Meta-analysis of the role of zinc in coordinating absorption of mineral elements in wheat seedlings

Zinc (Zn) is an important nutrient for human beings, which is also an essential micronutrient for crop growth. This study investigated the role of Zn in coordinating the mineral elements absorption in modern wheat (Triticum aestivum L.) cultivars with a new developed method. A method was developed, and showed a robust capability to simultaneously investigate seven mineral elements uptake in wheat seedling. With this method, we found low Zn supply (<  1 μM) promoted the absorption of potassium (K), magnesium (Mg) and manganese (Mn) in wheat seedling, while high Zn supply (>  1 μM) significantly inhibited the absorption of these elements. Cultivars with the green genes (Rht-B1b and Rht-D1b) showed a higher uptake capability on ammonium (NH4+), and cultivars with Rht-B1b allele can uptake more phosphors (P), K, calcium (Ca), Mn and Zn compared to cultivars with Rht-D1b. Further analysis indicated higher uptake capability of NH4+ in cultivars contained Rhts was independent of Zn. The key role of Zn in coordinating for mineral elements absorption was identified in modern wheat cultivars, providing the reference for Zn application in wheat. Meanwhile, this study provides a robust method for quantifying the absorption of mineral elements, which may be adopted into the broadly investigations on the coordinated nutrients absorption of plant.


Background
To enhance the yield, chemical fertilizer has become the main source of mineral elements for all kinds of crops [1,2]. While these inputs help to meet the demand for crop yields, then overuse of fertilizers also causes serious environmental damage, such as the greenhouse effect and water eutrophication [3]. Therefore, it is important to understand the mineral element absorption rules of crops for rational fertilization.
Zn is an essential nutrient element for plants and humans, which is widely involved in biochemical and physiological processes, including enzyme activity, cell division, photosynthesis, carbohydrate and lipid metabolism, detoxification of oxygen radicals and protein synthesis [4][5][6][7]. Zn deficiency is common in most crops, which causes leaf chlorosis, curling and wilting, and stunted and thin stems [8][9][10], and ultimately leads to low grain yields and poor grain quality [11,12]. Foliar application of Zn can be absorbed through the cuticle and/or the stomates, and then transported to leaves, stems, reproductive tissues and grains, to rescue the symptom of Zn deficiency  [13,14]. Proper supply Zn can promote plant growth and development by enhancing photosynthesis and carbohydrate metabolism [15]. On the other side, excess Zn can lead to leaf necrosis [16], biomass decline [17], affect the absorption of other elements [18] and many adverse effects on crop growth and development [5,19]. Previous studies focused on the effects of other elements on Zn uptake in crops [20][21][22]. However, the effect of Zn in regulating other mineral elements absorption is still largely unknown, which hinders our understanding of the rational use of Zn fertilizer in agricultural production.
The commonly application of dwarfing genes Rht-B1b and Rht-D1b significantly increases modern wheat yield and the high-input of nitrogen fertilizers is the key to achieve the maximum its yield potential [23][24][25][26]. Previous studies have shown that Rhts allele significantly altered iron (Fe), Zn, copper (Cu) and Mg content in wheat grains [24,27], then the underlying mechanism still keeps in largely unknown.
At present, the research on nutrient elements absorption in crops mainly adopts tissue digestion method to determine the content of mineral elements in plant tissues [24,28,29]. However, this method is relatively tedious and also has the risk because of the strong acid application. Therefore, the aim of this study was to develop a new method for quantifying the absorption of mineral elements and identify the role of Zn in the coordinated absorption of mineral elements in wheat seedling.

A new method for quantifying mineral elements uptake in wheat seedling
To investigate the mineral elements uptake in wheat seedling, a nutrient solution quantification in the test tube was developed. Briefly, hydroponic wheat seedling (14 days after germination) was transferred to the tube containing 1/2 Hoagland nutrient solution with different treated time (Fig. 1), then quantifying the reduction of mineral elements in solution by atomic absorption spectrophotometer and continuous flow analyzer, to represent the uptake by the wheat seedling.
To evaluate the reliability of this method and determine the optimal treatment time, two wheat cultivars and four treatment times (12 h, 36 h, 60 h, 84 h) were selected for the evaluation. The absorption of the all three elements (NH 4 + , P and K) increased with the treatment duration, and showed significant difference in absorption between two tested cultivars (Fig. 2a). Meanwhile, the contents of three elements in the control tube remained constant at any quantification time (Additional file 1: Figure S1), indicating the mineral elements were absorbed in wheat seedlings, and the quantification procedure is reliable. To determine the optimal treatment time point, the absorption rates of three elements were analyzed (Fig. 2b) and showed that the absorption rate of three elements gradually decreased with the treatment duration, and then plateaued at 60 h. In addition, the absorption of Mg, Ca, Mn and Zn in wheat seedling could also showed a stable value after 60 h treatment (Additional file 1: Figure S2). Therefore, 60 h treatment was selected for subsequent investigations.

Correlation of absorption of seven mineral elements in wheat seedling
To understand the coordinated absorption of different elements in wheat seedling, a correlation analysis was performed (Fig. 3), and showed a significant positive correlation among the five macronutrients (N, P, K, Mg and Ca), consistent with the previous reports [30,31]. A new finding is that trace element Zn is significantly positive correlated with macronutrients N, K, Mg and trace element Mn, indicating that Zn plays an important role in mediating nutrient absorption of wheat.

Zn significantly affects the uptake of other mineral elements in wheat seedling
To further clarify the role of Zn in mediating mineral elements absorption, five Zn concentrations were performed on seven wheat cultivars. Results showed the uptake of Zn in all seven cultivars is significantly increased with the increase of Zn concentration (Additional file 1: Figure S3). In different Zn concentrations, the absorption of K, Mg and Mn in all seven wheat cultivars is promoted by low concentration Zn (< 1 μM) but is inhibited by the high concentration (10 μM), and Wheat seedlings that had been hydroponic for two weeks after germination were transferred to tubes containing nutrient solution for treatment. The test tube mouth is covered with a film to reduce the loss of water and nutrients. Nutrient solution in test tube without treatment of seedlings were used as control suggesting 0.01-0.1 μM Zn in solution have the optimal effect to promote the uptake of other mineral elements (Fig. 4). However, the uptake of NH 4 + and P seems not be affected by different Zn supply (Fig. 4). These results displayed the role of Zn in regulating the absorption of specific mineral elements in wheat seedling.
Interestingly, the uptake of Ca showed distinct response among different cultivars with the alterations of Zn concentration (Fig. 4), indicating the interaction between Zn and Ca uptake is highly variable in different wheat cultivars, which may be determined by the different genetic background of different wheat cultivar.

The potential role of dwarfing gene Rhts in mineral elements uptake
Previous studies suggested that the dwarfing gene Rhts significantly increases the uptake for nitrogen fertilizer in crops [26,32]. To further test our method, eight new wheat cultivars were added into our investigation. All fifteen wheat cultivars (see methods) were classified into three groups: Rht-B1b allele contained group, Rht-D1b allele contained group and Control group (without either Rht-B1b or Rht-D1b allele). We found the uptake of NH 4 + is significantly increased in both Rht-B1b and Rht-D1b group (Fig. 5) with medium Zn supply (1 μM), consistent with previous conclusion [23], further supporting the special role of green gene in mineral elements absorption.
In addition, Rht-B1b showed a significantly higher uptake of P, K, Ca and Mn compared to control group, and a higher absorption in P and Zn compared to Rht-D1b group (Fig. 5). However, neither Rht-B1b nor Rht-D1b significantly affected Mg absorption (Fig. 5). These results indicate that Rhts alleles contribute a broad effect in the absorption of mineral elements in addition to nitrogen, and suggest a different mineral element uptake potential between Rht-B1b and Rht-D1b.

The interaction between Zn supply and Rhts alleles on uptake of mineral elements
In low Zn concentration (< 1 μM), both Rht-B1b and Rht-D1b group showed a significantly increased absorption on NH 4 + with the Zn supply increase, and Rht-B1b and Rht-D1b group showed enhanced absorption of P and Mg, respective (Additional file 1: Figure S4). While the Rht-D1b group showed a significantly decreased absorption on Ca and Mn with Zn supply increase (Additional file 1: Figure S4).
In high Zn concentration (10 μM), Rht-B1b and Rht-D1b group still maintained the higher absorption on NH 4 + compared control group (Fig. 6), indicating that the higher NH 4 + absorption is independent of Zn concentration. Similarly, the absorption of P, K, Fig. 2 The absorption values (a) and absorption rates (b) of various elements in wheat seedlings under different treatment times. The absorption rate of wheat seedlings was indicated by the absorption value of per unit fresh weight per hour. UFWAV: unit fresh weight absorption value. Values are means of at last six replications ± SE. *t test significant difference. *P < 0.05, **P < 0.01 Ca, Mg, Mn was not significantly inhibited by high Zn concentration in Rht-D1b group (Fig. 6), compared to the absorption in medium Zn concentration (Fig. 5). In contrast, the absorption of these elements was significantly inhibited in Rht-B1b and control group, highlighting a special interaction between Zn and Rht-D1b in mineral elements absorption.

Discussion
In this study, we developed a new method to quantify the absorption of mineral elements in wheat seedling. Our data with this method demonstrated highly stable and reproducible, indicating it is a robust method, which could be applied to study the role of different elements in wheat nutrient coordinated absorption, and may be extend to other crops.
Although its robustness, there are still some disadvantages that should not be overlooked. Firstly, the NO 3 − ions, another important nitrogen form [33], failed to be quantified in this method. Secondly, few trace elements cannot be effectively detected due to their low content nature. Thirdly, the soil environments and related microorganism context played the core role in determining the absorption of soil mineral elements [34,35], thus this method definitely need further evaluate and improve to reflect the real status of nutrients uptake by crop in field condition. Then based on this initial method here, it is promised to develop a comprehensive and precise method for futural study in plant nutrients absorption. Pearson correlation analysis matrix between elements. *Significant correlation. *P < 0.05, **P < 0.01, ***P < 0.001. The fitting function are shown in Additional file 2: Table S1 In this study, our results (Fig. 4) indicate that Zn broadly affects K, Mg, and Mn uptake, supporting low concentration of Zn limited the absorption of K and Mg [12,36] and high concentration of Zn significantly inhibits the absorption of Mn and Ca [37][38][39]. This result further confirms that excessive Zn caused a negative interferences to others nutrients uptake [16]. It should be noted that the absorption of NH 4 + in different Zn concentrations keeps stable in this study (Fig. 4d), which is different with the positive interaction relationship between Zn and nitrogen in previous report [40]. We assume the difference may result from the quantification on uptake of nitrate is missed in our study, but this hypothesis needs further comprehensive investigation. Importantly, a general optimum concentration of Zn was proposed, the 0.01-0.1 μM of Zn in solution can promote the uptake of most mineral elements, providing a reference concentration for futural Zn fertilizer application.
Consistent with the previous study [23], both Rht-B1b and Rht-D1b significantly increased NH 4 + uptake by wheat seedling. Moreover, in our results Rht-B1b and Rht-D1b contribute a distinct influence in the uptake of mineral elements, and distinct response to alteration of Zn concentrations. Although limited wheat cultivars were investigated in this study, then these distinct responses between the Rht-B1b and Rht-D1b implying that our method may provide important clues for studying the effect of different genes on the mineral element absorption in plant.

Conclusions
In summary, the new method developed in this study have a robust capability to investigate the coordinated mineral elements absorption in wheat seedling, and promisingly extend to other crops. With this method, we identified the key role of Zn in coordinated absorption of mineral elements in wheat seedling, which provides a reference to properly apply Zn for enhancing wheat production.

Plant culture and treatments
Seeds of the wheat were surface-disinfected in 5% sodium hypochlorite for 10 minutes, then rinsed in distilled water for six times, and soaked in a low-temperature (4 °C) incubator for vernalization 3 days. The vernalized seeds were transferred into germinating box (15 × 15 cm, 16 seedlings per box) with filter paper and cultured in sterile distilled water. In addition, replacing 15 ml distilled water every two days. During this period, no mineral nutrients were added. These seedlings were cultured in a growth chamber with 22 °C/18 °C (day/night), 16 h/8 h (light/ dark, light intensity, 2000 lx), and 50% humidity.
After 14 days of germination, well-grown wheat seedlings from each cultivar were selected and transferred into a test tube containing 1/2 Hoagland nutrient solution (Fig. 1) . Notably, to avoid failure detection of trace elements, two seedlings were put into each test tube. In addition, the opening of the tube is sealed with plastic film to prevent the evaporation of nutrient solution. Then, these seedlings were transferred to the growth chamber with the same growth parameters for different treatment time.

Time gradient treatment
Two cultivars were selected for treatment at four time points (12 h, 36 h, 60 h, 84 h). The treatment time is calculated from the time when the seedlings are placed in a test tube containing nutrient solution to the time when they are removed. At least 6 replicates were performed for each cultivar under each time treatment, with one test tube as a replicate. In addition, test tubes without treatment of seedlings were used as controls and at least 6 replicates were also performed under each time treatment.  Table S2 Zn concentration gradient treatment In this study, five Zn concentration gradients of 0, 0.01, 0.1, 1, 10 μM were selected. Zn concentration was changed by changing the content of ZnSO 4 ·7H 2 O on the basis of 1/2 Hoagland nutrient solution, while the content of other elements remained unchanged. In addition, all treatments and culture conditions were consistent with those described above.

Determination of absorption value of mineral elements
After treatment, the wheat seedlings were taken out of the test tubes, and the fresh weight of seedlings and the volume of remaining nutrient solution were measured. Next, prepare standard solutions for each element, and then use Flame/Graphite Furnace Atomic Absorption Spectrometer "Z2000" (Hitachi, Japan) and continuous flow analyzer "FLOWSYS" (Systea, Italy) to detect the absorbance value of each element, and finally obtain the standard solution curve and curve equation of each element. Then detect the absorbance value of each element in the test tube of the control group and the treatment group, and calculate the concentration of each element through the curve equation.
Note: the absorbance value of total Zn, Mn, Cu, Mg, K and Ca in the nutrient solution in the test tube were measured by Atomic Absorption Spectrometer. Due to the limited detection range of the instrument, the nutrient solution must be diluted 10 times to measure the concentration of Mg, and diluted 100 times to measure the concentration of K and Ca. The absorbance value of total NH 4 + and P in the nutrient solution were measured by continuous flow analyzer, in which the measurement of element P requires dilution of the nutrient solution by 5 times. The concentrations of all elements in the nutrient solution of the untreated seedlings were as the control.
In order to avoid the influence of the different growth status of wheat seedlings on the measurement results, the absorption value per unit fresh weight was used as the index to evaluate the nutrient absorption capacity of wheat in this study. Unit fresh weight absorption value (UFWAV) = (control measurement value × volume − sample measurement value × volume)/fresh weight. Fig. 6 Effects of high Zn concentration (10 μM) on the absorption of mineral elements in wheat containing the Rhts allele. UFWAV: unit fresh weight absorption value. The control group contains seven cultivars, and the Rht-B1b and Rht-D1b groups respectively include four cultivars, and each cultivar has at least six replicates. Values are means of all replicates of all cultivar in each group ± SE.*t test and ANOVA significant difference. *P < 0.05, **P < 0.01. The results of ANOVA are shown in Additional file 2: Table S2