Boron tolerance and accumulation potential of four salt-tolerant plant species

Boron (B) is an essential element for plants, but excess B is phytotoxic. Since excess B often occurs along with high salinity in the environment, the purposes of the experiments are to screen plants that tolerate both excess B and high salinity for the remediation of B-contaminated saline water or soils. Here we tested the capacities of B tolerance and accumulation of four salt-tolerant plant species, Tripolium pannonicum, Suaeda glauca, Iris wilsonii, and Puccinellia tenuiflora using hydroponic culture systems, and compared their potential for application in phytoremediation. The maximum B supply concentrations for the survival of T. pannonicum, S. glauca, I. wilsonii, and P. tenuiflora are 40, 250, 700, and 300 mg/L, respectively. The maximum B concentrations in the shoot tissue of these plants are 0.45, 2.48, 15.21, and 8.03 mg/g DW, and in the root are 0.23, 0.70, 6.69, and 2.63 mg/g DW, respectively. Our results suggest that S. glauca, I. wilsonii, and P. tenuiflora are capable of tolerating and accumulating high levels of B, and I. wilsonii is a most promising candidate for the remediation of B-contaminated sites. This study will provide evidence in support of our future pilot studies (e.g., constructed wetlands) on the phytoremediation of B-contaminated water and soil.

Boron (B) is an essential element for plant growth, but it can be toxic when present in excess 1 . B toxicity in plants has been widely reported in North America, Southern Australia, the Middle East, Western Asia, North Africa, Malaysia, and China [2][3][4] . Soil B concentrations have been reported to be elevated by anthropogenic activities such as borate mining and processing, glass and ceramic production, as well as the use of B-enriched detergents, herbicides, fertilizers, and irrigation water 2,5-7 . B concentration is usually 0.1-0.5 mg/L in surface freshwaters, but the higher B concentrations are measured in some areas. For example, it has even been documented that B concentration in surface water of Rio Arenales and Loa River (two South American rivers) ranged between 4 and 26 mg/L in areas rich in B-containing soils 8 . High concentrations of boron in water are also very common in China. In an investigation in a B industry area, B concentrations in surface water and groundwater were up to 3.8 mg/L and 140 mg/L, respectively 9 .
Phytoremediation is considered as a green and sustainable technology for the treatments of B-contaminated soil or water, including the restoration of B-mining sites and the purification B-laden effluent 10,11 . When phytoremediation technologies are applied to B-contaminated soils, it is essential to find suitable plant species that can tolerate and/or accumulate high levels of B 12 . Previous studies have recorded some extremely B-tolerant plant species. For example, Gypsophila sphaerocephala, Chrysopogon zizanioides L., and Puccinellia distans were observed to survive up to 227 mg B/kg soil, 750 and 1250 mg B/L hydroponic solution, respectively [13][14][15] . In addition to B tolerance, some plant species have been reported to accumulate B efficiently. For example, Phytolacca americana, Ambrosia trifida L., and Commelina communis were reported to grow in the soil with 480-550 mg B/kg and obtain tissue B concentrations 2-3 folds greater than the soil 16 . High B-accumulation capacity was also recorded in Poplar sp., the leaves of which were observed to accumulate up to 845 mg B/kg dry weight 17 . Apart from terrestrial plants, some aquatic species are reported to be able to accumulate B. For example, a floating aquatic plant, Lemna gibba, was reported to accumulate 930 to 1900 mg B/kg dry weight in the tissue 18 .
In the soils of arid and semi-arid regions, excess B often presents simultaneously with excess salt (mostly sodium chloride), which may aggravate B toxicity in plants or affect B tolerance of the plants 12,19 . To remediate the soils with high B and salinity using plants, therefore, it is necessary to screen plant species that tolerate both B B treatment. B was added to the half-Hoagland's solution in the form of boric acid to obtain desired B concentrations. The pH of the solution was adjusted to 6.5. The B supply concentrations ranged from 0.25 to 40 mg/L for T. pannonicum, from 0.25 to 250 mg/L for S. glauca, from 0.25 to 700 mg/L for I. wilsonii, and from 0.25 to 300 mg/L for P. tenuiflora. These four ranges of B concentration were established according to preliminary tests for each species. In preliminary tests, the threshold of B concentrations for the four species was determined. And preliminary experimental conditions such as light and pH in the process of the experiment are the same as the formal experiment. The lowest concentration of B (0.25 mg/L) was the "control" treatment (assumed sufficient to prevent B deficiency). Each treatment was triplicated. The solutions were replaced once a week throughout the experiment.

Plant analysis.
After treated with B for 2 weeks, the plants were harvested. The plant samples were rinsed with tap water and then separated into shoot and root. The samples were dried at 75 °C for 24 h and then weighed to determine the dry weight (DW). The dried tissue was ground into powder and then digested using nitric acid/ hydrogen peroxide microwave digestion, and the B concentrations in the tissue were quantified using inductively coupled plasma atomic emission spectroscopy (ICP-AES) 15 .
The bioconcentration factor (BCF) of B in plant shoot and root was calculated using the following equation 27 : where C p is the concentration of B in plant tissue (mg/g DW) and C w is the concentration of B in the culture solution (mg/ml). The translocation factor (TF) of B was calculated using the following equation 27 : where C i is the concentration of B in shoot tissue (mg/g DW) and C r is the concentration of B in root tissue (mg/g DW).
Statistics. The data in the figures and tables were reported as the mean ± standard deviation (SD). All the data were analyzed using the program SPSS 17 (IBM Corp., Armonk, NY, USA). One-way ANOVA tests were conducted to determine the influence of B treatment on the dry weight and B concentration in plant tissue of the four species. Prior to ANOVA, the Levene score and significant values of dry weight and B concentration in plant shoot and root were analyzed. The homogeneity of the normal score of variances was verified with Levene's test. All the tests were conducted with a 95% confidence interval (α = 0.05). Duncan's multiple range tests were used to calculate the significant differences between the means of different treatments at the level of p < 0.05. All figures were produced using Origin 8.5 software (OriginLab Corporation, Northampton, MA, USA).

Results
Plant growth. According to the preliminary tests, the seedlings of S. glauca, I. wilsonii, and P. tenuiflora were able to survive at 250, 700, and 300 mg B/L, respectively, while T. pannonicum was unable to survive B concentrations higher than 40 mg/L. With increasing B concentrations, dry weight of the four species increased initially and then decreased progressively (Fig. 1 www.nature.com/scientificreports www.nature.com/scientificreports/ B concentrations in plant tissue. Shoot B concentrations of the four test species increased with increasing B supply concentrations. Although root B concentration in P. tenuiflora at B supply concentration of 100 mg/L was higher than that at 200 mg/L, the data of the two groups have no significant difference. But the general trend is that root B concentrations increased with increasing B supply concentrations. B concentrations of the four tested species in shoot were much higher than those in root (Fig. 2).
At B supply concentrations of 30, 250, 700, and 300 mg/L, shoot B concentrations in T. pannonicum, S. glauca, I. wilsonii, and P. tenuiflora increased to the maximum values, 0.45, 2.48, 15.21, and 8.03 mg/g DW, respectively. Similarly, at the same B supply concentrations, their maximum B concentrations were recorded in root as 0.23, 0.70, 6.69, and 2.63 mg/g DW, respectively. Compared with the other three species, over the whole range of B concentrations for plant survival, T. pannonicum showed the lowest capacity of B accumulation. For example, the maximum accumulation of B in the shoot of I. wilsonii was 15.21 mg/g DW, which was 33.8 times greater than that of T. pannonicum. And the maximum accumulation of B in the root of I. wilsonii was 29.09 times greater than that of T. pannonicum.
When tissue B concentration of the four plant species is plotted against B supply concentration, there is a high degree of correlation between the two parameters (Fig. 3). Because S. glauca, I. wilsonii, and P. tenuiflora were all able to grow over the range of 0.25-250 mg B/L, changes in tissue B concentrations between the three species can be compared. Over this range, shoot B concentrations in I. wilsonii and P. tenuiflora were similar and greater than those in S. glauca. In root, B concentrations of P. tenuiflora were greater than those in I. wilsonii and S. glauca, especially when B supply concentrations exceeded 100 mg/L.  www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
To remediate the soil with excess B and salt, it is important to find candidate plants that are able to tolerate both B and salt and have great capacities of B accumulation. Here we show that three salt-tolerant plant species (S. glauca, I. wilsonii, and P. tenuiflora) have the capacities of B tolerance and accumulation, especially for I. wilsonii (tolerates up to 700 mg B/L and accumulates up to 15.21 mg B/g DW in shoot). Our findings have provided potential candidates for the phytoremediation of high-B soils with high salinities.
As a candidate for the remediation of high-B soils, the plant must be able to survive at high B concentrations. We have observed that T. pannonicum was not able to survive at B concentrations greater than 40 mg/L, while S. glauca, I. wilsonii, and P. tenuiflora were able to survive 250, 700, and 300 mg B/L, respectively. So far, there are no criteria for the assessment of B tolerance of plants. In general, crop plants are more sensitive to B toxicity than other species. For some tolerant species (e.g., carrot, alfalfa, sugar beet, etc.), 2-4 mg B/L in irrigation water may be harmful concentrations 29 . Some species used for the phytoremediation of B-contaminated soils have exhibited greater B tolerance. For example, Puccinellia, a genus of alkali grass, has been demonstrated to tolerate high concentrations of B. Some species of this genus, P. frigid and P. distans, have been recorded to survive over 500 mg/ L 30 and 1250 mg B/L 15 under hydroponic conditions, respectively. In the present study, P. tenuiflora, a species of Puccinellia, was also observed to survive at high B concentrations (up to 300 mg/L).
Besides the survival abilities, the changes in plant biomass also reflect the capacities of plants adapting to B toxicity. Previous studies have demonstrated that B-tolerant species, cultivars, or genotypes exhibit less reduction in biomass or yield than sensitive ones when exposed to B toxicity 31,32 . Our results show that the optimal B concentrations for biomass accumulation were 10 mg/L for T. pannonicum and 50 mg/L for S. glauca, I. wilsonii, and P. tenuiflora, rather than 0.25 mg/L (B concentration of half Hoagland's solution) (Fig. 1). These results are consistent with previous observations in some B-tolerant plant species, which obtained the highest biomass at 2.5 to 50 mg B/L 15,33,34 . The data of the four species in the present study suggest that these species require more B for growth than those provided by the half-Hoagland's solution. In the present study, when B supply concentrations exceeded 50 mg/L, the biomass of S. glauca, I. wilsonii, and P. tenuiflora all decreased. The significant drop of shoot biomass for S. glauca, I. wilsonii, and P. tenuiflora was observed at B concentrations of 200, 150, and 100 mg B/L, respectively, which were quite different with the survival concentrations of B for the three species (250, 700, and 300 mg/L, respectively). These results suggest that the plant species surviving at higher B concentrations may not maintain greater capacities for biomass accumulation at high B concentrations.
A mechanism of B tolerance for plant is to restrict B transfer from root to shoot which restrict the accumulation of B in shoot 13,15,35 . In the present study, tissue B concentrations of S. glauca, I. wilsonii, and P. tenuiflora were observed to increase with increasing B supply. Compared with B supply concentrations of 50 mg/L, there was no significant increase in shoot B concentrations of S. glauca, I. wilsonii, and P. tenuiflora until B supply   www.nature.com/scientificreports www.nature.com/scientificreports/ concentrations reached 150, 150, and 200 mg/L, respectively (Fig. 2). These results indicate that the three species are all able to restrict B accumulation in shoot in response to increasing B supply. Over the range of B supply concentrations of 50 to 200 mg/L, I. wilsonii and P. tenuiflora obtained higher shoot B concentrations than S. glauca, indicating less B was accumulated in S. glauca (Fig. 3). Since B concentrations in the root of S. glauca were also lower than those in the root of I. wilsonii and P. tenuiflora, S. glauca is likely to have the ability to restrict B uptake or B efflux from the root.
As is known, greater bioconcentration factor (BCF) and translocation factor (TF) indicate the higher potential of the plant for element accumulation and greater transfer capacity of an element from root to shoot, respectively 27 . In the present study, over the range of 50-200 mg/L of B supply, the BCF values in the shoot of I. wilsonii (41. 30-26.47) and P. tenuiflora (56. 31-26.44) were much greater than those of S. glauca (18.77-8.78) ( Table 1). These results demonstrate that I. wilsonii and P. tenuiflora have a greater accumulation capacity than S. glauca. Along with high BCF values, high TF values were also observed in I. wilsonii, indicating that, at extremely high B concentrations, I. wilsonii did not restrict B uptake and its translocation from root to shoot. According to previous studies, for the same plant species, the tolerant varieties usually accumulate less B in shoot than sensitive varieties 31 . However, this rule may not be suitable for the comparison between different species. Under high B conditions, I. wilsonii seems to have a different mechanism of B tolerance, which remains to be determined. Nevertheless, these findings have provided evidence that I. wilsonii will be a most promising candidate for the phytoremediation of high-B soil. Although I. wilsonii has been used for removing nitrogen and phosphorus from water as candidate plant of constructed wetland 36 , the removal of B by I. wilsonii has not been investigated in constructed wetlands. In our future research, I. wilsonii will be applied to the treatment of high-B saline water using constructed wetland systems. Plants have been proved to play a direct role in B removal process in constructed wetlands 37,38 . Therefore, I. wilsonii might be a promising candidate for constructed wetlands in saline areas.
Many species of Puccinellia, such as P. distans and P. frigida, are known for their extremely high tolerance to B, and they also have been widely considered as potential candidates for the phytoremediation of B-contaminated sites 6,30,34,39,40 . In the present work, P. tenuiflora also has been proved to be tolerant to B and capable of B accumulation, confirming that Puccinellia is a B-tolerant genus. The other three plants, S. glauca, I. wilsonii, and P. tenuiflora, have not yet been studied for B tolerance and B enrichment. Since many species of Puccinellia are also tolerant to salt, more species of this genus should be screened for the remediation of soils with high salinities. In our laboratory, we have confirmed that the other three plants S. glauca, I. wilsonii, and P. tenuiflora are also more salt-tolerant species. Although most species of Puccinellia have small biomass, which is a disadvantage for B accumulation, their high growth rate may allow them as an initial cover for the restoration of B-contaminated soil.
The four species we used are all seedlings which are more sensitive to B than adult individuals. Although two weeks test period is not so long, it is enough to evaluate B toxicity in plant and to screen the species with high B tolerance and accumulation. It is important to note that our data are obtained under room conditions with plants cultivated in hydroponic systems. Therefore, it is hard to identify these three species as hyperaccumulators of B. But we believe that the present study will provide the scientific theoretical basis for controlling B-contaminated soils and water by phytoremediation technology in reality. Absolutely, more research and further verification in practical applications were required. In our future studies, plant growth rate will be determined to know whether the candidate plant species can absorb B within a reasonable time and whether the accumulation is decreased by extreme growth reduction 34 . In addition, because B toxicity and salt stress often occur simultaneously, the interaction of B and salt should be considered. Despite the four test species are all salt-tolerant, it should be emphasized that they should be evaluated under the combined stresses of B and salt before being applied in the phytoremediation of B-contaminated soils.

Conclusions
Among the four tested plant species, I. wilsonii is the most tolerant species to B toxicity and has the highest B accumulation capacity. S. glauca and P. tenuiflora show similar B tolerance, but P. tenuiflora has a greater B accumulation capacity than S. glauca. T. pannonicum has the lowest tolerance to B and the lowest accumulation capacity. Our results suggest that S. glauca, I. wilsonii, and P. tenuiflora are suitable for the phytoremediation of high-B soil with high salinities. We propose that I. wilsonii as a most promising candidate for B-phytoremediation because of its great capacity for B tolerance and accumulation.