−: 15 mmol/L); 1 M, low N stress treatment (NO3−: 1 mmol/L); 15 M + IAA, the normal N supply solution contain auxin (IAA, 10−.8 mol/L); 1 M + NPA, plants grown in low N stress solution with 5 μmol/L N-1-naphthylphthalamic acid (NPA, an inhibitor of polar auxin transport) applied to the rootstocks. Polar transport contribution of root IAA increment in 1 M compared to 15 M (%) = (root IAA content in 1 M − root IAA content in 1 M + NPA) / (root IAA content in 1 M − root IAA content in 15 M) × 100%. Contribution of in situ synthesis of auxin in root (%) = 1—Polar transport contribution. Dissimilar letters were significantly different at p < 0.05 according to the Duncan test. Bars on the columns stands for ± standard error (SE (n = 6))

Full size image

In the shoots, the IAA content of 1 M was significantly higher than that of 15 M by 19.71%. The IAA content of 15 M + IAA was 5.56% higher than that of 15 M, and the difference reached to a significant level. There was no significant difference between 1 M and 1 M + NPA (Fig. 3a).

3.3 Response of IAA Biosynthesis and Polar Transport to the Low N Stress

In the root, the relative expression of TAA1, YUC2, YUC4, YUC6 of 1 M were significantly higher than that of 15 M by 83.87%, 407.67%, 38.99%, 210.75% respectively (Fig. 4a). It indicated that low N stress significantly promoted IAA biosynthesis in the root of Brassica napus L. There was no significant difference on the relative expression of TAR2 between 1 M and 15 M (Fig. 4a).

Fig. 4
figure 4

The expressions of auxin synthesis genes in root (a) and shoot (b) of different treatments. 15 M, normal N treatment (NO3−: 15 mmol/L); 1 M, low N stress treatment (NO3−: 1 mmol/L). Different letters were significantly different at p < 0.05 according to the Duncan test. Bars on the columns stands for ± standard error (SE (n = 6))

Full size image

In the shoot, there was no significant difference on the relative expression of TAR2, YUC2, and YUC6 between 1 M and 15 M. The relative expression of TAA1 in 1 M was significantly lower than 15 M. However, the relative expression of YUC4 in 1 M was significantly higher than 15 M (Fig. 4b).

The IAA polar transport was promoted under N stress environment. The relative expression of AUX1 and PIN1 was 8.74 times and 4.14 times in 1 M compared to 15 M (Fig. 5).

Fig. 5
figure 5

The expressions of auxin polar transport genes of different treatments. 15 M, normal N treatment (NO3−: 15 mmol/L); 1 M, low N stress treatment (NO3−: 1 mmol/L). Different letters were significantly different at p < 0.05 according to the Duncan test. Bars on the columns stands for ± standard error (SE (n = 6))

Full size image

3.4 Effects of Low N Stress and NPA Treatment on the BnNRT1.1 and PM H.+-ATPase

The relative expression of BnNRT1.1 was significantly enhanced in low N stress environment, and the relative expression of BnNRT1.1 was 2.23 times in 1 M compared to 15 M. NPA treatment significantly inhibited the relative expression of BnNRT1.1, and the relative expression of BnNRT1.1 decreased by 46.99% in 1 M + NPA compared to 1 M. There was no significant difference on the relative expression of BnNRT1.1 between 15 M and 1 M + NPA (Fig. 6a).

Fig. 6
figure 6

The expressions of BnNRT1.1 (a) and PM H+-ATPase activity (b) under different treatments. 15 M, normal N treatment (NO3−: 15 mmol/L); 1 M, low N stress treatment (NO3−: 1 mmol/L); 1 M + NPA, plants grown in low N stress solution with 5 μmol/L N-1-naphthylphthalamic acid (NPA, an inhibitor of polar auxin transport) applied to the rootstocks. Different letters were significantly different at p < 0.05 according to the Duncan test. Bars on the columns stands for ± standard error (SE (n = 6))

Full size image

The PM H+-ATPase was significantly enhanced in low N stress environment, which was 79.31% higher in 1 M compared to 15 M (p < 0.05). NPA treatment significantly inhibited the PM H+-ATPase, which decreased by 32.47% in 1 M + NPA compared to 1 M. There was no significant difference on the PM H+-ATPase between 15 M and 1 M + NPA (Fig. 6b).

4 Discussion

The IAA content in root was increased under low N supply, which was consisted with previous studies (Asim et al. 2020; Caba et al. 2000; Tian et al. 2008). The increment of IAA was mainly due to the polar transport from shoot to root. However, previous study showed that auxin overproduction in shoots can not rescue auxin deficiencies in Arabidopsis roots (Chen et al. 2014). With auxin polar transport inhibited, there is still more IAA in the root of Brassica napus L under low N stress compared with normal N supply, which indicated that the in situ synthesis of auxin in root played an essential role to the low N stress. The in situ synthesis of auxin in root and auxin polar transport from shoot to root jointly increased the IAA gradient of Brassica napus L. root under low N stress (Fig. 7).

Fig. 7
figure 7

The conceptual model of NO3− uptake rate response to low N stress modulated by auxin in Brassica napus L

Full size image

Several auxin synthesis and polar transport genes were expressed upregulated in low N stress. In the root, the upregulation of TAA1 accelerated the transformation of tryptophan into IPA, and the increased expression of YUCs, which has been considered as the rate-limiting enzyme for auxin biosynthesis (Zhao et al. 2001), such as YUC2, YUC4, and YUC6, promoted the biosynthesis of auxin. The expression of TAR2 in the root of Brassica napus L. under low N stress was not increased compared to normal N supply, probably because of the increased expression of BnNRT1.1, which acts as a negative regulator of TAR2 in the root (Maghiaoui et al. 2020).

In the shoot, the expression of AUX1 and PIN1, which participate in polar auxin transport, were significantly increased as well. Studies showed that AUX1 and PIN1 play an important role in the nitrate deficiency-induced asymmetric root growth (Band et al. 2014; Boutté et al. 2007; Chai et al. 2020). The upregulated expressed AUX1 and PIN1 enhanced auxin polar transport from shoot to root, plus the promoted in situ synthesis of auxin in root caused by YUCs, leading to a higher IAA gradient in the root of Brassica napus L under low N stress.

Auxin plays an important role in NO3− absorption. Previous studies demonstrated that root architecture was regulated by auxin under low N stress (Girondé et al. 2015; Jia and von Wirén 2020; Krouk et al. 2010; Maghiaoui et al. 2020; Sun et al. 2018), that is, the lateral root growth was repressed by promoting basipetal auxin transport out of these roots, which was dependent on NRT1.1 (Krouk et al. 2010). The NRT1.1 not only served in the auxin transport in the roots but also was widely realized as a dual-affinity nitrate transporter and contributed to both low and high affinity nitrate uptake (Guo et al. 2002). The expression of NRT1.1 was induced by auxin. With higher auxin content, the expression of NRT1.1 was higher in low N stress compared with normal N supply. The expression of NRT1.1 was declined once the auxin content was reduced caused by auxin polar transport being inhibited. The results consisted with the study of Guo et al. (2002), which showed that the AtNRT1.1 expression was activated within 30 min of auxin treatment. A similar phenomenon was observed on PM H+-ATPase activities, which provide energy for NO3− uptake. It indicated that, under low N stress, the elevated auxin promoted the expression of NRT1.1 and the activity of PM H+-ATPase, thus promoted the absorption of NO3−, leading to a higher NO3− influx, and resulting a higher N use efficiency. It increases the adaptability of Brassica napus L. in low N environment.

5 Conclusion

The NO3− uptake rate was increased to deal with low nitrogen (N) stress (1 M) in Brassica napus L. Low N stress stimulated auxin polar transport and its in situ synthesis in root, leading to a higher auxin gradient in the root of Brassica napus L., and then promoted NO3− transporter and the energy of NO3− trans-membrane transport, finally accelerated NO3− absorption. It suggested an efficient N utilization mechanism mediated by auxin, further enhanced the adaptability of crops to low N stress.