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

Uptake, transport and distribution of molybdenum in two oilseed rape ( Brassica napus L.) cultivars under different nitrate/ammonium ratios 不同NO 3 − :NH 4 + 比对两种甘蓝型油菜钼吸收、转运和分布的影响 1 2 3 first-author 1 2 3 1 2 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:hucx@mail.hzau.edu.cn †E-mail:hucx@mail.hzau.edu.cn hucx@mail.hzau.edu.cn 1 2 3 1 2 3 Microelement Research Center, Huazhong Agricultural University, Wuhan 430070, China Microelement Research Center, Huazhong Agricultural University, Wuhan 430070, China Hubei Provincial Engineering Laboratory for New-Type Fertilizer, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China Hubei Provincial Engineering Laboratory for New-Type Fertilizer, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China MOA Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Wuhan 430070, China MOA Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Wuhan 430070, China Objectives: 2 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: 2 A hydroponic culture experiment was conducted with four nitrate/ammonium (NO 3 − :NH 4 + ) 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: 2 Both the cultivars showed maximum biomass and Mo accumulation at the 7.5:7.5 ratio of NO 3 − :NH 4 + 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 NO 3 − :NH 4 + 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 NO 3 − :NH 4 + ratio was more beneficial for L0917. On the contrary, a lower NO 3 − :NH 4 + 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 NO 3 − :NH 4 + ratio. Conclusions: 2 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 NO 3 − :NH 4 + ratio conditions. 目的 2 采用不同NO 3 − :NH 4 + 比的营养液，探索不同氮源对钼元素在两种甘蓝型油菜（L0917和ZS11）中的吸收、转运和分布的影响及品种间的差异。 创新点 2 在不同氮源条件下，从组织分布、汁液运输、亚细胞分布以及品种等方面研究了甘蓝型油菜的钼营养状况。 方法 2 将甘蓝型油菜L0917和ZS11的幼苗在正常营养液培养20天后，分别转移至4种NO 3 − :NH 4 + 比（14:1、9:6、7.5:7.5、1:14）且总氮为15 mmol/L的营养液中培养15天后收获。采用原子吸收分光光度计-石墨炉法测定根、茎和叶不同部位钼含量，木质部和韧皮部液钼含量，叶肉细胞细胞壁组分、细胞器组分和可溶性组分钼含量。 结论 2 在高或低NO 3 − :NH 4 + 比条件下，高的钼吸收能力、木质部转运、韧皮部再迁移以及叶片亚细胞钼储存在甘蓝型油菜钼积累上扮演重要角色。 Brassica napus L. Nitrogen source Transport Subcellular distribution Xylem Phloem 甘蓝型油菜 氮源 运输 亚细胞分布 木质部 韧皮部 Introduction 1 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 2011 ; Hille et al., 2011 2011 ). Generally in China, soils are acidic and poor availability of Mo in soils is widespread (Ye et al., 2011 2011 ; Cao et al., 2012 2012 ; Liu et al., 2012 2012 ), especially in Southeast China (Shi et al., 2006 2006 ). However, oilseed rape ( Brassica napus L.), an important oil crop, is predominantly grown in Mo-deficient soils (Yin et al., 2009 2009 ). Moreover, oilseed rape requires plenty of nitrogen (N) fertilizer for growth and yield (Behrens et al., 2002 2002 ; Barłóg and Grzebisz, 2004a 2004a ; 2004b 2004b ; Rathke et al., 2006 2006 ; Leiva-Candia et al., 2013 2013 ) and Mo deficiency often decreases the activity of nitrate reductase which leads to N deficiency in plants (Chatterjee and Nautiyal, 2001 2001 ; Ide et al., 2011 2011 ), poor seedling growth and lower grain yield (Yang et al., 2009 2009 ; Liu et al., 2010 2010 ). Furthermore, winter wheat subjected to higher rate of N-fertilization is prone to Mo deficiency (Wang et al., 1999 1999 ) while supplying NH 4 NO 3 to plants stimulates the remobilization of Mo in black gram (Jongruaysup et al., 1997 1997 ). Mo plays an important role in the N metabolism of plants, including nitrogen fixation and the transformation of nitrate (NO 3 − )-N to ammonium (NH 4 + )-N (Kovács et al., 2015 2015 ). However, little information is available about the Mo uptake, transport, or distribution under different N sources. NH 4 + and NO 3 − are two forms of inorganic N available for plant uptake. Studies on NH 4 + stress showed that a low NO 3 − :NH 4 + ratio inhibits plant growth and changes ion balance in the plant (Liu et al., 2014 2014 ; Babalar et al., 2015 2015 ; Sokri et al., 2015 2015 ). Esteban et al. ( 2016 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 2015 ) reported that the Mo content of Chinese cabbage declined significantly with a decreasing NO 3 − :NH 4 + ratio. Moreover, the molybdate transporter 1 (CrMOT1) of Chlamy-domonas reinhardtii is activated by nitrate but not by molybdate (Tejada-Jiménez et al., 2007 2007 ), while molybdate transporter 2 (CrMOT2) is activated by molybdate, but not by the N source (Tejada-Jiménez et al., 2011 2011 ). The responses of plants particularly of different cultivars to variable NO 3 − :NH 4 + 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 NO 3 − :NH 4 + ratios under hydroponic culture, mainly by tissue Mo, sap flow and subcellular Mo concentrations. Materials and methods 1 Plants and growth conditions 2 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(NO 3 ) 2 ·4H 2 O, 6 mmol/L KNO 3 , 1 mmol/L NH 4 H 2 PO 4 , 2 mmol/L MgSO 4 ·7H 2 O, 46.2 μmol/L H 3 BO 3 , 9.1 μmol/L MnCl 2 ·4H 2 O, 0.8 μmol/L ZnSO 4 ·7H 2 O, 0.3 μmol/L CuSO 4 ·5H 2 O, 100 μmol/L EDTA-Fe, and 1 μmol/L (NH 4 ) 6 Mo 7 O 24 ·4H 2 O. After 20 d, the seedlings were treated with four different NO 3 − :NH 4 + ratios with four replications. Plants were cultured in primary solution (NO 3 − :NH 4 + 14:1) or in primary solution with NH 4 Cl (NO 3 − :NH 4 + 9:6, 7.5:7.5, 1:14) according to Nimptsch and Pflugmacher ( 2007 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. Exudation techniques 2 The xylem sap was collected by the method described by Ueno et al. ( 2011 2011 ) and Wu et al. ( 2015 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 2013 ). Shoots were incubated in a conical flask filled with 65 ml 25 mmol/L EDTA-Na 2 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. Cell wall, organelle and vacuole isolation 2 The frozen plant fresh leaves (1 g) were homogenized and the subcellular fractions were separated according to Su et al. ( 2014 2014 ) and Li et al. ( 2016 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 MgCl 2 , and 10 mmol/L cysteine using a chilled mortar and pestle. The mixture was centrifuged at 2057 g 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 12 857 g 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. Mo determination 2 Plant materials and dried xylem and phloem sap samples were digested with HNO 3 /HClO 4 (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 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. Statistical analysis 2 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. Results 1 Biomass 2 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 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 NH 4 + and NO 3 − , but a higher proportion of NH 4 + 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. Data were presented as mean±SE ( n =4). Different letters above the bar mean significant differences at P < 0.05 by LSD-test Mo distribution in tissues 2 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 2a ), suggesting that high NH 4 + 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 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 NO 3 − :NH 4 + ratio, but the proportion of Mo in stalks decreased under the same conditions. 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 3a ). However, Mo concentrations of L0917 shoots showed no significant difference among the NO 3 − :NH 4 + ratios, while Mo concentrations of ZS11 significantly decreased with decreasing NO 3 − :NH 4 + (Fig. 3b 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 NO 3 − :NH 4 + but then increased to the highest value under the 1:14 treatment. The TFs of cultivar ZS11 decreased with the decreasing NO 3 − :NH 4 + ratio (Fig. 4 4 ). The results indicated that Mo concentrations of the roots of both cultivars have the same absorption tendency treated with NO 3 − :NH 4 + ratios, while Mo transportation from roots to shoots was inhibited particularly in cultivar ZS11. Data were presented as mean±SE ( n =4). Different letters above the bar mean significant differences at P < 0.05 by LSD-test Data were presented as mean±SE ( n =4). Different letters above the bar mean significant differences at P < 0.05 by LSD-test Mo transport in xylem and phloem 2 Mo contents of xylem and phloem sap were measured to further study the effects of NO 3 − :NH 4 + ratio on Mo transport in the oilseed rape (Fig. 5 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 NO 3 − :NH 4 + 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 NO 3 − :NH 4 + 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 NO 3 − :NH 4 + ratio or higher NH 4 + concentration. Data were presented as mean±SE ( n =4). Different letters above the bar mean significant differences at P < 0.05 by LSD-test Subcellular Mo distribution in leaves 2 Mo concentrations and Mo percentages in subcellular fractions of leaves under different treatments are shown in Figs. 6 6 and 7 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 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 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 NO 3 − :NH 4 + with 2.2% and 1.5% in cultivars L0917 and ZS11, respectively (Fig. 7 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 NO 3 − :NH 4 + ratios. High NO 3 − :NH 4 + ratios increased the Mo distribution in organelle fractions of L0917, but low NO 3 − :NH 4 + ratios increased the Mo distribution in cell wall fractions and soluble fractions of both the cultivars. Data were presented as mean±SE ( n =4). Different letters above the bar mean significant differences at P < 0.05 by LSD-test Discussion 1 In recent years, the understanding with regard to plant responses to the NO 3 − :NH 4 + ratio has increased, whereas the effects of the ratio on Mo status are still unknown. The significant symptom of higher NH 4 + concentrations is the suppression of plant growth (Jampeetong and Brix, 2009 2009 ). In this work, we found that oilseed rape grows better under a NO 3 − :NH 4 + ratio of 7.5:7.5 than under 14:1, 9:6 or 1:14. Similar results were reported by Lu et al. ( 2009 2009 ) in tomato and Tabatabaei et al. ( 2008 2008 ) in strawberry plants, and their results indicated that combined application of NO 3 − and NH 4 + 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 NH 4 + stress. Therefore, combined and particularly equal applications of NO 3 − and NH 4 + were the most beneficial to both oilseed rape cultivars and the low Mo accumulator cultivar L0917 was more sensitive to higher concentration of NH 4 + . Although the uptake mechanisms underlying ammonium inhibition are not fully understood, previous studies have found that the uptake of many inorganic cations (K + , Mg 2+ , Ca 2+ ) is reduced under NH 4 + nutrition and consequent changes in plant ion balance (Britto and Kronzucker, 2002 2002 ; Dickson et al., 2016 2016 ). There is evidence that the effects of NH 4 + nutrition are predominantly upon root growth and compete with or pass through the same protein channels (Li et al., 2010 2010 ; Zheng et al., 2015 2015 ). In the present study, on two oilseed rape cultivars, Mo accumulation was only decreased under the NO 3 − :NH 4 + ratio of 1:14 (Fig. 2 2 ), suggesting that higher NH 4 + concentrations restrain Mo absorption in oilseed rape. Similarly, Zhu et al. ( 2015 2015 ) reported that shoot Mo contents were decreased significantly with a decreasing NO 3 − :NH 4 + 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 NO 3 − :NH 4 + ratio, suggesting that Mo transport from root to shoot was inhibited by decreasing NO 3 − :NH 4 + 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 2011 ; Wu et al., 2015 2015 ) and phloem is a good indicator for the nutritional conditions in leaves (Peuke, 2010 2010 ). Yu et al. ( 2002 2002 ) also reported that higher phloem mobility was a key factor for leaf Mo accumulation. The present study suggested that at low NO 3 − :NH 4 + 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 1994 ; 1997 1997 ) also explained that variability of Mo mobility in phloem was found in Mo-adequate plants and NH 4 NO 3 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 6 ). The percentages of Mo in cell wall and solution fractions for L0917 were increased but in the organelle fraction were decreased with decreasing NO 3 − :NH 4 + 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 NO 3 − :NH 4 + ratio was toxic to organelle and cell wall fractions (Küpper et al., 1999 1999 ; Hale et al., 2001 2001 ; Britto and Kronzucker, 2002 2002 ; Zhao et al., 2015 2015 ). Plant cell wall binding and soluble fractions are the important mechanisms for metal accumulation (Li et al., 2006 2006 ; Fu et al., 2011 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 NO 3 − :NH 4 + ratio sharply decreased the Mo distribution in the organelle fractions of L0917, indicating that organelle damage could be the integrated effect of NH 4 + excess. This could also be associated with the biosynthesis of the Mo cofactor which is localized in the cytosol (Mendel, 2011 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 NO 3 − :NH 4 + ratio of 7.5:7.5, and the Mo uptake and Mo transport from roots to shoots were inhibited by low NO 3 − :NH 4 + ratios or higher NH 4 + . 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1600249 Project supported by the National Key Technologies R&D Program of China (No. 2014BAD14B02) and the &ldquo;948&rdquo; Project of the Ministry of Agriculture, China (Nos. 2016-X41 and 2015-Z34) 2016-06-01 2017-05-26 2016-08-26 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 18