Soil-applied selenite increases selenium and reduces cadmium in roots of Moringa oleifera

Deficiency of selenium (Se) will lead to malnutrition and decreased immune function of the body. There is a common phenomenon of Se deficiency in foods. In this study, different concentrations of sodium selenite (Na2SeO3) were applied to Moringa oleifera grownin soil. The purpose was to explore the feasibility of Se biofortification of M. oleifera root. The effect of exogenous Se on the accumulation of Se and cadmium (Cd) in the roots of M. oleifera was studied by inductively coupled plasma mass spectrometry, and the mechanism of exogenous Se on the accumulation of Se and Cd in the roots was studied by Fourier transform infrared spectroscopy (FTIR) combined with principal component analysis and partial least squares regression analysis. The results showed that Na2SeO3 significantly affected the accumulation of Se and Cd in the roots (p < 0.05). The increase in Se was highest when Na2SeO3 was around 4.0 mg/kg, which increased by 315% compared with the control. The decrease in Cd was the lowest when Na2SeO3 was around 2.0 mg/kg, which decreased by 80% compared with the control. The results of FTIR analysis showed that Na2SeO3 treatment changed the carboxylate, phosphate radical, hemicellulose and protein in roots of M. oleifera, while the increase of Se was related to hemicellulose, protein, polysaccharide and lignin, and the decrease of Cd was related to hemicellulose and protein. The results showed that exogenous Se increased the accumulation of Se and inhibited the absorption of Cd. Therefore, the roots of M. oleifera can be used in Se biofortified products.


Results
Cd and Se accumulation in M. oleifera roots. ICP-MS results ( Fig. 1) showed that the application of all concentration of Na 2 SeO 3 significantly reduced the concentration of Cd (p < 0.05) whereas significantly enhanced concentration of Se in roots of M. oleifera (p < 0.05) than control sample. When Na 2 SeO 3 was added to the soil less than 4.0 mg/kg, the concentration of Se accumulation in the root increased with the increase of the added amount. The concentration of Se decreases significantly (p < 0.05) while added above 4.0 mg/kg. Compared with the control group, Se increased 27-315% and Cd decreased 30-80% in the roots of M. oleifera with treatment of Na 2 SeO 3 in soil.
FTIR spectroscopy analysis. FTIR spectra of M. oleifera roots are shown in Fig. 2

. O-H or N-H stretch-
ing vibration was at 3390 cm −1 . The peaks around 2927 and 2884 cm −1 were assigned to methyl and methylene stretching vibrations 25 . The absorption peak of the vibration of the saturated ester groups compounds appeared around 1740 cm −126 and the peak at 1643 cm −1 which was ascribed to the C = O stretching vibrations of carboxylic anions, hemicelluloses or amide groups in proteins 27 . The peak around 1517 cm −1 was attributed to lignin 28 . Carboxylate vibration was at 1421 cm −129 . The peaks around 1241 and 859 cm −1 were due to the S-O stretching vibrations 30 . The peaks at 1160, 1079 and 1020 cm −1 suggested the presence of hemicelluloses 28 . The peak around 929 cm −1 was ascribed to the vibration of β-glycoside 23 and the peak at 765 cm −1 due to the vibration of α-glycoside 31 . The peak of S-O in the control sample was at 1238 cm −1 . After the addition of exogenous Se, the peaks hifted to 1242 cm −1 . Therefore, the addition of exogenous Se may affect S in roots of M. oleifera.
PCA of spectra. PCA was used to reduce the dimensionality of the spectral data to understand the possible sources of the explained differences. From the PCA score plot (Fig. 3a), it can be seen that the FTIR spectra of M. oleifera root cultivated under different conditions could be accurately separated.
The loadings plot was examined to establish possible sources of variance within the spectra, and several areas of high variance were identified. Therefore, the loading plot of PCA can be used to explain the changes in the composition of M. oleifera roots cultivated under different conditions. According to PC 1 loadings plot (Fig. 3b), there are strong positive weighted peaks around 3604, 3120 and 2884 cm −1 , which are related to the stretching vibration of carboxyl and methyl groups. A positively weighted peak at 1681 cm −1 , was due to hemicelluloses and    www.nature.com/scientificreports/ amide 27 . The positively weighted peaks around 1421 and 1347 cm −1 were related to carboxylate radical 29 , and the positively weighted peak at 1176 cm −1 due to hemicelluloses 28 . Therefore, the loadings plot of PC 1 is related to the changes of carboxylate and hemicellulose in the roots, and proteins may also be involved. The load of PC 2 Fig. 3c has a strong positive weighted peak around 3430 cm −1 , which is related to O-H or N-H stretching vibration. The negatively weighted peak at 1245 cm −1 , was due to amide III, and the negative weighted peaks around 1132 and 960 cm −1 were related to the PO 4 3− stretching vibration 31,32 . Therefore, the loadings plot of PC 2 is related to the changes of PO 4 3− and proteins in roots of M. oleifera. The results of PCA showed that the main sources of FTIR difference in M. oleifera roots were carboxylate, PO 4 3− , hemicellulose and proteins.
PLSR analysis. In order to understand the effect of related components in the root of M. oleifera on the accumulation of Cd and Se, the PLSR analysis of Cd and Se was established by infrared spectra (4000 ~ 400 cm −1 ), as shown in Fig. 4. The determination coefficients (R c 2 ) for the PLSR analysis of Cd and Se were 0.9181 and 0.7479 respectively. Figure 5a shows the loadings plot of the wavenumber weight in the PLSR analysis of Cd. There are positive weighted peak around 1660 cm −1 and negative weighted peak around 1616 cm −1 , the two peaks are related to amide I in the protein. The positive weighted peak around 1542 cm −1 is related to amide II. There are positive weighted peaks around 1176, 1108 and 1062 cm −1 which are related to hemicellulose. Therefore, the

Discussion
In this study, the cultivation of M. oleifera root by adding Na 2 SeO 3 significantly increased the Se concentration.
Previous studies have shown that Moringa has the exceptional ability to extract Se from the soil and accumulate it in the leaves, which is significantly higher than other plants 34,35 , and it probably also applies to roots. Plants absorb Se from the environment mainly via the roots. Selenite absorbed into the roots will be transported to various parts of the plant, but the selenite retained in the roots was higher than those transported to other parts, so the Se content in the roots was generally higher 36 . Plant plasma membrane can maintain normal intracellular homeostasis and nutrition, and participate in the perception and response to various environmental stimuli, while the protein of plant plasma membrane plays an important role in response to the external environment. The results of FTIR and PCA showed that when different concentrations of Na 2 SeO 3 were used to cultivate M. oleifera, the PO 4 3− and protein in roots of M. oleifera were changed. This is due to the perception of plant roots to the external environment, mainly by reversible phosphorylation of the sensing protein, and using the membrane related G-protein, polyphosphoinositide signal pathway and other signal pathways for transmission 37 . Some studies have also found that the active absorption of Se is regulated by phosphate transporters 38 . Se can bind to sulfhydryl groups in certain proteins and inhibit Cd from entering cells 39 . Therefore, adding different concentrations of exogenous Se can change the growth environment of roots of M. oleifera. By regulating the abundance of proteins related tochannels, transporters and membrane vesicles transport, the root cells can promote or inhibit the absorption and transport of substances.
The absorption and accumulation of elements in plants are influenced by external environment (such as pH, humidity and temperature of soil), the regulation of transcription factors and the expression of related genes. bHLH transcription factors and jasmonic which are widely found in plants, play an important role in plant growth and secondary metabolite synthesis 40 . The bHLH transcription factors can increase the tolerance of Previous studies have found that exogenous Se can up regulate the gene expression of hormone synthetase in plants, promote the synthesis of hormones such as jasmonate or methyl jasmonate, thereby inducing plants to absorb Se externally and increase the amount of Se 43 . Na 2 SeO 3 enabled strawberry plants to improve the activity of antioxidant enzyme glutathione reductase and the activity of l-galactono-1, 4-lactone dehydrogenase responsible for the biosynthesis of ascorbate, to fight against cadmium stress 10 . When Na 2 SeO 3 was added to M. oleifera, the carboxyl group, hemicellulose and lignin in Moringa root changed, and the concentration of Cd in the root was significantly reduced (p < 0.05). According to the analysis of PLSR, the concentration of Cd was related to hemicellulose, which was consistent with the results of Guo et al. 28 . Root Cd mainly exists in the polysaccharides of the cell wall, which is attributed to the binding effect of the carboxyl and carboxylate groups in hemicellulose on Cd ions 44 . Therefore, exogenous Se caused the changes of hemicellulose and protein in the roots of M. oleifera, and effectively reduce the absorption of Cd. It has been reported that exogenous Na 2 SeO 3 changes the number of cells per unit area of xylem in the root 45 , and increases the content of pectin and hemicellulose in the cell wall of the root 46 , the results of these studies were consistent with the results of our study. Exogenous Se changed the polysaccharides in roots, and affected the concentration of Cd in the roots.

Materials and methods
Cultivation of M. oleifera and experimental design. M. oleifera cultivation experiments were conducted from March 2017 to July 2018 in Xishuangbanna, Yunnan, China (101°25′N, 21°41′E). The soil for cultivation was taken from the acidic red soil locally in Xishuangbanna and collected from the 0 to 20 cm soil layer. The plant residue were removed from the soil and passed through a 10 mesh sieve after air-drying. Each pot used for cultivation was filled with 5 kg of soil. The basic properties of soil were shown in Table 1.
M. oleifera seed was obtained from Yunnan Manze Biotechnology Co., Ltd., Chian. Since selenite tends to accumulate more selenium in the roots of plants, this study applied selenite to cultivate M. oleifera. Eight experimental groups with Na 2 SeO 3 and one blank control group were set up, the concentrations of Na 2 SeO 3 in soil were 0, 0.1, 0.3, 0.5, 1.0, 1.5, 2.0, 4.0 and 6.0 mg/kg, respectively. Each treatment had three parallel experiments, 27 pots in total. Two seeds were planted in each pot and growing under natural light and temperature conditions. After 16 months, the plants were harvested and divided into leaves, stems and roots. The roots were washed with tap water and deionized water and dried to constant weight in a drying oven (50 °C), and then digested and analyzed.

Measurement of total Se and Cd in dry matter.
Three parallel experiments were carried out for each M. oleifera, and three blank groups of samples were set. 10 mL 69% HNO 3 and 1 mL 70% HClO 4 were used for digestion of samples (0.50 g). For digestion, a high-performance graphite furnace digestion system (DigiBlock ED54-iTouch, China) equipped with advanced composite PTFE vessels was used. The decomposition of organic matter was carried out at atmospheric pressure. When the rest digested solutions were clear with a volume of about 3 mL, 1 ml of 2% HNO 3 and deionized water was used to adjust the samples to constant volume (25 mL). The total Cd and Se concentration was measured by ICP-MS (Elan DRC-e, Perkin Elmer, USA). In order to validate the methods, the standard reference materials soybean (GBW10013, China) were used as reference materials to assess the experimental procedures. The standard values of Cd and Se in reference materials were 0.011 and 0.022 mg/kg, respectively. The Cd and Se values of the reference materials measured by ICP-MS were 0.012 ± 0.003 and 0.024 ± 0.004 mg/kg (n = 3), respectively. Therefore, recoveries of Cd and Se in samples ranged from 92 to 109% and 95 ~ 112% respectively.
Detection and analysis methods of FTIR spectroscopy. Infrared spectra were acquired using FTIR Spectroscopy (Frontier, Perkin Elmer, USA) equipped with a DTGS detector. All spectra were recorded in the range of 4000-400 cm −1 with a 4 cm −1 resolution and 16 scans. All samples were measured by KBr pellet method. The interferences of H 2 O and CO 2 as well as KBr background were subtracted automatically when scanning. Quadruplicate spectra were collected for each sample. The average spectra were used for PCA and PLS regression analysis performed by using The Unscrambler X 10.4 software.

Conclusion
In conclusion, exogenous Se significantly increased the content of Se in roots of M. oleifera, while significantly reduced the content of Cd. As our present study was only utilized the roots of M. oleifera, the effects of exogenous Se on the leaves of M. oleifera may be different. Therefore, it is necessary to further study the effect of exogenous Se on Cd and Se accumulation in M. oleifera. Even so, the results of this study still provide information for the roots of M. oleifera as a Se-enriched product.