Introduction

Salinization of agricultural land has been a major challenge for sustainable development of agriculture. The entire saline soil area is about 9.5 billion ha, which accounts for 10% of arable land in the world1. China is in possession of 1/16 of the globe saline-alkali land that constitutes about 21% of the cultivated land in China2. Canola (Brassica napus L.) is one of the most widely cultivated oil crops in the world because of its healthy fatty acid composition in oil and high protein content in meal3,4. Currently, the planting area of canola in China is about 7 million hectares, and canola oil owns the largest proportion of domestic edible vegetable oils in the market5.

The adverse effect of salt stress on reducing growth rate, which results in smaller leaves, shorter stature and sometimes fewer leaves6,7, is functionally carried out by its initial and primary effects on osmotic pressure8. Under the action of osmosis, the uptake of water by root is reduced, and furthermore, salt stress may cause excessive uptake of ions9. The excessive ions in plant may cause ion toxicity and metabolism disturbance, which may interfere plant ion uptake and intracellular ion balance, and subsequently inhibit photosynthesis and growth10,11. Seed germination and seedling emergence are sensitive to salt stress12,13,14. It has been reported that there are great differences in seed germination among canola varieties under salt condition15,16.

Nitrogen (N) as an essential nutrient is important for crop yield achievement17. It is a structural component of amino acids and a constituent of all enzymes and involved in many physiological processes18. Salt stress usually inhibits N absorption and accumulation in canola, resulting in decreased plant height, stunted growth process and ultimately reduced yield19. Besides, carbon (C) is the largest component of plant biomass and constitutes a stable 50% of plant dry weight20. C and N assimilation are the two most important physiological processes related to plant growth and productivity21. Studies have demonstrated that C assimilation including photosynthetic carbon assimilation and carbohydrate transport and utilization are adversely affected in plant exposed to salt stress22. Nevertheless, little attention was paid to the effects of salt stress on balance of C and N assimilation in canola. N physiologically translocation from the vegetative organs to the reproductive organs after anthesis stage is the important resource of seed N accumulation23,24. It was reported that the N translocation from vegetative organs accounts for 50% of seed N accumulation25. However, little was known about the N translocation in canola under salt stress.

In the past, researches about salt stress were mainly conducted on pot experiment. The salt stress in these experiments was artificial and not fully reflect the physicochemical properties of saline soil in natural environment. And these researches mainly focus on seedling stage, rarely on flowering stage and maturity stage, due to the limitation of experiment condition. Nevertheless, seed yield is the key to agricultural production. Therefore, in this study, to improve the knowledge of the effects of salt stress on the growth of canola, a field experiment was carried out to investigate the effects of salt stress on N accumulation and translocation, C accumulation, biomass and seed yield. We hypothesize that: (a) salt stress could decrease seed yield in canola plants; (b) N and C assimilation would be inhibited and the balance between N and C assimilation would be changed under salt stress; (c) salt stress would inhibit N translocation from vegetative organs to seed.

Results

Growth process

The results of (Table 1) indicated that soil salt-ion concentration affect the growth process after sowing. Specially, as the soil salinity concentration increased from LSSC to HSSC, the time of early flowering stage after sowing of Yanyouza 3 and Ningza 1818 was postponed by 4 ~ 5 days during two growing seasons. The time of maturity stage after sowing was postponed by 6 ~ 8 days.

Table 1 Days of early flowering stage and maturity stage after sowing.
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Seed yield

The ANOVA results indicated that soil salt-ion concentration, cultivar and their interaction significantly affected seed yield. The seed yield, ranging from 1679.76 to 3232.34 kg ha−1, decreased dramatically with the soil salt-ion concentration increasing from LSSC to HSSC. More specially, the seed yield of Yanyouza 3 under HSSC was reduced by 45.78 and 44.92% during 2019–2020 and 2020–2021 growing seasons, respectively, as compared with that under LSSC. For Ningza 1818, both the reductions of seed yield during two growing seasons were 46.11%. Moreover, Ningza1818 showed greater yield performance than Yanyouza 3 under both LSSC and HSSC conditions (Fig. 1).

Figure 1
figure 1

The ANOVA results and seed yield under different treatments. LSSC and HSSC represent low and high soil salt-ion concentration. Probability levels are performed by ns, and ** for not significant, and 0.01. Different letters indicate significant difference at p = 0.05 between different treatments during two growing seasons. Data are mean ± SE (n = 3).

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Biomass accumulation

The ANOVA results (Table 2) indicated that soil salt-ion concentration and cultivar significantly affected biomass accumulation in different parts at both early flowering stage and maturity stage, except cultivar effect on root biomass accumulation at both two growth stages; however, year and the interactions showed no significant effect on most of biomass accumulation. The increasing soil salt-ion concentration decreased biomass accumulation in all parts at both growth stages. Averagely over two growing seasons, HSSC treatment significantly decreased biomass accumulation of different pars in Yanyouza 3 and Ningza 1818 by 21.28% and 24.55% (root), 33.03% and 33.92% (stem), 25.64% and 24.99% (leaf), and 36.86% and 34.79% (per-anthesis deciduous leaf) at early flowering stage, as compared with LSSC treatment. These reductions in maturity stage were 26.93% and 26.70% (root), 32.27% and 29.93% (stem), 22.31% and 20.86% (post-anthesis deciduous leaf), 39.25% and 42.64% (pod), and 46.35% and 46.11% (seed), respectively.

Table 2 The ANOVA results and biomass accumulation (103 kg ha−1) under different soil salt-ion concentration.
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N characters

The ANOVA results (Table 3) showed that soil salt-ion concentration significantly affected N content of all parts at both two growth stages; cultivar significantly affected N content of most parts (except root) at maturity stage and stem N content at early flowering stage. Year and the interactions between two factors or three factors showed no significant effect mostly. As the soil salt-ion concentration increased from LSSC to HSSC, the N content of all parts in Yanyouza 3 and Ningza 1818 was significantly increased at both early flowering stage and maturity stage. For example, as compared with LSSC, HSSC averagely increased the N content of root, stem and leaf in Yanyouza 3 and Ningza 1818 over two growing seasons by 11.86% and 10.95%, 11.47% and 16.25%, 5.29% and 4.52% at early flowering stage. Similarly, HSSC treatment increased N content of root, stem, pod and seed by 21.01% and 21.70%, 33.22% and 29.79%, 18.40% and 14.91%, 8.00% and 9.09% at maturity stage.

Table 3 The ANOVA results and N content (%) under different treatments.
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However, the effect of soil salt-ion concentration on N accumulation amount was different from that on N content (Table 4). As compared with LSSC, HSSC significantly reduced the N accumulation amount of different parts. The HSSC treatment averagely decreased N accumulation amount of root, stem, leaf and pre-anthesis deciduous leaf in Yanyouza 3 and Ningza 1818 over two growing seasons by 11.94% and 12.97, 25.36 and 23.18%, 21.73% and 21.58%, 31.56% and 28.85% at early flowering stage. These reductions at maturity stage were 11.58% and 10.83% for root, 9.76% and 9.03% for stem, 10.10% and 5.04% for post-anthesis deciduous leaf, 28.08% and 34.08% for pod, 40.99% and 41.21% for seed, respectively. The ANOVA results indicated that soil salt-ion concentration significantly affected N accumulation of all parts, cultivar affected N accumulation at early flowering stage (except root) and stem N accumulation at maturity stage. Year and the interaction between two factors or three factors showed no significant effect mostly.

Table 4 The ANOVA results and N accumulation (kg ha−1) under different treatments.
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The ANOVA results (Table 5) showed that soil salt-ion concentration significantly affected N translocation efficiency of stem and leaf and N utilization efficiency; cultivar significantly affected N translocation efficiency of stem and N utilization efficiency; year and interactions had no significant effect. Similarly, the effects of soil salt-ion concentration on N translocation efficiency followed the same change tendency as N accumulation amount. As compared with LSSC, HSSC averagely decreased the N translocation efficiency of stem and leaf in Yanyouza 3 and Ningza 1818 over two growing seasons by 17.11% and 18.38%, 4.61% and 6.17%, respectively. The HSSC treatment also decreased the N utilization efficiency by 19.03% in Yanyouza 3 and 20.58% in Ningza 1818, as compared with LSSC treatment. Moreover, the correlation analysis indicated that seed yield and seed N accumulation were significantly and positively related with stem and leaf N translocation efficiency.

Table 5 The ANOVA and correlation analysis results and N translocation efficiency (%) and N utilization efficiency under different treatments.
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C characters and C/N

The ANOVA results (Table 6) showed that soil salt-ion concentration significantly affected C content in all organs at two growth stages. The range of C content in root, stem, leaf and pre-anthesis deciduous leaf at earling flowering stage were 40.16–41.52%, 38.16–39.83%, 40.18–41.75% and 35.23–37.12%, respectively. At maturity stage, the range of C content in root, stem, post-anthesis deciduous leaf, pod and seed were 40.12–41.32%, 40.43–41.38%, 34.03–35.88%, 38.65–41.09% and 57.06–58.32% respectively. For the one cultivar during one growing seaon, the C content in specific organ under HSSC treatment mostly had no significant difference from that under LSSC treatment. However, the C content under HSSC treatment showed mild decline than those under LSSC treatment.

Table 6 The ANOVA results and C content (%) under different treatments.
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The ANOVA results (Table 7) indicated that soil salt-ion concentration showed significant effect on C accumulation; cultivar significantly mainly affected C accumulation at maturity stage; year and the interactions rarely exerted significant effect on C accumulation. Similar to N accumulation, the increase in soil salt-ion concentration reduced the C accumulation. In contrast to LSSC treatment, the HSSC treatment averagely decreased the C accumulation of root, stem, leaf and pre-anthesis deciduous leaf in Yanyouza 3 and Ningza 1818 at early flowering stage by 23.37% and 22.55%, 35.08% and 35.81%, 27.81% and 26.53%, and 38.58% and 36.94%, respectively. At maturity stage, the reduction of root, stem, post-anthesis deciduous leaf, pod and seed in Yanyouza 3 and Ningza 1818 were 28.16% and 27.85%, 33.03% and 31.37%, 24.04% and 23.96%, 40.32% and 44.78%, and 46.33% and 46.98%, respectively.

Table 7 The ANOVA results and C accumulation (kg ha−1) under different treatments.
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The ANOVA results (Table 8) showed that soil salt-ion concentration significantly affected C/N. At early flowering stage, the C/N in pre-anthesis deciduous leaf was the largest (38.06–44.21), followed by root (30.38–36.30) and stem (19.79–25.25), the least one was leaf (9.67–10.63). At maturity stage, the C/N in root, stem and pod were relatively higher (all above 50), followed by post-anthesis deciduous leaf, the least one is seed (14.25–16.52). as the soil salt-ion concentration increasing, the C/N in all organs were significantly deceased. At early flowering stage, the C/N of root, stem, leaf and pre-anthesis deciduous leaf in Yanyouza 3 and Ningza 1818 under HSSC treatment were 12.97% and 11.02%, 13.06% and 16.41%, 7.85% and 6.32%, 10.26% and 11.40% lower than those under LSSC treatment. At maturity stage, the C/N of root, stem, post-anthesis deciduous leaf, pod and seed in Yanyouza 3 and Ningza 1818 under HSSC treatment were 18.83% and 19.11%, 25.79% and 24.54%, 15.50% and 19.88%, 17.04% and 16.27%, 9.03% and 9.81% lower than those under LSSC treatment.

Table 8 The ANOVA results and C/N under different treatments.
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Photosynthetic rate and leaf area index

The ANOVA results (Fig. 2)showed that soil salt-ion concentration significantly affected photosynthetic rate and leaf area index; cultivar, year and interactions between two factors or three factors showed no significant effect except interactions between three factors on leaf area index.

Figure 2
figure 2

The ANOVA results and photosynthetic rate and leaf area index at early flowering stage under different soil salt-ion concentration. (a,c): photosynthesis rate during 2019–2020 and 2020–2021 growing seasons; (b,d): leaf area index during 2019–2020 and 2020–2021 growing seasons. LSSC and HSSC represent low and high soil salt-ion concentration. Different letters indicate significant difference at p = 0.05 between different treatments. Data are mean ± SE (n = 3).

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The variation of photosynthetic rate at early flowering stage between LSSC and HSSC was little but reached a significant level. As compared to LSSC, HSSC averagely decreased the photosynthetic rate at early flowering stage in Yanyouza 3 and Ningza 1818 over two growing seasons by 9.13% and 8.03%, respectively. The leaf area index, ranging from 3.30 to 4.82, was also decreased under HSSC. HSSC treatment decreased leaf area index in Yanyouza 3 and Ningza 1818 by 24.25% and 25.69%, respectively.

Discussion

In this study, we found that high soil salt-ion concentration decreased the N and C accumulation with more decrement observed in C accumulation than in N accumulation, and inhibited photosynthetic rate and leaf area index, and reduced N translocation efficiency and N utilization efficiency, resulting in reduced biomass accumulation and seed yield.

In our study, the increase in soil salt-ion concentration postponed the time of early flowering stage and maturity stage. Generally speaking, plants exposed to abiotic stress usually accelerate senescence and shorted growth period26. Zirgoli et al. found that drought stress can result in the flowering and maturity advancing in canola, and the days to flowering stage and maturity stage under drought stress were decreased by 3.27 and 1.29%, respectively, compared to those under normal condition27. In addition, low temperature stress promoted floral initiation, as reported by Luo et al.28. However, in the current study, salt stress delayed the early flowering stage and maturity stage and prolong the whole growth period. Meanwhile, salt stress decreased the biomass and N accumulation in canola, with more reduction in biomass accumulation than in N accumulation. Therefore, the plant N content in canola under salt stress was increased. The increased N content in canola may resulted in the prolonged growth period.

N and C are two important elements in plant growth and crop yield formation. Salt stress usually inhibits N and C metabolism in plants. In our study, as compared with LSSC, HSSC significantly decreased the N and C accumulation amount at both early flowering stage and maturity stage. Similar results were reported previously29. The decrease in N accumulation may be attributed to the limited activities of enzymes in N metabolism. Previous studies had demonstrated that the activities of nitrate reductase and glutamine synthetase which are the key enzymes in N accumulation are reduced under salt stress30. The plant N content in our study was increased due to high soil salt-ion concentration. Additionally, the C/N under HSSC was decreased. These results suggested that C assimilation was more sensitive to salts stress than N assimilation. C and N assimilation together constitute the structure of plants, and the balance of these two physiological processes are vital for plant growth and development. The photosynthesis of plant is a important way to assimilate and accumulate C, and it requires a large amount of N which is closely related to synthesis of photosynthetic enzymes31,32. It is generally believed that photosynthesis rate is positively correlated with plant N content. According to Kumar et al., under sulfur optimum application, the leaf photosynthesis rate in canola under enough N supply was 48% higher than that with N-limited treatment33. Kuai et al. reported that as N application increasing from 0 to 270 kg N ha−1, the leaf photosynthesis rate in canola was increased34. Gammelvind et al. also demonstrated that the leaf photosynthesis rate in canola responded linearly to increasing N content in leaf35. In current study, although N content increased under salt stress, the photosynthetic rate was decreased. This negative effect on photosynthesis under salt stress may be due to lower activities of photosynthetic enzymes. Generally, photosynthesis depends not only on the amount of these enzymes of photosynthesis but also on the activities of these enzymes36. Therefore, the increase in enzyme amount per leaf area due to increasing N content under salt condition was not enough to compensate for the decrease in activities of enzyme, resulting in the decrease in photosynthesis rate. In turn, the N accumulation under salt stress is decreased because of lower energy and carbon skeletons provided by C assimilation37. In conclusion, the balance between C and N assimilation of canola plant is destroyed by salt stress, with stronger negative effect on C assimilation. In addition, leaf acts as the main photosynthetic organ of canola at early flowering stage. We found that the leaf area index was also decreased due to HSSC, with more decline than photosynthetic rate, suggesting that the decrease in photosynthetic area is the main reason for the inhibited synthesis of carbohydrate through photosynthesis.

Most of the seed N accumulation in canola are derived from remobilization of N in vegetative organs. A great capacity of N translocation is related to seed yield formation. In our study, the N translocation efficiency in stem and leaf was significantly and positively related with seed N accumulation and seed yield, suggesting that N translocation in vegetative organs is an important source of seed N. However, the N translocation efficiency in all organs decreased with the increase of soil salt-ion concentration, with the most reduction observed in stem. Results agreed with ours were reported that salt stress reduced N translocation in canola29, in rice38 and barley39, suggesting that salt stress prefer to fix N into vegetative organs rather than to transport it into reproductive organs. Ultimately, N utilization efficiency was declined because of lower N translocation efficiency.

Conclusions

In this field study, HSSC reduced seed yield and postponed the time of early flowering stage and maturity stage. Besides, HSSC decreased N and C accumulation at both growth stages and reduced the C/N, suggesting that salt stress breaks the balance between N and C assimilation and shows stronger negative effects on C assimilation than on N assimilation. Moreover, although the plant N content was increased under salt stress, the photosynthesis rate was reduced. The leaf area index under HSSC was also reduced, with more reduction than photosynthesis rate. In addition, HSSC reduced the N translocation efficiency in all vegetative organs and N utilization efficiency. This means HSSC tend to fix N into vegetative organs rather than transport it into reproductive organs. The findings from this study would help further to understand that salt stress decrease canola seed yield by affecting N and C assimilation and N translocation.

Materials and methods

Experimental materials, site and soil conditions

During 2019–2020 and 2020–2021 growing seasons, two hybrid canola cultivars (Yanyouza 3 and Ningza 1818) were planted at the experimental field of Jiangsu Golden Agriculture Shareholding Co., Ltd., Jiangsu, China (33°24′ N, 120°35′ E). The two hybrids were popular winter canola in Middle-Lower Yangtze Area, China. The soil in experiment had a texture of sandy loam. Soils were sampled for the measurement of soil salt-ion concentration, soil organic matter content and soil pH prior to sowing in 2019. The soil chemical properties of the plough layer (0–20 cm) were listed in (Table 9). The soil was sampled and then air-dried at room temperature. Then the soil samples were passed through a 2 mm sieve for measurement (1 soil:5 water). The soil salt-ion concentration in the leachate was determined. Na+, K+, Ca2+ and Mg2+ were determined by atomic absorption spectrophotometry. Cl− was determined by silver nitrate titration. HCO3− were determined by sulfuric acid titration, SO42− was determined by barium sulfate turbidimetric method. The soil salt-ion concentrations between two treatments were different significantly for some ions, donated by the low soil salt-ion concentration (LSSC) and the high soil salt-ion concentration (HSSC). The difference in the soil salt-ion concentration between LSSC and HSSC was attributed to their difference at altitude of 0.9 and 1.1 m, respectively.

Table 9 Soil basic properties in the study.
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Experimental design

A split plot design was arranged with two soil salt-ion concentrations as main plots and two cultivars as subplots, in three replicates. The plot size was 18 m in length by 2.4 m in width. The canola seeds were manually sown on October 11th in each year and seedling density was adjusted at planting density of 45 × 104 plants ha−1 at the fourth-leaf growth stage for all plots (row spacing 0.4 m and plant spacing 0.055 m). Urea (N, 46%), diammonium hydrogen phosphate compound fertilizer (N-P2O5, 18–46%), potassium sulfate fertilizer (K2O, 52%) and boron fertilizer (B, 12%) were applied pre-sowing at rate of 166.0 kg ha−1, 326.1 kg ha−1, 144.2 kg ha−1 and 4.5 kg ha−1, respectively, as basal fertilizers. Urea was applied at rate of 293.5 kg ha−1 at bolting stage.

Sampling and measurement

Seed yield and biomass accumulation

Ten plants were sampled from each plot at early flowering stage (about 25% of plants begins to blossom). The samples were separated into root, stem and leaf, and dried in an oven for 30 min at 105 °C to deactivate enzymes then again at 80 °C until constant weight to determine the dry weight. Canola was harvested when approximately 90% of pods were yellow. Ten plants were again sampled from each plot. The samples were separated into root, stem, pod and seed. Next, the samples were aired, threshed, dried at 80 °C and weighted. The seed yield was calculated by multiplying the seed yield per plant by density.

Throughout experiment, the deciduous leaves were collected, dried and weighted, at a fixed site (six consecutive rows and in 2 m length each row) from each plot, once every 2 weeks. All the collected deciduous leaves were later separated into pre-anthesis and post-anthesis weights.

N accumulation amount, C accumulation amount, N/C and plant N content

The N and C content in different organs was determined using the elemental analyzer (Vario MAX CN, Elementar Co., Germany). The N (C) accumulation amount in specific organ was calculated by multiplying the dry weight by N content in this organ. The C/N was the ratio of C accumulation to N accumulation.

N translocation efficiency and N utilization efficiency

The various parameters referring to N translocation efficiency and utilization efficiency of canola in this study were calculated as follows:

  • N translocation amount of specific vegetative organ = N accumulation amount of specific organ at early flowering stage−N accumulation amount of specific organ at maturity stage;

  • N translocation efficiency of specific vegetative organ = N translocation amount of specific vegetative organ/N accumulation amount of specific vegetative organ at early flowering stage × 100;

  • N utilization efficiency = Seed yield/N accumulation amount at maturity (kg kg−1).

Photosynthetic rate and leaf area index

The photosynthetic rate was measured at early flowering stage using a portable photosynthetic system (LI-COR, Lincoln, NE, USA). The data was obtained from the second and third fully expanding top leaves from 9:00 to 12:00 AM on sunny day. The measurement was performed under a light-saturating photosynthetic photon flux density of 1200 μmol m−2 s−1. The CO2 concentration in the leaf chamber was set at 400 μmol mol−1.

Leaf area was measured at early flowering stage using leaf area meter (Model LI-3100, Lincoln, Nebraska). Leaf area index was calculated as the below.

$$\mathrm{Leaf \; area\; index} = \frac{\mathrm{leaf \; area \; per \; plant}\times \mathrm{density}}{\mathrm{planting \; area}}$$

Statistical analysis

The observation data were compiled with Microsoft Excel 2007, and the analysis of variance (ANOVA) and significance test were conducted using SPSS statistical 20 software (SPSS Inc., Chicago, IL, USA). The mean difference between treatments were separated by Duncan’s multiple range test at significance level of p < 0.05. Graphs were performed using Origin 9.0 software (Origin Lab Corp, Northampton, MA, USA).

Ethical approval and consent to participate

The seeds were kindly provided by Yangzhou Academy of Agricultural Sciences, Yangzhou, China and Jiangsu Academy of Agricultural Sciences, Jiangsu, China. In this study, the experimental research and field studies on plants, including collection of plant material, complied with relevant institutional, national, and international guidelines and legislation.