Genotypic Variation in Nickel Accumulation and Translocation and Its Relationships with Silicon, Phosphorus, Iron, and Manganese among 72 Major Rice Cultivars from Jiangsu Province, China

Nickel (Ni) is a ubiquitous environmental toxicant and carcinogen, and rice is a major dietary source of Ni for the Chinese population. Recently, strategies to decrease Ni accumulation in rice have received considerable attention. This study investigated the variation in Ni accumulation and translocation, and also multi-element (silicon (Si), phosphorus (P), iron (Fe), and manganese (Mn)) uptake and transport among 72 rice cultivars from Jiangsu Province, China, that were grown under hydroponic conditions. Our results showed a 2.2-, 4.2-, and 5.3-fold variation in shoot Ni concentrations, root Ni concentrations, and translocation factors (TFs) among cultivars, respectively. This suggests that Ni accumulation and translocation are significantly influenced by the genotypes of the different rice cultivars. Redundancy analysis of the 72 cultivars revealed that the uptake and transport of Ni were more similar to those of Si and Fe than to those of P and Mn. The Ni TFs of high-Ni cultivars were significantly greater than those of low-Ni cultivars (p < 0.001). However, there were no significant differences in root Ni concentrations of low-Ni and high-Ni cultivars, suggesting that high-Ni cultivars could translocate Ni to shoots more effectively than low-Ni cultivars. In addition, the cultivars HD8 and YD8 exhibited significantly lower levels of Ni accumulation than their parents (p < 0.05). Our results suggest that breeding can be an effective strategy for mitigating excessive Ni accumulation in rice grown in Ni-contaminated environments.


Introduction
Nickel (Ni) is a ubiquitous trace metal that has both natural and anthropogenic sources (e.g., vehicle emissions, as well as the Ni mining, smelting, cement manufacture, metallurgical, and electroplating industries), and its environmental accumulation has become a concern worldwide [1,2]. In recent years, fertilizer and organic manure use have increased Ni concentrations in cropland soils, exacerbating the problem of Ni pollution [2,3]. A recent nationwide Chinese soil survey revealed that 19.4% of the cropland soil samples were polluted. Ni was the second most abundant (4.8%) potentially toxic element (PTE) in soil and a key pollutant of Chinese farmlands [4]. In Jiangsu Province, located in the eastern coastal region of China, the surface soil (0-20 cm) background concentrations of Ni varied from 1.6 to 238 mg·kg −1 , with a geometric mean of 32.9 mg·kg −1 [5]. Although the average soil Ni concentration

Experimental Design
Uniform 20-day-old rice seedlings at the three-leaf stage were selected and cultured in 400-mL plastic cups. Each cup contained three seedlings and 350 mL of 0.5-strength nutrient solution. The seedlings were treated with 0 and 10 µmol·L −1 Ni [7], and each treatment was replicated three times. A Ni stock solution was prepared from nickel sulfate hexahydrate (NiSO 4 ·6H 2 O). After exposure to 10 µmol·L −1 Ni for 3 days (short-term experiment), the rice samples were collected, rinsed three times with deionized water, and then separated into shoots and roots. In addition, 2 mL aliquots of the nutrient solution from each replicate were filtered using a 0.45-µm syringe filter and then stored at 4 • C to determine the total Ni concentrations. After oven-drying the sample tissues for 2 days at 60 • C, the dry weights of the shoots and roots were recorded. Then, the tissues were ground using zirconia beads in a high-throughput sample grinder (CK-2000; Thmorgan, Beijing, China). The translocation factor (TF) was calculated as the shoot Ni concentration/root Ni concentration. The bioconcentration factor (BCF) of Ni from the culture medium to shoots or roots was calculated as follows: BCF = shoot or root Ni concentration/Ni concentration in the medium.

Statistical Analyses
SigmaPlot software (ver. 12.5; Systat, San Jose, CA, USA) was used to create the figures. Independent samples t-test, one-way analysis of variance, and post-hoc multiple comparisons (Tukey's test) were used to determine the significance (p < 0.05 or 0.01) of the results using SPSS software (ver. 20.0; IBM Corp., Armonk, NY, USA). Pearson's correlation analysis was also performed using SPSS (ver. 20.0). Redundancy analysis (RDA) was performed to analyze the relationships among the accumulation and translocation of Ni and other elements in the rice cultivars using Canoco software (ver. 4.5; Microcomputer Power, Ithaca, NY, USA) [43].

Accumulation and Translocation of Ni in 72 Rice Cultivars
As shown in Figure 1, we investigated the genotypic variation among 72 rice cultivars in shoot and root Ni concentrations and root-to-shoot Ni translocation. After exposure to 10 µmol·L −1 Ni for 72 h, significant differences were observed among the rice cultivars.
The BCFs were calculated to investigate the Ni accumulation capacities of shoots and roots among the rice cultivars ( Figure S1). In general, the BCFs of shoots and roots differed significantly among the cultivars. The shoot Ni BCFs varied from 17.8 to 38.8, whereas the root Ni BCFs varied from 513 to 2,136. HD5 had the lowest and LJ6 (early rice) had the highest shoot Ni BCFs ( Figure  S1A). YD8 had the lowest and NJ34 had the highest root Ni BCFs ( Figure S1B). Table 2 shows the 72 rice cultivars separated into 11 subgroups based on their locations within Jiangsu Province. To characterize the variation in Ni accumulation among these subgroups, we defined the 20 lowest and 20 highest shoot Ni-accumulating genotypes among the 72 cultivars as The BCFs were calculated to investigate the Ni accumulation capacities of shoots and roots among the rice cultivars ( Figure S1). In general, the BCFs of shoots and roots differed significantly among the cultivars. The shoot Ni BCFs varied from 17.8 to 38.8, whereas the root Ni BCFs varied from 513 to 2136. HD5 had the lowest and LJ6 (early rice) had the highest shoot Ni BCFs ( Figure S1A). YD8 had the lowest and NJ34 had the highest root Ni BCFs ( Figure S1B). Table 2 shows the 72 rice cultivars separated into 11 subgroups based on their locations within Jiangsu Province. To characterize the variation in Ni accumulation among these subgroups, we defined the 20 lowest and 20 highest shoot Ni-accumulating genotypes among the 72 cultivars as low-Ni and high-Ni cultivars, respectively. Most of the low-Ni cultivars were found in four subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). low-Ni and high-Ni cultivars, respectively. Most of the low-Ni cultivars were found in four subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). low-Ni and high-Ni cultivars, respectively. Most of the low-Ni cultivars were found in four subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). low-Ni and high-Ni cultivars, respectively. Most of the low-Ni cultivars were found in four subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). low-Ni and high-Ni cultivars, respectively. Most of the low-Ni cultivars were found in four subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). low-Ni and high-Ni cultivars, respectively. Most of the low-Ni cultivars were found in four subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). low-Ni and high-Ni cultivars, respectively. Most of the low-Ni cultivars were found in four subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). low-Ni and high-Ni cultivars, respectively. Most of the low-Ni cultivars were found in four subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). low-Ni and high-Ni cultivars, respectively. Most of the low-Ni cultivars were found in four subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05). low-Ni and high-Ni cultivars, respectively. Most of the low-Ni cultivars were found in four subgroups (i.e., HD, LJ, W-J, and YJ; Table 2). In addition, the proportions of high-Ni cultivars in these four subgroups were lower compared to the proportions of low-Ni and mid-Ni cultivars (22.2%, 22.2%, 25%, and 25% of the total cultivars, respectively). Furthermore, most of the cultivars in the TJ subgroup were low-Ni cultivars, and this subgroup also had the lowest geometric mean (18.9 mg·kg −1 ). In contrast, there were no low-Ni cultivars in the XD subgroup, which had the highest geometric mean (24.5 mg·kg −1 ). Note: The low Ni-accumulating (low-Ni) and high Ni-accumulating (high-Ni) cultivars are defined as the 20 lowest and 20 highest shoot Ni-accumulating genotypes among the 72 cultivars, respectively. The other 32 cultivars are defined as middle Ni-accumulating (mid-Ni) cultivars. The subgroup W-J comprised two WJ, five WYJ, and one WXJ cultivars. Data are means ± standard deviation (n = 3).

Variation in Shoot Ni Concentrations among Different Rice Subgroups
We also compared the shoot Ni concentrations of the rice cultivars with those of their parents ( Table 3). The shoot Ni concentrations of HD11 and ZD16 were similar to those of their parents. In contrast, the shoot Ni concentrations of HD8 and YD8 were significantly lower than those of their parents (p < 0.05), whereas the shoot Ni concentration of YJ2 was significantly higher than that of its parent (p < 0.05).

Relationships between Accumulation and Translocation of Si, P, Fe, Mn, and Ni in the Rice Cultivars
Shoot Ni concentrations were positively correlated with Ni, P, and Fe TFs (p < 0.01), but negatively correlated with root Fe concentrations (p < 0.01) (Table 4). Root Ni concentrations were positively correlated with Mn TFs (p < 0.05), but negatively correlated with Ni TFs (p < 0.01), shoot Fe (p < 0.01), and Si concentrations in rice shoots and roots (p < 0.05). The correlation analysis also found a positive relationship between Ni TFs and P TFs, and among the concentrations of Si, Ni, and Fe in shoots (p < 0.01), but a negative relationship between Ni TFs and root Ni concentrations (p < 0.01).  Among the 20 low-Ni cultivars, we observed a positive relationship between shoot Ni concentrations and Ni TFs (p < 0.05; Table S1). However, there was no such relationship among the 20 high-Ni cultivars (Table S2). In these high-Ni cultivars, shoot Ni concentrations were positively correlated with Mn TFs (p < 0.05). Root Ni concentrations were negatively correlated with Ni TFs in both low-Ni and high-Ni cultivars (p < 0.01). In addition, root Ni concentrations were positively correlated with Fe TFs and Mn TFs in low-Ni and high-Ni cultivars, respectively (both, p < 0.05; Tables S1 and S2). The Ni TFs were positively correlated with shoot Si concentrations in both low-Ni and high-Ni cultivars (p < 0.05). Furthermore, the Ni TFs were positively correlated with shoot Fe concentrations in high-Ni cultivars (p < 0.05), but the correlation coefficients were not significant for the low-Ni cultivars.

Relationships among the Accumulation and Translocation of Ni and Other Elements in the Rice Subgroups
Both the first canonical axis and all canonical axes explained a significant amount of the variation (p = 0.004 and p = 0.002, respectively) based on the Monte Carlo permutation test (number of permutations = 499; Figure 2). The first and second axes contributed 29.1% and 9.7% of the total variation, respectively. For Ni accumulation (displayed as shoot Ni and root Ni concentrations) and translocation (displayed as Ni TFs), shoot Si (p = 0.002; F = 14.39) and root Fe (p = 0.008; F = 6.00) concentrations accounted for the variation among the 72 rice cultivars. Shoot Si concentrations explained the greatest proportion of this variation (17%), followed by root Fe concentrations (7%).

Relationships among the Accumulation and Translocation of Ni and Other Elements in the Rice Subgroups
Both the first canonical axis and all canonical axes explained a significant amount of the variation (p = 0.004 and p = 0.002, respectively) based on the Monte Carlo permutation test (number of permutations = 499; Figure 2). The first and second axes contributed 29.1% and 9.7% of the total variation, respectively. For Ni accumulation (displayed as shoot Ni and root Ni concentrations) and translocation (displayed as Ni TFs), shoot Si (p = 0.002; F = 14.39) and root Fe (p = 0.008; F = 6.00) concentrations accounted for the variation among the 72 rice cultivars. Shoot Si concentrations explained the greatest proportion of this variation (17%), followed by root Fe concentrations (7%). An RDA of the relationships among accumulation and translocation of Ni and multi-element concentrations in the 20 lowest and 20 highest Ni-accumulating rice cultivars is shown in Figure 3. The high-Ni rice cultivars mainly clustered in the left upper and lower quadrants, whereas the low-Ni rice cultivars clustered in the lower right and left quadrants. The first and second RDA axes accounted for 21.9% and 17.0% of the total variation, respectively (both, p = 0.002). The shoot Si and P TFs accounted for 12% and 15% of the Ni accumulation and translocation variation, respectively (both, p < 0.01). An RDA of the relationships among accumulation and translocation of Ni and multi-element concentrations in the 20 lowest and 20 highest Ni-accumulating rice cultivars is shown in Figure 3. The high-Ni rice cultivars mainly clustered in the left upper and lower quadrants, whereas the low-Ni rice cultivars clustered in the lower right and left quadrants. The first and second RDA axes accounted The first and second axes accounted for 75.7% and 4.5% of the total variation among the 20 lowest Ni-accumulating rice cultivars, respectively (p = 0.002; Figure S2). The Si TFs, root Fe and shoot Fe concentrations, and P TFs accounted for 40%, 11%, 9%, and 6% of the variation in Ni accumulation and translocation, respectively (all, p < 0.05). In contrast, an RDA analysis of the relationships among Ni accumulation, translocation and multi-element concentrations in the 20 high-Ni rice cultivars is not shown because the Monte Carlo tests of the first canonical axis and all canonical axes were not significant (p = 0.218 and p = 0.23, respectively).

Effects of Ni Exposure on Rice Seedling Growth
We also investigated the effects of Ni exposure on rice growth, under hydroponic conditions. The five lowest and highest shoot Ni accumulating genotypes were selected from among the 72 cultivars, and the effect on biomass (dry weight) of providing 10 μmol·L −1 Ni was analyzed ( Figure S3). No significant differences in shoot ( Figure S3A,B) or root ( Figure S3C,D) biomass were observed in any of the ten cultivars. Similar results were observed for the remaining 62 cultivars (data not shown). These results suggest that exposure to Ni for 3 days did not significantly affect rice seedling growth. The first and second axes accounted for 75.7% and 4.5% of the total variation among the 20 lowest Ni-accumulating rice cultivars, respectively (p = 0.002; Figure S2). The Si TFs, root Fe and shoot Fe concentrations, and P TFs accounted for 40%, 11%, 9%, and 6% of the variation in Ni accumulation and translocation, respectively (all, p < 0.05). In contrast, an RDA analysis of the relationships among Ni accumulation, translocation and multi-element concentrations in the 20 high-Ni rice cultivars is not shown because the Monte Carlo tests of the first canonical axis and all canonical axes were not significant (p = 0.218 and p = 0.23, respectively).

Effects of Ni Exposure on Rice Seedling Growth
We also investigated the effects of Ni exposure on rice growth, under hydroponic conditions. The five lowest and highest shoot Ni accumulating genotypes were selected from among the 72 cultivars, and the effect on biomass (dry weight) of providing 10 µmol·L −1 Ni was analyzed ( Figure S3). No significant differences in shoot ( Figure S3A,B) or root ( Figure S3C,D) biomass were observed in any of the ten cultivars. Similar results were observed for the remaining 62 cultivars (data not shown). These results suggest that exposure to Ni for 3 days did not significantly affect rice seedling growth.

Effects of Ni Exposure on Multi-Element Uptake
To further investigate the effects of Ni exposure on the uptake of nutrients, we analyzed the presences of multiple elements in rice shoots and roots after exposure to 10 µmol·L −1 Ni for 3 days. In general, the uptake of Si, P, Fe, and Mn in rice shoots and roots decreased in response to Ni exposure. In addition, Ni exposure had a much greater effect on multi-element uptake in the shoots of the five highest Ni-accumulating cultivars than in those of the five lowest Ni-accumulating cultivars ( Figures  S4 and S5). Among the low-Ni cultivars, the uptakes of Si and Fe were considerably more affected by Ni exposure than those of P and Mn ( Figure S4). In addition, the Mn uptake in shoots and roots of the five high-Ni cultivars decreased significantly ( Figure S5). Furthermore, in roots exposed to Ni, Fe concentrations were much higher than Mn concentrations in both low-Ni and high-Ni cultivars. Conversely, in rice shoots, Mn concentrations were much higher than Fe concentrations ( Figures S4  and S5).

Discussion
Ni is the major PTE pollutant present in rice grains [9,20], and rice is a major dietary source of Ni for the Chinese population, particularly children aged 2-11 years and Ni-sensitive individuals [20]. However, strategies to decrease Ni accumulation in rice have received little attention until now. Many studies have shown that the concentrations of PTEs (e.g., Cd, As, Pb, and Ni) in grains from various rice subpopulations differ significantly [12,20,44,45]. Therefore, it may be possible to identify cultivars that accumulate significantly lower levels of Ni. In China, approximately 7.4% of the total rice-planting area is located in Jiangsu Province and 90% of the rice grown is of the japonica variety [46]. In this study, we investigated Ni accumulation and translocation in 72 major rice cultivars, as well as multi-element uptake and translocation in different rice tissues. Our results may be used to prevent excessive Ni accumulation in rice grown in Ni-contaminated soil and to improve food safety.
There were significant differences in Ni accumulation and translocation across the 72 major rice cultivars after 3 days of Ni exposure (Figure 1). Shoot Ni concentrations were positively correlated with Ni TFs (p < 0.01), but not with root Ni in all rice cultivars (Table 4). Therefore, differences in shoot Ni concentrations were explained by the different Ni-transport capacities of the rice genotypes rather than the immobilization of Ni in roots in response to Ni exposure. Previous studies have shown that phytochelatins (PCs) are important for PTE detoxification in plants [47,48]. However, in contrast to responses induced by PTEs such as Cd and As (including arsenite and trivalent methylarsonous acid) [48], PC synthesis was not strongly induced by Ni [47,49], suggesting that PC synthesis and subsequent Ni sequestration in roots are less important for Ni detoxification. Similar results were also observed in low-Ni (20 lowest shoot Ni BCFs; Figure S1A) and high-Ni (20 highest shoot Ni BCFs; Figure S1A) cultivars. The geometric means of shoot Ni concentrations (26.5 ± 1.33 mg·kg −1 ) and Ni TFs (0.037 ± 0.006) in high-Ni cultivars were significantly (p < 0.001) greater than those of low-Ni cultivars (18.0 ± 1.99 mg·kg −1 and 0.025 ± 0.007, respectively; Figure 1A,C). However, the root Ni concentrations of low-Ni (792 ± 278 mg·kg −1 ) and high-Ni cultivars (738 ± 142 mg·kg −1 ) were not significantly different ( Figure 1B). These results suggest that the high-Ni cultivars translocate Ni to shoots more effectively than low-Ni cultivars. In addition, although root Ni sequestration is not the most important factor affecting shoot Ni concentrations, root Ni concentrations were negatively correlated with Ni TFs in both low-Ni and high-Ni cultivars (p < 0.01; Tables S1 and S2). These observations suggest that cultivars that sequester greater concentrations of Ni in their roots can decrease Ni translocation to their shoots.
To further identify the major factors that affect the uptake and translocation of Ni in different rice genotypes, RDA was used to analyze the relationships among the accumulation and translocation of Ni and other elements in rice tissues (Figure 2, Figure 3 and Figure S2). The results showed that shoot Si and root Fe concentrations significantly affected Ni accumulation and translocation in the 72 cultivars studied (p < 0.01; Figure 2). Therefore, the uptake and transport of Ni is closely associated with Si and Fe concentrations. Si is an important nutrient for rice growth [25,50] and also protects plants from toxic metals such as Cd, As, Ni, and Zn by enhancing growth and photosynthetic carbon fixation, and suppressing the uptake of toxic metals [31,[51][52][53]. Therefore, rice cultivars with a greater capacity for Si assimilation may also accumulate less Ni in their shoots and roots. In addition, the geometric mean of root Si concentrations was significantly greater in the five low-Ni cultivars than in the five high-Ni cultivars, regardless of whether the plants were exposed to Ni (p < 0.05; Figures S4 and S5). Furthermore, root Fe concentrations were correlated with Ni uptake and transport in rice (p < 0.01; Figure 2 and Figure S2). Interestingly, Fe plaque deposits, which are visible as a reddish-brown coating on the surface of roots, can sequestrate PTEs such as Cd, As, and Ni and reduce their toxicity [34,38,54]. However, we found no evidence of Ni and Fe co-precipitation on the surface of roots, and no significant correlations between root Fe and root Ni concentrations were observed (Table 4, Tables S1, and S2). The excess P in the culture medium (i.e., 5 mg L −1 P for 3 days) might be responsible for the results because previous studies have demonstrated that iron plaque is induced by P starvation [33]. Nonetheless, Fe accumulation in rice roots was correlated with the uptake and transport of Si in the 72 cultivars (p < 0.01; Table 4), and this may have an indirect effect on the uptake and transport of Ni in rice. Similarly, indirect effects of P TFs on the uptake and transport of Ni among 20 low-Ni and high-Ni cultivars were also observed ( Figure 3), because P TFs were correlated with the uptake and transport of Si and Fe but not Ni (Tables S1 and S2).
Our results indicated that the uptake of nutrient elements was inhibited in rice under Ni stress. Figures S4 and S5 show that the concentrations of most nutrient elements in rice shoots and roots decrease after exposure to Ni for 3 days in both low-Ni and high-Ni cultivars. Differences in shoot or root element concentrations in response to Ni exposure were probably due to Ni rather than a difference in rice growth, because no differences were observed in shoot ( Figure S4A,B) or root ( Figure S4C,D) biomass after 72 h of Ni exposure. Similar results were obtained from other rice studies, which showed decreased K, Ca, Mg, Fe, and Mn uptake and distribution [49,55,56]. This may be due to alterations in root membrane permeability in response to excessive exposure to Ni [55]. Furthermore, our data suggest that the high-Ni rice cultivars were much more sensitive to Ni than the low-Ni cultivars, especially with respect to accumulation of Si and Mn in rice shoots ( Figures S4 and S5). This may be crucial for the enhanced shoot Ni accumulation in high-Ni cultivars because these nutrients are essential for rice growth and are also important for alleviating PTEs toxicity [31,57].
We showed that genotype has a significant effect on shoot Ni concentrations among the different rice subgroups (Table 2). In addition, significant differences among cultivars were found within the same subgroup. For example, among the 72 cultivars, those with the lowest (HD5 and LJ6) and highest (HD10 and LJ6: early rice) concentrations of Ni in their shoot were found in the same subgroups (HD and LJ). Genetic differences in the parent plants may be responsible for differences in the PTE accumulation capacities of different rice cultivars [20,58]. However, it is unclear how genotype affects Ni accumulation. Some studies have suggested that progeny plants inherit genes that decrease the accumulation of PTEs from parent plants that also show low levels of PTE accumulation [59,60]. Although cultivar YJ2 exhibited a greater capacity for Ni accumulation than its parent, other cultivars (e.g., HD8 and YD8) exhibited a similar capacity for decreased Ni accumulation to their parents ( Table 3), indicating that it should be possible to breed rice that accumulates lower levels of Ni. The four subgroups with the greatest number of low-Ni cultivars (i.e., HD, LJ, W-J, and YJ) are shown in Table 2 and these may be used to further investigate the genetics underpinning low-Ni accumulation.

Conclusions
This study demonstrates that genotype had a significant effect on Ni accumulation and translocation in a population of 72 rice cultivars. The variation in the shoot Ni concentrations was explained by different capacities for Ni transport in different rice genotypes rather than by the immobilization of Ni in roots exposed to high Ni concentrations. In general, the Ni TFs of high-Ni cultivars were significantly greater than those of low-Ni cultivars. The RDA of the 72 rice genotypes suggested that the uptake of Si and Fe was the major factor affecting the accumulation and translocation of Ni. However, significant differences were also observed between 20 low-Ni and 20 high-Ni rice cultivars. Among the 20 low-Ni cultivars, Si TFs accounted for most of the variation in Ni accumulation and translocation. However, P TFs accounted for most of the variation in Ni uptake and translocation observed between the 20 low-Ni and 20 high-Ni cultivar groups. Mn was less important than Si, Fe, and P in influencing Ni accumulation and translocation in these cultivars. The results of this study may be used to prevent excessive Ni accumulation in rice grown in Ni-contaminated environments. Further studies are needed to investigate how genotypes influence Ni uptake and transport in rice.

Supplementary Materials:
The following are available online at http://www.mdpi.com/1660-4601/16/18/3281/s1, Figure S1: Nickel (Ni) bioconcentration factors in the rice shoots (A) and roots (B). The bioconcentration factor (BCF) of Ni from culture medium to shoots or roots were calculated as: BCF = Cshoots Ni or roots Ni/CNi in culture medium, Figure S2: RDA ordination diagrams of the relationships between accumulation and translocation of Ni and multi-element (Si, P, Fe, and Mn) in 20 lowest Ni-accumulating rice cultivars, Figure S3: The biomass (dry weights) of rice seedlings after exposure to 0 and 10 µmol·L −1 Ni for 3 days under hydroponics condition, Figure S4: The amount of nutrients in rice shoots and roots with or without addition of 10 µmol·L −1 Ni for 3 days under hydroponics condition, Figure S5: The amount of nutrients in rice shoots and roots with or without addition of 10 µmol·L −1 Ni for 3 days under hydroponics condition, Table S1: Pearson correlation coefficients between accumulation and translocation of multi-element (Si, Ni, P, Fe, and Mn) in 20 lowest Ni-accumulating rice cultivars, Table S2: Pearson correlation coefficients between accumulation and translocation of multi-element (Si, Ni, P, Fe, and Mn) in 20 highest Ni-accumulating rice cultivars.

Conflicts of Interest:
The authors declare no conflict of interest.