Uptake, transport and distribution of molybdenum in two oilseed rape (Brassica napus L.) cultivars under different nitrate/ammonium ratios

Abstract

Objectives: To investigate the effects of different nitrate sources on the uptake, transport, and distribution of molybdenum (Mo) between two oilseed rape (Brassica napus L.) cultivars, L0917 and ZS11. Methods: A hydroponic culture experiment was conducted with four nitrate/ammonium (NO₃ ⁻:NH₄ ⁺) ratios (14:1, 9:6, 7.5:7.5, and 1:14) at a constant nitrogen concentration of 15 mmol/L. We examined Mo concentrations in roots, shoots, xylem and phloem sap, and subcellular fractions of leaves to contrast Mo uptake, transport, and subcellular distribution between ZS11 and L0917. Results: Both the cultivars showed maximum biomass and Mo accumulation at the 7.5:7.5 ratio of NO₃ ⁻:NH₄ ⁺ while those were decreased by the 14:1 and 1:14 treatments. However, the percentages of root Mo (14.8% and 15.0% for L0917 and ZS11, respectively) were low under the 7.5:7.5 treatment, suggesting that the equal NO₃ ⁻:NH₄ ⁺ ratio promoted Mo transportation from root to shoot. The xylem sap Mo concentration and phloem sap Mo accumulation of L0917 were lower than those of ZS11 under the 1:14 treatment, which suggests that higher NO₃ ⁻:NH₄ ⁺ ratio was more beneficial for L0917. On the contrary, a lower NO₃ ⁻:NH₄ ⁺ ratio was more beneficial for ZS11 to transport and remobilize Mo. Furthermore, the Mo concentrations of both the cultivars’ leaf organelles were increased but the Mo accumulations of the cell wall and soluble fraction were reduced significantly under the 14:1 treatment, meaning that more Mo was accumulated in organelles under the highest NO₃ ⁻:NH₄ ⁺ ratio. Conclusions: This investigation demonstrated that the capacities of Mo absorption, transportation and subcellular distribution play an important role in genotype-dependent differences in Mo accumulation under low or high NO₃ ⁻:NH₄ ⁺ ratio conditions.

1. Introduction

Molybdenum (Mo) is an essential micronutrient for plant growth and it plays an important role in carbon, sulfur, and nitrogen metabolism, which is mainly via metalloenzymes in plants (Havemeyer et al., 2011; Hille et al., 2011). Generally in China, soils are acidic and poor availability of Mo in soils is widespread (Ye et al., 2011; Cao et al., 2012; Liu et al., 2012), especially in Southeast China (Shi et al., 2006). However, oilseed rape (Brassica napus L.), an important oil crop, is predominantly grown in Mo-deficient soils (Yin et al., 2009). Moreover, oilseed rape requires plenty of nitrogen (N) fertilizer for growth and yield (Behrens et al., 2002; Barłóg and Grzebisz, 2004a; 2004b; Rathke et al., 2006; Leiva-Candia et al., 2013) and Mo deficiency often decreases the activity of nitrate reductase which leads to N deficiency in plants (Chatterjee and Nautiyal, 2001; Ide et al., 2011), poor seedling growth and lower grain yield (Yang et al., 2009; Liu et al., 2010). Furthermore, winter wheat subjected to higher rate of N-fertilization is prone to Mo deficiency (Wang et al., 1999) while supplying NH₄NO₃ to plants stimulates the remobilization of Mo in black gram (Jongruaysup et al., 1997). Mo plays an important role in the N metabolism of plants, including nitrogen fixation and the transformation of nitrate (NO₃ ⁻)-N to ammonium (NH₄ ⁺)-N (Kovács et al., 2015). However, little information is available about the Mo uptake, transport, or distribution under different N sources.

NH₄ ⁺ and NO₃ ⁻ are two forms of inorganic N available for plant uptake. Studies on NH₄ ⁺ stress showed that a low NO₃ ⁻:NH₄ ⁺ ratio inhibits plant growth and changes ion balance in the plant (Liu et al., 2014; Babalar et al., 2015; Sokri et al., 2015). Esteban et al. (2016) showed that complementation with nitrate at low doses, which has long been practised in agriculture, can counterbalance the ammonium toxicity in the laboratory. Zhu et al. (2015) reported that the Mo content of Chinese cabbage declined significantly with a decreasing NO₃ ⁻:NH₄ ⁺ ratio. Moreover, the molybdate transporter 1 (CrMOT1) of Chlamy-domonas reinhardtii is activated by nitrate but not by molybdate (Tejada-Jiménez et al., 2007), while molybdate transporter 2 (CrMOT2) is activated by molybdate, but not by the N source (Tejada-Jiménez et al., 2011). The responses of plants particularly of different cultivars to variable NO₃ ⁻:NH₄ ⁺ ratios are still not well known. In this study, we evaluated the variations in Mo uptake, transport, and distribution between two B. napus genotypes treated with different NO₃ ⁻:NH₄ ⁺ ratios under hydroponic culture, mainly by tissue Mo, sap flow and subcellular Mo concentrations.

2. Materials and methods

2.1. Plants and growth conditions

Seeds of oilseed rape cultivars ZS11 (high Mo accumulating cultivar) and L0917 (low Mo accumulating cultivar) were sterilized with 10% (v/v) NaClO for 30 min and washed by deionized water for 5–10 min. The sterilized seeds were cold-treated at 4 °C for 2 d and then were germinated on moistened gauze that was fixed to a black plastic tray filled with deionized water at room temperature. After one week, 15 uniform seedlings were transferred to black polyethylene boxes containing 10 L 1/2-strength modified Hoagland solution for 6 d and subsequently full-strength solution. The modified Hoagland solution contains 4 mmol/L Ca(NO₃)₂·4H₂O, 6 mmol/L KNO₃, 1 mmol/L NH₄H₂PO₄, 2 mmol/L MgSO₄·7H₂O, 46.2 μmol/L H₃BO₃, 9.1 μmol/L MnCl₂·4H₂O, 0.8 μmol/L ZnSO₄·7H₂O, 0.3 μmol/L CuSO₄·5H₂O, 100 μmol/L EDTA-Fe, and 1 μmol/L (NH₄)₆Mo₇O₂₄·4H₂O. After 20 d, the seedlings were treated with four different NO₃ ⁻:NH₄ ⁺ ratios with four replications. Plants were cultured in primary solution (NO₃ ⁻:NH₄ ⁺ 14:1) or in primary solution with NH₄Cl (NO₃ ⁻:NH₄ ⁺ 9:6, 7.5:7.5, 1:14) according to Nimptsch and Pflugmacher (2007). The pH of solutions was adjusted to 6.5±0.2 with 1 mol/L NaOH. The solutions were renewed every 4 d during the 35-d experiment. At the end of the experiment, plants were harvested and separated into fresh samples (leaves immediately frozen in dry ice and kept frozen until use) and dry samples (roots, stems and leaves were dried at 80 °C until a constant weight was achieved). Meanwhile, xylem sap and phloem sap were collected.

2.2. Exudation techniques

The xylem sap was collected by the method described by Ueno et al. (2011) and Wu et al. (2015). Briefly, plant shoots (two plants) were cut at 3 cm above the roots and the xylem sap was collected from the cut surface by the root pressure method (the initial 1–2 μl of exudates were discarded). The volume of xylem sap was recorded and 4 ml xylem sap was placed in a 50-ml volumetric flask and dried on a hot plate for Mo determination. The phloem sap was collected as described by Tetyuk et al. (2013). Shoots were incubated in a conical flask filled with 65 ml 25 mmol/L EDTA-Na₂ in a growth chamber for 24 h at 20 °C and 95% humidity in dark. The collected solution was dried on a hot plate for Mo determination.

2.3. Cell wall, organelle and vacuole isolation

The frozen plant fresh leaves (1 g) were homogenized and the subcellular fractions were separated according to Su et al. (2014) and Li et al. (2016). Each plant sample was homogenized in 12-ml precooled extraction buffer containing 250 mmol/L sugar, 50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L MgCl₂, and 10 mmol/L cysteine using a chilled mortar and pestle. The mixture was centrifuged at 2057g for 10 min with a refrigerated centrifuge (Eppendorf 5810 R, Germany), and the precipitate was designated as the cell wall fraction mainly consisting of cell walls and cell wall debris. The supernatant was recentrifuged at 12 857g for 50 min, and the precipitate was designated as the organelle fraction which consisted of membrane and organelle components, and the resultant supernatant solution is referred to as the soluble fraction which mainly includes vacuoles and cytoplasm. All the above mentioned steps were performed at 4 °C.

2.4. Mo determination

Plant materials and dried xylem and phloem sap samples were digested with HNO₃/HClO₄ (4:1, v/v) mixture at 190 °C for 2 h and then at 205 °C. The samples were then dissolved in 10 ml water, and Mo concentration (C _Mo) was determined using a graphite furnace atomic absorption spectrometer (Z-2000 series, Hitachi, Japan) method (Nie et al., 2014). The translocation factor (TF) is calculated by C _Mo in leaf/C _Mo in stalk or C _Mo in stalk/C _Mo in root, and Mo accumulation is calculated by C _Mo×weight of dry matter.

2.5. Statistical analysis

Data were statistically analyzed using SPSS 13.0. One-way analysis of variance (ANOVA) with a least significance difference (LSD) test was performed to analyze the significant differences between treatments (P<0.05). Data were presented as mean±standard error (SE) and Sigma Plot v12.0 was used for the graphs.

3. Results

3.1. Biomass

All the biomass (fresh weight (FW)) of roots and shoots of the two rape cultivars reached a maximum at the 7.5:7.5 ratio, but they declined to a minimum at the ratio of 1:14 (Fig. 1). The biomass decreased by 43.8% and 33.9% for cultivar L0917 and by 33.5% and 34.0% for cultivar ZS11 for roots and shoots, respectively, by changing the ratio from 7.5:7.5 to 1:14. The average biomass of cultivar ZS11 roots and shoots was 1.86 and 1.31 times higher than that of cultivar L0917, respectively. This suggests that the two rape cultivars produce the highest biomass under the same concentrations of NH₄ ⁺ and NO₃ ⁻, but a higher proportion of NH₄ ⁺ results in significant diminution of the biomass of both cultivars particularly the root biomass of L0917 (P<0.05). The high Mo accumulator ZS11 not only had the higher biomass but also had less growth inhibition at the 1:14 ratio than the low Mo accumulator L0917.

Fig. 1.

Fresh weights of roots (a) and shoots (b) at different NO₃ ⁻:NH₄ ⁺ ratios in hydroponic culture

Data were presented as mean±SE (n=4). Different letters above the bar mean significant differences at P<0.05 by LSD-test

3.2. Mo distribution in tissues

Mo accumulation by both cultivars reached a maximum at the 7.5:7.5 ratio, while the high Mo accumulator ZS11 exhibited a 24.8% (P<0.05) decrease in Mo accumulation at the 1:14 ratio (Fig. 2a), suggesting that high NH₄ ⁺ inhibited Mo accumulation in the plant. The accumulation percentages of Mo by L0917 were 12.4%, 66.6%, and 21.0% in roots, stalks and leaves, respectively, while these were respectively 14.2%, 36.8%, and 49.0% by ZS11 (Fig. 2b), suggesting that the majority of Mo occurs in the stalks of L0917 and the leaves of ZS11. The proportion of Mo in leaves of both cultivars was increased with the decreasing NO₃ ⁻:NH₄ ⁺ ratio, but the proportion of Mo in stalks decreased under the same conditions.

Fig. 2.

Total plant Mo accumulations (a) and tissue Mo accumulation percentages (b) at different NO₃ ⁻:NH₄ ⁺ ratios in hydroponic culture

Data were presented as mean±SE (n=4). Different letters above the bar mean significant differences at P<0.05 by LSD-test

The root Mo concentrations of both the cultivars treated with the 7.5:7.5 ratio were higher than those under the other ratios (Fig. 3a). However, Mo concentrations of L0917 shoots showed no significant difference among the NO₃ ⁻:NH₄ ⁺ ratios, while Mo concentrations of ZS11 significantly decreased with decreasing NO₃ ⁻:NH₄ ⁺ (Fig. 3b). Root and shoot Mo concentrations were higher in cultivar ZS11 than in cultivar L0917 at all treatments. The shoot/root Mo TFs of cultivar L0917 initially declined with the decreasing NO₃ ⁻:NH₄ ⁺ but then increased to the highest value under the 1:14 treatment. The TFs of cultivar ZS11 decreased with the decreasing NO₃ ⁻:NH₄ ⁺ ratio (Fig. 4). The results indicated that Mo concentrations of the roots of both cultivars have the same absorption tendency treated with NO₃ ⁻:NH₄ ⁺ ratios, while Mo transportation from roots to shoots was inhibited particularly in cultivar ZS11.

Fig. 3.

Mo concentrations in roots (a) and shoots (b) of oilseed rape grown at different NO₃ ⁻:NH₄ ⁺ ratios in hydroponic culture

Data were presented as mean±SE (n=4). Different letters above the bar mean significant differences at P<0.05 by LSD-test

Fig. 4.

Mo translocation factors (TFs) for shoot/root of L0917 (a) and ZS11 (b) at different NO₃ ⁻:NH₄ ⁺ ratios in hydroponic culture

Data were presented as mean±SE (n=4). Different letters above the bar mean significant differences at P<0.05 by LSD-test

3.3. Mo transport in xylem and phloem

Mo contents of xylem and phloem sap were measured to further study the effects of NO₃ ⁻:NH₄ ⁺ ratio on Mo transport in the oilseed rape (Fig. 5). The Mo concentration in xylem sap of L0917 decreased by 23.40% (P<0.05) but that of ZS11 increased by 49.65% (P<0.05) at the ratio of 1:14 as compared to 14:1. Similarly, Mo accumulation in phloem sap of cultivar L0917 decreased by 59.48% (P<0.05) when the NO₃ ⁻:NH₄ ⁺ ratio was decreased from 14:1 to 1:14. However, phloem sap Mo accumulation in ZS11 increased to a maximum at the ratio of 9:6 before decreasing again with a further reduction in the NO₃ ⁻:NH₄ ⁺ ratio. At the ratio of 1:14, the phloem sap Mo accumulation was 36.78% (P<0.05) higher than that at 14:1. The Mo concentrations and accumulations in xylem and phloem sap of ZS11 were 2.02-and 3.59-fold (P<0.05) higher than those of L0917, respectively. These results showed that ZS11 exhibits notably higher Mo loads in the xylem and phloem transport systems than L0917. In particular, Mo transport in xylem sap of L0917 was inhibited while that of ZS11 was considerably promoted by a low NO₃ ⁻:NH₄ ⁺ ratio or higher NH₄ ⁺ concentration.

Fig. 5.

Mo concentration in xylem sap (a) and Mo accumulation in phloem sap (b) of oilseed rape grown at different NO₃ ⁻:NH₄ ⁺ ratios in hydroponic culture

Data were presented as mean±SE (n=4). Different letters above the bar mean significant differences at P<0.05 by LSD-test

3.4. Subcellular Mo distribution in leaves

Mo concentrations and Mo percentages in subcellular fractions of leaves under different treatments are shown in Figs. 6 and 7. Mo concentrations in cell wall fractions and soluble fractions of both the cultivars under the 1:14 ratio were higher than those under other ratios (Figs. 6a and 6c). In contrast, the Mo concentration in the organelle fraction of L0917 under the 14:1 ratio was 33 times higher than that under the 1:14 ratio (P<0.05), while there was no significant difference in the organelle Mo concentrations of the ZS11 cultivar among the treatments (Fig. 6b). Overall, the average Mo concentrations of both cultivars in the cell wall, organelle, and soluble fractions were 0.915, 0.204, and 1.151 mg/kg, respectively. The percentages of Mo in the organelle fraction of both cultivars were at a minimum with the 1:14 ratio of NO₃ ⁻:NH₄ ⁺ with 2.2% and 1.5% in cultivars L0917 and ZS11, respectively (Fig. 7). The Mo concentrations in the cell wall fractions of leaves of ZS11 were 2.80, 5.05, 3.94, and 4.28 times higher than those of L0917 under the 14:1, 9:6, 7.5:7.5, and 1:14 treatments, respectively. The Mo concentrations in the soluble fractions of leaves of ZS11 were 6.21, 5.67, 3.70, and 3.63 times higher than those of L0917 cultivar under the 14:1, 9:6, 7.5:7.5, and 1:14 treatments, respectively. While the Mo concentration in the organelle fractions of leaves of L0917 were 18.08, 4.05, 2.01, and 0.39 folds higher than those of ZS11 under the 14:1, 9:6, 7.5:7.5, and 1:14 treatments, respectively. These results clearly indicated that more Mo was distributed in cell wall and soluble fractions of ZS11, while Mo distribution in L0917 was much greater in the organelle fractions particularly under high NO₃ ⁻:NH₄ ⁺ ratios. High NO₃ ⁻:NH₄ ⁺ ratios increased the Mo distribution in organelle fractions of L0917, but low NO₃ ⁻:NH₄ ⁺ ratios increased the Mo distribution in cell wall fractions and soluble fractions of both the cultivars.

Fig. 6.

Mo concentrations in cell wall (a), organelle (b), soluble fractions (c) of oilseed rape grown at different NO₃ ⁻:NH₄ ⁺ ratios in hydroponic culture

Data were presented as mean±SE (n=4). Different letters above the bar mean significant differences at P<0.05 by LSD-test

Fig. 7.

Mo percentages in subcellular fractions for L0917 (a) and ZS11 (b) at different NO₃ ⁻:NH₄ ⁺ ratios in hydroponic culture

4. Discussion

In recent years, the understanding with regard to plant responses to the NO₃ ⁻:NH₄ ⁺ ratio has increased, whereas the effects of the ratio on Mo status are still unknown. The significant symptom of higher NH₄ ⁺ concentrations is the suppression of plant growth (Jampeetong and Brix, 2009). In this work, we found that oilseed rape grows better under a NO₃ ⁻:NH₄ ⁺ ratio of 7.5:7.5 than under 14:1, 9:6 or 1:14. Similar results were reported by Lu et al. (2009) in tomato and Tabatabaei et al. (2008) in strawberry plants, and their results indicated that combined application of NO₃ ⁻ and NH₄ ⁺ significantly enhanced the biomass as compared to sole application of either form of N. Compared with the 7.5:7.5 ratio, the decrease in biomass of L0917 cultivar at the 1:14 ratio was greater than that of ZS11 cultivar, indicating that the high Mo accumulating cultivar ZS11 had a better resistance to NH₄ ⁺ stress. Therefore, combined and particularly equal applications of NO₃ ⁻ and NH₄ ⁺ were the most beneficial to both oilseed rape cultivars and the low Mo accumulator cultivar L0917 was more sensitive to higher concentration of NH₄ ⁺.

Although the uptake mechanisms underlying ammonium inhibition are not fully understood, previous studies have found that the uptake of many inorganic cations (K⁺, Mg²⁺, Ca²⁺) is reduced under NH₄ ⁺ nutrition and consequent changes in plant ion balance (Britto and Kronzucker, 2002; Dickson et al., 2016). There is evidence that the effects of NH₄ ⁺ nutrition are predominantly upon root growth and compete with or pass through the same protein channels (Li et al., 2010; Zheng et al., 2015). In the present study, on two oilseed rape cultivars, Mo accumulation was only decreased under the NO₃ ⁻:NH₄ ⁺ ratio of 1:14 (Fig. 2), suggesting that higher NH₄ ⁺ concentrations restrain Mo absorption in oilseed rape. Similarly, Zhu et al. (2015) reported that shoot Mo contents were decreased significantly with a decreasing NO₃ ⁻:NH₄ ⁺ ratio at 15 mmol/L N on Chinese cabbage. The percentages of Mo accumulated in the roots of both oilseed rape cultivars increased, whereas the shoot Mo concentrations and the shoot/root TFs for cultivar ZS11 decreased with decreasing NO₃ ⁻:NH₄ ⁺ ratio, suggesting that Mo transport from root to shoot was inhibited by decreasing NO₃ ⁻:NH₄ ⁺ ratio. The Mo accumulation of ZS11 was respectively 64.9%, 72.2%, 59.2%, and 49.6% higher than that of L0917 at the 14:1, 9:6, 7.5:7.5, and 1:14 treatments. Therefore, it is probable that efficient xylem loadings play an important role in shoot mineral accumulation (Ueno et al., 2011; Wu et al., 2015) and phloem is a good indicator for the nutritional conditions in leaves (Peuke, 2010). Yu et al. (2002) also reported that higher phloem mobility was a key factor for leaf Mo accumulation. The present study suggested that at low NO₃ ⁻:NH₄ ⁺ ratios Mo concentrations in xylem and phloem sap of ZS11 were all higher than those of L0917. These results also suggested that ZS11 exhibited a higher Mo transport in xylem, as well as an enhanced ability for remobilization of Mo through phloem transport. Jongruaysup et al. (1994; 1997) also explained that variability of Mo mobility in phloem was found in Mo-adequate plants and NH₄NO₃ stimulated the remobilization of Mo in the tissues. Therefore, the high Mo concentrations in xylem and phloem sap indicate good uptake in the root and well-supplied leaves.

Compartmentalization of Mo is vital for Mo homeostasis in plant cells. The average Mo concentrations in the cell wall, organelle and soluble fractions of leaves of the two rapeseed cultivars across the four treatments were 0.366, 0.204, and 1.151 mg/kg respectively (Fig. 6). The percentages of Mo in cell wall and solution fractions for L0917 were increased but in the organelle fraction were decreased with decreasing NO₃ ⁻:NH₄ ⁺ ratios. It also appears that Mo was mostly accumulated firstly by the soluble fraction and then by the cell wall, which could be a good reason why the high NO₃ ⁻:NH₄ ⁺ ratio was toxic to organelle and cell wall fractions (Küpper et al., 1999; Hale et al., 2001; Britto and Kronzucker, 2002; Zhao et al., 2015). Plant cell wall binding and soluble fractions are the important mechanisms for metal accumulation (Li et al., 2006; Fu et al., 2011). We showed that the higher Mo accumulator ZS11 distributed more Mo firstly in the soluble fractions and secondly in the cell wall than cultivar L0917. It could be accordingly concluded that the soluble fraction in the high accumulation cultivar is more inclined to retain Mo than that in the low accumulation cultivar. Low NO₃ ⁻:NH₄ ⁺ ratio sharply decreased the Mo distribution in the organelle fractions of L0917, indicating that organelle damage could be the integrated effect of NH₄ ⁺ excess. This could also be associated with the biosynthesis of the Mo cofactor which is localized in the cytosol (Mendel, 2011).

In conclusion, this study indicated that the two rapeseed cultivars produced more biomass and accumulated more Mo while the nitrogen was supplied at the NO₃ ⁻:NH₄ ⁺ ratio of 7.5:7.5, and the Mo uptake and Mo transport from roots to shoots were inhibited by low NO₃ ⁻:NH₄ ⁺ ratios or higher NH₄ ⁺. The high Mo accumulator ZS11 not only exhibited higher Mo concentrations in all the tissues, cell-organs, xylem and phloem sap, but also accumulated more Mo firstly in the soluble fraction and secondly in the cell wall than L0917.