TcOPT3, a Member of Oligopeptide Transporters from the Hyperaccumulator Thlaspi caerulescens, Is a Novel Fe/Zn/Cd/Cu Transporter

Background Thlaspi caerulescens is a natural selected heavy metal hyperaccumulator that can not only tolerate but also accumulate extremely high levels of heavy metals in the shoots. Thus, to identify the transportors involved in metal long-distance transportation is very important for understanding the mechanism of heavy metal accumulation in this hyperaccumulator. Methodology/Principal Findings We cloned and characterized a novel gene TcOPT3 of OPT family from T. caerulescens. TcOPT3 was pronouncedly expressed in aerial parts, including stem and leaf. Moreover, in situ hybridization analyses showed that TcOPT3 expressed in the plant vascular systems, especially in the pericycle cells that may be involved in the long-distance transportation. The expression of TcOPT3 was highly induced by iron (Fe) and zinc (Zn) deficiency, especially in the stem and leaf. Sub-cellular localization showed that TcOPT3 was a plasma membrane-localized protein. Furthermore, heterogonous expression of TcOPT3 by mutant yeast (Saccharomyces cerevisiae) complementation experiments demonstrated that TcOPT3 could transport Fe2+ and Zn2+. Moreover, expression of TcOPT3 in yeast increased metal (Fe, Zn, Cu and Cd) accumulation and resulted in an increased sensitivity to cadmium (Cd) and copper (Cu). Conclusions Our data demonstrated that TcOPT3 might encode an Fe/Zn/Cd/Cu influx transporter with broad-substrate. This is the first report showing that TcOPT3 may be involved in metal long-distance transportation and contribute to the heavy metal hyperaccumulation.


Introduction
Metal hyperaccumulators can not only tolerate high concentration of heavy metals in the soils but also take them up actively and accumulate and distribute them to appropriate tissues at extreme high levels, thus make them very attractive for the remediation of heavy metal polluted soils [1]. A large amount of different metal hyperaccumulators have been recognized in different regions all over the world [2], among them, Thlaspi caerulescens, a Cd/Zn/Ni hyperaccumulator, has been used as a model plant to study the physiological and molecular mechanisms of heavy metal hyperaccumulation [3,4].
The efficient metal loading and unloading ability in the vascular systems was regarded as the key step of hyperaccumulating process for hyperaccumulators [5]. Nonetheless, knowledge about the mechanisms and proteins involved in transporting heavy metals from soil via the roots and stems into their storage sites is much limited. Transporter families, including ZIP (ZRT/IRT like Protein), Nramp (Natural Resistance and Macrophage Protein), MTP (Metal Tolerance Protein), HMA (Heavy Metal transporting P-type ATPase), ABC-type (ATP-binding cassette), COPT family CTR/COPT (high-affinity Cu transporters) as well as YSL (Yellow strip1-Like transporters) families have been porved to involve in transit metal movements, they either act at the plasma membrane (PM) to transport metals into cytoplasm or redistribute metals from intracellular compartments into the cytoplasm [6,7,8,9]. Besides the above transporters families, novel transporters related to metal homeostasis are continuously under discovery, oligopeptide transporter (OPT) family is among those being focused recently [10,11,12]. OPT family is one of three families responsible for peptide transport; the other two are the ATPbinding cassette (ABC-type) transporters superfamily and the peptide transporter (PTR) superfamily [10]. Peptides, existing abundantly in xylem and phloem saps, are important for plant growth, development and signaling. Transport of peptides is taken as a more efficient means of nitrogen distribution than transport of individual amino acids, implying the important role of peptide transporters in long-distance transport of nutrients [10]. Besides the peptides, inorganic nutrients and transit metals have been reported as the substrates of peptide transporters in the recent studies. For intances, the ABC transporters AtMRP3 [13], AtATM3 [14] and AtPDR8 [15] are proposed to involve in heavy metal (Cd, Pb) resistance and movement.
The first OPT family is identified in the pathogenic yeast Candida albicans [16] and subsequently in Schizosaccharomyces pombe [17] and Saccharomyces cerevisiae [18]. Later, it is also found in bacteria, plants, and archaea [11], but still not in animals. OPT family is grouped into two distinct subfamilies, the yellow stripe (YS) clade and the peptide transport (PT) clade [11,12]. The function of YS1-like (YSL) transporter on transportation of metalcomplex has been documented well in planta. For example, YSLs mediate long-distance transport of specific Fe species [19,20]. ZmYS1 cloned from Zea mays is the founding member of YSL family, which functions in root Fe-phytosiderophore (Fe-PS) uptake from the soil [21]. HvYS1 encoding a YSL transporter is a specific transporter for iron (III)-PS in barley roots [22]. Since non-Poaceae species do not synthesize phytosiderophores, the substrates of YSL transporters in dicotyledonous plant are suggested to be Fe(II)/Fe(III)-nicotianamine (Fe-NA) functioning in the long-distance transporting system [23,24,25]. NA is a chelator for several micronutrient metals and involves in the movement of micronutrients and heavy metals throughout the plant [26]. Three YSL genes cloned from T. caerulescens express at higher levels as compared with their Arabidopsis thaliana homologs with distinct patterns. TcYSL3 was further confirmed as a Fe/Ni-NA influx transporter [19]. Functional analysis in yeast demonstrated that TcYSL3 can transport both Ni-NA and Fe-NA complexes into yeast, indicating that TcYSL3 may be involved in the long-distance translocation of Ni in T. caerulescens.
In Arabidopsis, nine putative OPT orthologues (AtOPT1 to AtOPT9) have been identified [27,28]. OPT transporters predominantly recognize tetrapeptides and pentapeptides, thus they are also called peptide transporters, but this is challenged recently as AtOPTs have been proved to play a role in metal homeostasis and movements. Transcript of AtOPT2 is induced in root by iron and zinc deficiency, and that of AtOPT3 by iron, copper and manganese deficiency [28,29]. AtOPT3 is further proved to involve in the movement of iron to the developing seeds [30]. Moreover, both AtOPT6 and AtOPT7 can transport cadmium or cadmium-glutathione conjugates when heterologously expressed in yeast [31,32]. All these studies indicate that OPT family might play important roles in metal uptake and transportation.
In the present study, we isolated a member of the OPT family from a metal hyperaccumulator, Thlaspi caerulescens, named TcOPT3. We characterized its expression in different tissues, and conducted heterologous expression in yeast to investigate the role of TcOPT3 in metal uptake. We demonstrated that TcOPT3 was involved in Fe/Zn/Cd/Cu transportation and it should be a very important component in the metal long-distance transport system, and contribute to heavy metal hyperaccumulation in the shoots.

Identification of TcOPT3 Gene
Degenerated primers were designed from the most conserved regions according to the AtOPT3, BjGT1 and ZmGT mRNA sequence. We isolated the full-length cDNA from Thlaspi caerulescens by RACE PCR, and designated it as TcOPT3 (Genebank accession no. HQ69984). The coding region of TcOPT3 was 2211 bp, and a corresponding 737 amino-acid sequence was predicted.
TcOPT3 exhibited 79% identity with its homologous AtOPT3, and the deduced protein TcOPT3 showed 95% identity with AtOPT3 ( Fig. 1). It contained 14 putative transmembrane domains (TM I-TM XIV, Fig. 1) and two highly conserved motifs (NPG motif and KIPPR motif, Fig. 1) of OPT family, thus TcOPT3 is likely to be localized to membrane and owns the structure of a transporter protein.
From the rooted phylogenetic tree to compare TcOPTs with OPTs and YSLs in A. thaliana and the related species. We can see that TcOPT3 belongs to the PT clade, moreover, it clusters together in one branch with the OPTs from T.caerulescens, B. juncea, A. lyrata and A. thaliana and is most closely related to BjGT1 (Fig. 2).

Tissue-and Organ-Specific Analysis of TcOPT3
The expression of TcOPT3 was investigated by real-time RT-PCR, where ubiquitin-conjugating enzyme gene (UBQ10) was used as a control. The transcript level of TcOPT3 mRNA was 2fold more in the leaf and stem tissues than that in the root (Fig. 3).
To further identify the spatiotemporal expression of TcOPT3 in vivo, we performed in situ hybridization on the longitudinal and transverse sections of root, stem and leaf using DIG-labeled TcOPT3 antisense and sense RNA as probes (Fig. 4). Senseoriented probe was used as the negative control ( Fig. 4 B, D, F, H, J). In roots, more intense signals were detected in the pericycle and the vascular tissues, including xylem and phloem (Fig. 4A). This pattern of localization was further confirmed in the cross-section (Fig. 4C). In the stem, TcOPT3 transcripts were strong in almost all the central cylinder cells (Fig. 4E, I). Additionally, in the leaf, extremely strong signals were detected in the vein of the mesophyll (Fig. 4G). All these suggest that as a transporter protein, TcOPT3 most probably plays a role in the long-distance transporting system.

TcOPT3 Expression in Response to Metal Deficiency
To see whether the gene responses to metal deficiency, we measured the expression of TcOPT3 in root, stem and leaf tissues (Fig. 5) and found it was also highly induced by Fe and Zn deficiency, especially in the stem and leaf. But the expression of TcOPT3 responded to Fe and Zn deficiency faster in the root as the enhancement was detected in 1-day treatment, while 2-day in the aerial parts.

Sub-Cellular Localization of the TcOPT3 Protein
To investigate the sub-cellular localization, TcOPT3 fused with a green fluorescent protein (GFP) under the control of the cauliflower mosaic virus 35 S promoter and pm-rk (Plasma membrane marker) were co-expressed in the onion epidermis cells. The confocal microscopy observation showed that the green fluorescence and red pm-rk were both confined to the plasma membrane ( Fig. 6) and can be merged perfectly, indicating that TcOPT3 encoded a plasma membrane-localized protein.

Functional Characterization of TcOPT3 by Heterologous Expression in Yeast
To study the function of TcOPT3 in Fe and Zn transport, we tested whether expression of a TcOPT3 cDNA could restore the growth of yeast double mutants fet3fet4 (strain DEY1453) and zrt1zrt2 (strain ZHY3), which can not grow on the Fe-and Zn-limited medium respectively [33,34,35]. As expected, expression of TcOPT3 complemented the growth of mutants fet3fet4 and zrt1zrt2 (Fig. 7) when both metal sources in the medium were provided at a low concentration (10 mM Fe 2+ or 50 mM Zn 2+ ), suggesting that TcOPT3 can transport both Fe 2+ and Zn 2+ .

TcOPT3 Contributes to Fe/Zn/Cu/Cd Accumulation
The role of TcOPT3 in the metal accumulation was further investigated by expressing TcOPT3 and empty vector in the wildtype yeast line (stain DY1455) cultured in the solutions containing 50 mM FeCl 3 , 50 mM ZnSO 4 , 50 mM CuSO 4 or 20 mM CdSO 4 , respectively. Compared with those empty vector-expressing strains, the TcOPT3-expressing trains grew worse (Fig. 8), meanwhile the corresponding contents of these metal ions were significantly higher as well, suggesting that TcOPT3 can transport Fe/Zn/Cd/Cu into yeast cells (Fig. 9).

Discussion
The Oligopeptide transporters (OPTs) were initially characterized as small-peptide transporters. However, this has been challenged by their new functions in metal trafficking recently. The first report is from Wintz et al who found that heterologous expression of AtOPT3 in yeast can transport Cu, Mn and Fe [28]. Later, AtOPT6 and AtOPT7 were also proved to transport Cd or Cd-glutathione chelate in yeast [31]. Recently, AtOPT3 was further demonstrated to be involved in Fe homeostasis [30]. It was showed that heterologously expressed AtOPT3 increased Cd sensitivity of S. cerevisiae opt2 mutants and contributed to Cd accumulation [36]. All the above reports indicate the potential role of OPTs in transit metal transportantion. Thlaspi caerulescens, a Cd/ Zn/Ni hyperaccumulator, ecotype of Ganges, from southern France, can accumulate 10 000 mg Cd kg 21 at shoot dry weight base [3,37]. In this study, we cloned and elucidated the function of a novel member of OPT family, TcOPT3 from T. caerulescens.

Role of TcOPT3 in Metal Transport
AtOPT3 has been proved to play a role in Fe homeostasis in A. thaliana [30]. Here, we also found that heterologous expression of TcOPT3 in Saccharomyces cerevisiae rescued the growth of Fedepleted mutant fet3fet4 (Fig. 7), suggesting that TcOPT3 can transport Fe too. As T. caerulescens is also a Zn hyperaccumulator, we tested the possible role of TcOPT3 in transporting Zn. As expected, TcOPT3 restored the growth of Zn-uptake-defective mutant zrt1zrt2. Furthermore, heterologous expression of TcOPT3 negatively affected the growth of S. cerevisiae when treated with elevated Fe, Zn, Cd, or Cu (Fig. 8) as more Fe, Zn, Cd or Cu was accumulated in the transformed yeast ( Fig. 9), suggesting a broader substrates specificity for TcOPT3. The broad substrate specificity of cytoplasmic transition metal importers may ensure their universal potential to fulfill different developmental stages, without perpetuating the consequences of limited metal transporter specificity throughout the plant [7], while the specificity of transition metal export from cytoplasm may serve to establish specificity through the differential storage of transition metals in specific tissues of cell types. Therefore, the specificity of the efflux transporters appears to be more pounced than that of the influx transporters. For example, the P 1B -type ATPases HMA3 from T.caerulescens showed high specificity for Cd, that serves to efflux Cd into vacoule [38]. As T. caerulescens can also accumulate Ni, we tested the possibility of transporting nickel (Ni), and found that altough the growth was negatively affected by Ni addtion (Fig. S1), the content in the yeast did not change much (Fig. S2), suggeting that TcOPT3 does not transport Ni, and there must be other transporters responsible for Ni hyperaccumulation, such as TcYSL3 [19]. Futhermore, we still found that TcOPT3 did not transport Pb into yeast too (Fig. S1, S2).
Roles of membrane transporters that are responsible for hyperaccumulation include uptake, efflux, translocation and sequestration of transit metals. Metal transporters herein identified include ZIP (ZRT/IRT like Protein), Nramp (Natural Resistance and Macrophage Protein), MTP (Metal Resistance Protein), and HMA (Heavy metal/CPX-type ATPases) families in A.thaliana and hyeperaccmulators such as T. caerulescens and A. halleri [4,38,39,40,41]. Transporters of the HMA family and the MTP family that are involved in metal efflux from the cytoplasm, either by movement across the plasma membrane (PM) or into organelles, whereas metal uptake transporters include the NRAMP family and the ZIP families that are transporters either act at the PM to move metals into the cytoplasm or remobilize metals from intracellular compartments into the cytoplasm [6]. In this study, heterologous expression of TcOPT3 in S. cerevisiae increased the sensitivity of yeast by accumulating more metals, indicating that TcOPT3 function as an uptake transporter for heavy metals (Fig. 8, 9).

Potential Role of TcOPT3 in Plant Long-distance Metal Transport and Hyperaccumulation
In this study, qRT-PCR results showed that TcOPT3 was preferentially expressed in the aerial parts (stem and leaf) than roots both under normal condition or Fe/Zn deficient conditions (Fig. 3, 5). While in Arabidopsis, AtOPT3 expressed stronger in flower, leaf and root, but relatively lower in the stem [42]. It may be due to the different growth stage tested, but most probably, the different expression patterns between AtOPT3 and TcOPT3 may suggest their different roles in trafficking heavy metals within plants. It was reported that reduced expression of AtOPT3 in the Arabidopsis mutant opt3-2 resulted in the accumulation of very high levels of Fe in tissues except seeds [30]. Recently, it was showed that heterologously expressed AtOPT3 increased Cd sensitivity of S. cerevisiae opt2 mutants and contributed to Cd accumulation [36]. Consistent with the role of OPT3 in Cd detoxification, the early-stage seedlings of A. thaliana opt3-3 knockdown allele were extremely sensitive to Cd. In contrast, leaves of hydroponically-grown opt3-3 mature plants were more tolerant to Cd compared to the wild type [36]. We also found that heterologously expressed TcOPT3 increased heavy metal (Cu/Cd) sensitivity of S. cerevisiae and contributed to metal accumulation, suggesting a similar role of TcOPT3 in heavy metal trafficking in plant. However, little evidence from overexpression OPTs in metal distribution is reported to date. Given the different expression patterns between TcOPT3 and AtOPT3, it is interesting to investigate the potential role of TcOPT3 in hyperaccmulation in T. caerulescens. The tissue specific overexperssion of TcOPT3 is highly desirable in order to verify its potential role in creating metal hyperaccumulators for the purpose of phytoremediation of heavy metal polluted environments in future.  As proposed by Milner and Kochian [4], Zn hyperaccumulation of T.caerulescens appears to include at least five physiological events: (1) Increased Zn 2+ influx across the root-cell PM; (2) Reduced Zn sequestration in the root-cell vacuole; (3) Increased Zn transport into the xylem and via the xylem to the shoot; (4) Increased Zn 2+ influx into leaf mesophyll cells; and (5) Zn and Cd stored primarily in leaf epidermal cells. In situ hybridization results showed that, TcOPT3 was highly expression in vascular cells both in roots and shoots (Fig. 4). This expression pattern is similar to the previous report on the expression of AtOPT3 in A. thaliana, which is also highly expressed in the vascular tissues both in lightgrown seedlings and adult plants [42]. As efficient translocation of metals from root to shoot is one of the most important hallmarks of hyperaccumulators. Thus, efficient transporters expressed in loading and unloading tissues are fundamental for hyperaccumulation. For example, HMA4 is characterized metal transporter primarily localized in the root stele. It plays a critical role in loading heavy metals into xylem for long-distance transportation from root to shoot not only in T.caerulescens but also in another hyperaccumulating plant A.halleri [43,44,45]. TcHMA3, another member of HMA family was proved to be a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens [38]. Uptake transporters responsible for hyperaccumulation, TcIRT1, TcIRT2, TcZNT1 and TcZNT5 of ZIP families in T.caerulescens, for instance, were expressed only in roots but not in leaves [46]. The yellow-stripe 1-like (YSL) subfamily is included in the OPT superfamily, some of which was proved to be involved in loading and unloading of nicotianamine-metal chelates from the vascular tissues. TcYSLs of YSL transporters from T. caerulescens, especially for TcYSL3 and TcYSL7, were expressed in xylem parenchyma and phloem [19]. Furthermore, TcYSL3 was shown to transport Ni-NA chelates. Here we demonstrated that TcOPT3 is a plasma membrane-localized protein, and can transport metals into yeast cells (Fig. 6, 8). TcOPT3 was expressed primarily in shoots, especially under nutrient deficiency, indicating their specificity in metal uptake and transport in shoots (Fig. 3, 5). As it is highly and constructively expressed in vascular system, including xylem, phloem and vein, it is reasonable to assume that it plays important roles in unloading of nutrients and heavy metals in those tissues, thus consists of an important component in the long-distance transportation system. However, it needs further investigation.
In this study, ecotype Ganges instead of ecotype Prayon was studied. Considered that heavy metal hyperaccumulation varies greatly among different ecotypes, it is interesting to further confirm the expression and functional analysis of TcOPT3 in two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens. We have analyzed the amino acids sequence and expression of TcOPT3-p preliminarily; however, there is no significant difference between TcOPT3-g and TcOPT3-p expression pattern. Further studies need to be conducted in the future.
In conclusion, we demonstrated that TcOPT3 in a metal hyperaccumulator, Thlaspi caerulescens, was an Fe/Zn/Cu/Cd influx transporter with non-specificity substrate. This is the first report showing that TcOPTs gene may be involved in metal longdistance transport systems that contribute to heavy metal hyperaccumulation.

Gene and cDNA Cloning and Sequencing
Total RNA was extracted from T. caerulescens roots or leaves by the Trizol reagent (Invitrogen, USA) and purified by DNase I (RNase Free) Kit (TaKaRa, China). First strand cDNA of T. caerulescens synthesis was performed using M-MLV reverse transcriptase (TaKaRa, China) and PCR-amplified with degenerated primers designed from conserved AtOPT3 sequence. One fragment was cloned into the pMD19-T Vector (TaKaRa, China), sequenced and identified as AtOPT3 orthologs by BLAST using the NCBI database. RACE PCR (59 and 39) were performed in order to obtain full-length sequences from uncomplete cDNA fragments (see Table 1 for primer sequences). The 59 and 39 regions of cDNA fragments were cloned with a SMART TM RACE cDNA Amplification Kit (Clontech, TaKaRa Bio, USA) according to the manufacturer's protocol. The PCR products were subcloned to pMD19-T Vector and sequenced. By sequencing alignment with its AtOPT3 ortholog from A. thaliana, it was observed that the 59 region of the isolated TcOPT3 cDNA contained the first initiating ATG codon and the 39 region containing the stop codon and the 39 UTR. The gene we cloned has been deposited in Genbank with the accession number of HQ699884.

Sequence Comparisons
The predicted amino-acid sequence from TcOPT3 cDNA and other selected OPT protein sequences were aligned using the CLUSTAL W program, version 1.8 (Thompson et al., 1994). The putative trans-membrane domains were predicted by the TMHMM (version 2.0; http://www.cbs.dtu.dk/services/ TMHMM/). The putative amino acid sequences were aligned with the program Clustal X Version 1.8 [47] and viewed by GeneDoc version 2.6 [48]. Phylogenetic trees were constructed with the neighbor-joining algorithm using the program with MEGA 4 (http://www.megasoftware.net) [49]. Hydrophilicity plots for TcOPT3 was generated based on the Kyte and Doolittle (1982) method using Protean sequence analysis software (DNAS-TAR) under default parameters.

Quantitative PCR Analysis of TcOPT3 Expression
Total RNA was isolated from roots, stems and leaves of onemonth-old T. caerulescens and treated by DNase I (RNase free) as described above. RT-PCR with oligo d(T)-anchor primer were performed with PrimeScript RT (Perfect Real Time) reagent kit (TaKaRa, China). For real-time RT-PCR, 2 ml of the diluted (1:5) cDNA products were used as templates with 5 ml SYBR Pre mix (Takara, China), 0.5 ml primers (10 mM each) and 2.5 ml water. Calculation of the DCp values was performed as described [50]. Primers were list in Table 1.

In Situ Hybridization
One-month-old seedlings grown on hydroponic culture were obtained. Plant materials were fixed in potassium phosphate buffer (0.1 M, pH 7.4) containing 4% paraformaldehyde overnight at 4uC, then dehydrated in ethanol series, and embedded in paraffin. A gene-specific fragment containing the 416 bp fragment cross 39UTR of TcOPT3 was amplified by PCR (primers were shown in Table 1) and cloned into pSPT19 vectors. Sense and antisense probes were synthesized using SP6 and T7 primers with DIG RNA Labeling kit according to the manufacturer's instructions (Roche, USA). In-situ hybridization was performed as described previously [19,51].

GFP Fusion and Subcellular Localization
To construct the TcOPT3-GFP fusion protein, the ORF without the stop codon of TcOPT3 cDNA fragment containing SmaI and SacI restriction sites (see Table 1 for primer sequences) was cloned into the modified pEGFP-N2 vector under the control of 35 S promoter. The final construct p35S::TcOPT3-EGFP and the pm-rk were transiently expressed in onion epidermal cells using a particle gun-mediated system (PDS-1000/He; Bio-Rad), where pm-rk was used as the plasma membrane-localizated maker [52]. Onion epidermal cells were bombarded with 1 mm gold particles coated with plasmid DNA and then incubated in the dark at 25uC for 20 h. Fluorescence was observed by confocal laser scanning microscopy (LSM700; Carl Zeiss).

Heavy Metal Tolerance in Yeast
Wild-type yeast DY1455 was transformed with TcOPT3-pFL61, or with pFL61, which served as a negative control. DY1455 transformants were grown on liquid SD-ura medium overnight at an OD 600 of 1. After washed by CaCl 2 and centrifugation, resuspended in SD-ura with 50 mM FeCl 3 , 50 mM ZnSO 4 , 20 mM CdCl 2 , or 50 mM CuSO 4 were grown for another 0, 6, 12, 24 hours. After measured the OD values and washed by CaCl 2 to remove adsorbed transit metals in apoplast cells, aliquots of yeast cells were taken for measurement of metal accumulation.

Elemental Analysis
For metal accumulation, yeast cells were concentrated at 5,000 rpm for 5 min and washed by 0.5 mM CaCl 2 for 5 min twice to remove adsorbed transit metals from the yeast cell walls. Then cell sediments were dried at 70uC for 2 days, and digested in HNO 3 . Metal concentration in the digested solution was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES; IRIS/AP optical emission spectrometer).

Statistics
Data were statistically analyzed using analysis of variance (ANOVA) in Origin 8, and tested for significant (P#0.05) treatment differences using Student's t-test.