Phosphate transporters, PnPht1;1 and PnPht1;2 from Panax notoginseng enhance phosphate and arsenate acquisition

Background Panax notoginseng is a medicinally important Chinese herb with a long history of cultivation and clinical application. The planting area is mainly distributed in Wenshan Prefecture, where the quality and safety of P. notoginseng have been threatened by high concentration of arsenic (As) from the soil. The roles of phosphate (Pi) transporters involved in Pi acquisition and arsenate (AsV) tolerance were still unclear in this species. Results In this study, two open reading frames (ORFs) of PnPht1;1 and PnPht1;2 separated from P. notoginseng were cloned based on RNA-seq, which encoded 527 and 541 amino acids, respectively. The results of relative expression levels showed that both genes responded to the Pi deficiency or As exposure, and were highly upregulated. Heterologous expression in Saccharomyces cerevisiae MB192 revealed that PnPht1;1 and PnPht1;2 performed optimally in complementing the yeast Pi-transport defect, particularly in PnPht1;2. Cells expressing PnPht1;2 had a stronger AsV tolerance than PnPht1;1-expressing cells, and accumulated less As in cells under a high-Pi concentration. Combining with the result of plasma membrane localization, these data confirmed that transporters PnPht1;1 and PnPht1;2 were putative high-affinity H+/H2PO4− symporters, mediating the uptake of Pi and AsV. Conclusion PnPht1;1 and PnPht1;2 encoded functional plasma membrane-localized transporter proteins that mediated a putative high-affinity Pi/H+ symport activity. Expression of PnPht1;1 or PnPht1;2 in mutant strains could enhance the uptake of Pi and AsV, that is probably responsible for the As accumulation in the roots of P. notoginseng.


Background
Panax notoginseng (Burk.) F.H. Chen is a rare and wellknown perennial herb, of which the main medicinal part is radix, and has been used for 600 years in clinical treatment with clearly medicinal actions of dissipating blood stasis, arresting bleeding, blood-activating, and inflammationdiminished, thereby promoting the elimination of swelling and relieving pain [1][2][3]. Wenshan Autonomous Prefecture in Yunnan Province is famous for the cultivation of P. notoginseng, where the arsenic (As) concentration in background soil is very high, and partially caused by mining activities and the use of As-containing pesticides [2,4]. Previous studies found that almost half of cultivated fields had a crisis of excessive As in the Wenshan area, of which 21 fields were analyzed in total [1]. Thus, As accumulation in P. notoginseng has a closed link with background value in soil. Investigations indicated that As content in the radix, stems, and flowers occasionally exceeded the threshold value (2.0 mg/kg, As standard of China Green Trade
PnPht1;1 and PnPht1;2 gene expression in the roots of P. notoginseng under Pi deficiency and As exposure An obvious phenomenon was uncovered: both PnPht1;1 and PnPht1;2 positively responded to the Pi deficiency or As exposure and were highly upregulated (Fig. 4). Actually, the upregulations of PnPht1;1 and PnPht1;2 were higher under the stress of Pi deficiency, rather than As exposure, presenting a significantly difference of PnPht1;2 under a low-Pi treatment with or without As, e.g., PnPht1; 1: 28.4-fold increase with lPnAs, 25.6-fold increase with lPhAs, 8.5-fold increase with mPhAs, and 10.8-fold increase with hPhAs; PnPht1;2: 105.6-fold increase with lPnAs, 67.2-fold increase with lPhAs, 5.4-fold increase with mPhAs, and 11.8-fold increase with hPhAs. Note that supplementation with AsV could decrease the expression level in lP groups (lPnAs and lPhAs), e.g., PnPht1;1: 28.4fold increase with lPnAs and 25.6-fold increase with of lPhAs; PnPht1;2: 105.6-fold increase with lPnAs and 67.2fold increase with lPhAs. Interestingly, compared with low phosphate (lP) treatment, the expression levels of PnPht1; 1 and PnPht1;2 sharply decreased under supplementation with sufficient Pi (0.7 mM and 1.4 mM).
In addition, growth rate coefficients were evaluated via exponential regression based on the logarithmic growth phase. Representative assays are shown in Fig. 6a-c, and the means of a number of independently obtained growth rate coefficients for each transporter (n = 4) are shown in Fig. 6d. Under a low Pi concentration (20 μM), the growth rate coefficient of MB192-YEplac112 was relatively low (0.0984), while the cells harboring PnPht1; 1 or PnPht1;2 had a higher coefficient (0.1594, 0.163). These results revealed that both PnPht1;1 and PnPht1;2 Pi transporters performed optimally in complementing the yeast Pi-transport defect, particularly in PnPht1;2.
Yeast cells expressing PnPht1;1 and PnPht1;2 improve As tolerance Current evidence show that the phosphate transport system is the main pathway for AsV uptake. However, AsV uptake is competitively inhibited by sufficient Pi [28]. As    (Fig. 7a, b, c). The As tolerance for each transgenic line was assessed by calculating the percentage of growth under As exposure relative to growth in the absence of As. The results revealed that the As tolerance of MB192-PnPht1;2 was significantly stronger than that of MB192-PnPht1;1 and MB192-YEplac112. Although As tolerance of MB192-PnPht1;1 was also larger than MB192-YEplac112, the difference was non-obvious (Fig. 7d).
As shown in Fig. 8a, the OD 600 of WT, MB192-PnPht1;1 and MB192-PnPht1;2 significantly increased with the elevation of Pi concentration from 20 μM to 100 μM, suggesting that high Pi concentration relieved the stress of AsV. However, the change of OD 600 of mutant strains were not obvious. Additionally, OD 600 of PnPht1;2-expressing cells was a little larger than MB192-PnPht1;1 without a significant difference under the same treatments containing 80 μM AsV. The phenomenon revealed that Pi addition may improve the probability, that Pi transporters assimilate Pi under the competition of AsV. Under a high level of Pi concentration, PnPht1;1 and PnPht1;2 preferred to combine Pi. The discovery was reinforced by the As accumulation in cells of WT, MB192-PnPht1;1 and MB192-PnPht1;2, which decreased with the addition of high Pi concentration (Fig. 8b). The decreased As of MB192-PnPht1;2 presented a significant difference from 20 μM to 100 μM Pi concentration, as well as WT. The As concentration of MB192-PnPht1;1 or MB192-PnPht1;2 was significantly less than WT under 20 or 100 μM Pi concentration, but significantly higher than mutant strains of MB192 and MB192-YEplace112 under 20 μM Pi (Fig. 8b). It is worth mentioning that it's still a significant difference between the As concentration of MB192-PnPht1;1 and mutant strains under 100 μM Pi. For MB192-PnPht1;1 and MB192-PnPht1;2, the difference was significant under 100 μM Pi concentration. MB192-PnPht1; 1 accumulated over 2.3-fold more arsenic than cells expressing PnPht1;2, suggesting that PnPht1;1 was likely to combine AsV compared to PnPht1;2. Combined with the results of As tolerance in Fig. 7d, it is concluded that the transporters PnPht1;1 and PnPht1;2 had different capacities of assimilating As, and the PnPht1;2-expressing cells had a stronger As tolerance. In addition, a high Pi concentration could alleviate As stress.

Discussion
P. notoginseng is an important Chinese medicinal plant, of which the rhizome are main medicinal portions containing active substances, e.g. notoginsenoside. However, the quality of P. notoginseng has been threatened by high concentration of As in primary producing areas [1]. The results of cultivation showed that As content in the roots of P. notoginseng gradually increased with elevated AsV concentration but significantly decreased with a high level of Pi concentration under the high-As treatment ( Figure S1). In the process, Pi transporters play a vital role in uptake and translocation [31,32].
Herein, we identified two Pi transporter-encoding genes, PnPht1;1 and PnPht1;2, from the fibrous roots of P. notoginseng under the treatments of Pi deficiency and AsV exposure. According to the bioinformatics and phylogenetic tree, both PnPht1;1 and PnPht1;2 belonged to subfamily Pht1 with the signature sequence "GGDYPLSATIxSE" and 11 transmembrane domains (Figs. 1 and 2), which is the primary pathway of Pi uptake and translocation [13].  Further evidence suggested that the Pi transporter was not only responsible for Pi uptake but also transport congeners of P, e.g., As [16,27]. This finding suggests a competitive relationship of substrates between Pi and AsV [17,33,34]. However, the affinity of Pi or As with the Pi transporter depends on the characteristics of the Pi transporters, the concentration, the duration or chemical speciation of Pi and As, and the tissue of plants [19,26,[35][36][37]. In this study, qPCR results showed that upregulation of PnPht1;1 and PnPht1;2 expression is induced via either Pi deficiency or AsV exposure. In contrast, the responses of PnPht1;1 and PnPht1;2 to Pi deficiency are more positive than AsV, of which the expression levels were increased by as much as 30-and 100-fold, respectively, suggesting that PnPht1;1 and PnPht1;2 would be high-affinity Pi transporters. Interestingly, an increasing concentration of AsV lowered the expression levels of PnPht1;1 and PnPht1;2 in the low-Pi treatment group (lPnAs and lPhAs), especially for PnPht1;2 (Fig. 4). Increasing evidence have illuminated the phenomenon that numerous Pht1 genes could significantly respond to the induction of Pi deficiency or As exposure, e.g., upregulation of OsPT1, OsPT2, OsPT4 and OsPT8 in O. sativa, CmPT1 in Chrysanthemum morifolium, and PvPht1;3 in P. vittata under Pi deficiency or As exposure [13,38,39], and downregulation of PvPht1;1 in P. vittata under As exposure [13]. Occasionally, Pi transporters showed little ability to transport AsV, e.g., PvPht1;2 in P. vittata [40]. Hence, further research is warranted to elucidate the properties that are likely to regulate PnPht1;1 and PnPht1; 2.
Subsequently, the properties of PnPht1;1 and PnPht1;2 were analyzed through complementation assays in yeast mutant MB192 knocked out for high-affinity Pi transporterencoding genes. Yeast cells expressing PnPht1;1 and PnPht1;2 could complement the defect of the loss of highaffinity Pi transporters, growing well under low-Pi concentrations (2 and 20 μM Pi) (Fig. 5). The results of pH-dependent and ACP activity assays showed that both PnPht1;1 and PnPht1;2 are H + dependent-type Pi transporters, which are driven by H + concentration gradients. Yeast cells expressing PnPht1;1 or PnPht1;2 were significantly inhibited in medium supplemented with CCCP or 2, 4-DNP, which are typical protonophores, resulting in the inhibition of anion uptake [41]. This finding is consistent with previous reports that many Pht1 proteins are usually H + / H 2 PO 4 − symporters and are involved in energy-dependent transport at the plasma membrane, mediating Pi uptake [9,12,39,42,43]. This confirmed our analysis of the localization of PnPht1;1 and PnPht1;2 (Fig. 3). However, the process of H + /H 2 PO 4 − symport in the membrane has not been determined, likely due to the mechanism of proton and glycerol-3-phosphate symport in E. coli [39,44,45].
As described above, there is a complicated relationship between AsV and Pi uptake and translocation. AsV in the cytoplasm competes with Pi, forming an unstable complex of ADP-AsV, thereby disrupting the energy flow [46,47]. Therefore, a high level of Pi supply to As-treated plants could decrease membrane damage by lowering oxidative injury [48]. In the plantation experiments, studies find that Pi supply could suppress As uptake by plants [26,[49][50][51], which is in line with our result as described in Figure S1. In addition, our results suggested that yeast cells expressing PnPht1;1 and PnPht1;2 improved As tolerance, particular in PnPht1;2 with a significant difference by comparison to MB192-vector, indicating by growth rate coefficients and the As tolerance index (Fig. 7). In addition, cells harboring PnPht1;2 had a stronger AsV tolerance, while PnPht1;1-expressing cells accumulated more arsenic (Fig. 8b). An assumption was concluded that PnPht1;1 preferred to combine AsV compared to PnPht1; 2. Besides, an interesting phenomenon was shown that As concentrations in transformants harboring PnPht1;1 or PnPht1;2 were significantly less than WT. It may be related to the difference of genetic characteristic between PHO84 knocked out in mutant and the two target genes. PHO84-overexpressing in Saccharomyces cerevisiae obviously enhanced the uptake of AsV [52]. In summary, as complementary mutant strains, the capability of MB192-PnPht1;1 and MB192-PnPht1;2 of assimilating Pi or AsV is relatively weaker by comparison with WT. These observations are in line with previous studies and collectively suggest that high-affinity Pi transporters have comparable specificities for AsV uptake and play important roles in the enhanced AsV uptake and tolerance, e.g., PvPht1;3 in P. vittata [13] and OsPT1 in O. sativa [15]. As a highaffinity Pi transporter, pht1;1-3 in A. thaliana displays a slow rate of AsV uptake that ultimately enables the mutant to accumulate two times the arsenic found in wildtype plants [35], while AtPht1;5 or AtPht1;7 also have a preference for Pi over AsV [13]. In contrast, although OsPT8 was found to have a high affinity for both Pi and AsV, Wu et al. considered that the Pi transporter contributed only slightly to As uptake [14]. Taken together, both PnPht1;1 and PnPht1;2 responded to the stresses of Pi deficiency or As exposure and improved the tolerance of AsV, particularly in a high level of Pi concentration. Many efforts need to be made to research the possibility of using the Pht genes in P. notoginseng to improve the adaptability to the stresses of Pi deficiency or As exposure, e.g., the construction of stable PnPht1;1-or PnPht1;2-overexpression system in P. notoginseng.

Conclusions
In this study, we uncovered the roles of PnPht1;1 and PnPht1;2 of P. notoginseng in the uptake of Pi and AsV. The results of qPCR showed that PnPht1;1 and PnPht1;2 responded to the Pi deficiency or As exposure and were highly upregulated. However, the expression levels of PnPht1;1 or PnPht1;2 decreased under supplementation with sufficient phosphate. Heterologous expression in Saccharomyces cerevisiae MB192 revealed that PnPht1;1 and PnPht1;2 performed optimally in complementing the yeast Pi-transport defect, particularly in PnPht1;2. Cells expressing PnPht1;2 had a stronger AsV tolerance than PnPht1;1-expressing cells, and accumulated less As in cells under a high-Pi concentration. In addition, Pi supply could suppress As accumulation in the roots of P. notoginseng. Taken together, we confirmed that PnPht1;1 and PnPht1;2 encoded functional plasma membrane-localized transporter proteins that mediated a putative high-affinity Pi/H + symport activity. Expression of PnPht1;1 or PnPht1;2 in mutant strains could enhance the uptake of Pi and AsV, that is probably responsible for the As accumulation of P. notoginseng.

P. notoginseng material and experimental setup
All P. notoginseng seedlings used in this study were bought from Wenshan Miaoxiang Sanqi Technology Co. LTD, and were identified by professor Ronghua Zhao, who is specialized in identification, cultivation and processing of Chinese herbs.
One-year-old P. notoginseng in good condition, cultivated in a standard planting garden were transplanted into garden pots. There was no significant difference in weight, height or leaf number of these seedlings. The cultivation medium was sandy loam texture, including 20% lightweight aggregate, 40% expanded vermiculite, 30% clay and 10% silt, modified according to Mandal et al. [53]. The concentration of dissolved P in the soil was decreased to 0.07 mM through double rinsing with 1% NaHCO 3 . The Pi concentration was adjusted to 0.07 mM, 0.7 mM and 1.4 mM (in dry weight) via adding KH 2 PO 4 , which are minimally limited, growth-promoting and excessive concentrations for P. notoginseng. The 3 concentration treatments were referred to as low phosphate (lP), middle phosphate (mP) and high phosphate (hP). Before planting, sodium arsenate (Na 3 AsO 4 ) was blended into mixed soil with the additive amount of 0.2 mM (in dry weight). The As concentration was as high as the background value of soil at the main producing areas in the Wenshan Autonomous Prefecture Yunnan Province [4]. In the experiment, 5 treatments were set up in total, as follows: lPnAs, lPhAs, mPhAs, hPnAs, and hPhAs. Meanwhile, mPnAs were taken as a control check (CK). Due to the absence of mineral nutrition in mixed soil, 50 mL 1/4 Hoagland's solution lacking phosphorus was used to nourish the plants every 3 days, in which KH 2 PO 4 was replaced by KNO 3 . P. notoginseng grew at 25°C, 85% relative humidity, avoiding direct sunlight and water-accumulated in the greenhouse. After 5 months, the fresh fibrous roots were harvested, rinsed with deionized water, frozen in liquid nitrogen, and stored at − 80°C. Each treatment had eight biological replicates and four plants in every replicate.

Analysis of total As concentration in the rhizome of P. notoginseng
The total As concentration in the rhizome of P. notoginseng was determined as described by Wu et al. [14] and Xu et al. [54]. Plant samples were ground to fine powders and digested with HNO 3 :H 2 O 2 (85:15, v/v). Then, the digestion solution was determined using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7500c, USA).

Clones of PnPht1;1 and PnPht1;2
The open reading frame (ORF)'s base-pair information of PnPht1;1 and PnPht1;2 were obtained from a transcript of P. notoginseng roots treated as described above. The primers of PnPht1;1 and PnPht1;2 for the ORF clone are listed in Table 1. First-strand cDNA was used as a template for ORF PCR amplification, which was reverse-transcribed from total RNA with a Primescript II 1st strand cDNA synthesis kit (Takara, Japan). Total RNA was extracted from the fibrous roots with the miniBEST plant RNA extraction kit (TaKaRa, Japan). PCR procedures comprised an initial denaturation step (94°C/5 min) followed by 35 cycles of 94°C/1 min, 58°C /30 s, 72°C/1 min, and holding at 4°C. The sequences have been submitted to NCBI, of which the GenBank accession numbers for PnPht1;1 and PnPht1;2 are MN420501 and MN420502.
qPCR Total RNAs in fibrous roots of 5 treatment groups (lPnAs, lPhAs, mPhAs, hPnAs, hPhAs) and control check (mPnAs) were extracted as described above. As a template, cDNA was reverse-transcribed with Primescript RT reagent kit with gDNA eraser (TaKaRa, Japan). All qPCRs were performed with TB Green Premix Ex Taq (Tli RNa-seH Plus), ROX plus (TaKaRa, Japan) with the genespecific primers (Table 1) according to the manufacturer's instructions. Each 20 μL reaction system contained 10 μL TB Green mix, 100 ng cDNA and 0.2 μM of each primer. 26S-2 was targeted as the reference gene and used for normalization of RT-qPCR data [55]. The primer pair is shown in Table 1. In the end, relative transcription levels were estimated using the 2 -ΔΔCt method [56].

Bioinformatics analysis
The ORF of the full-length cDNA was identified using online software at https://www.ncbi.nlm.nih.gov/orffinder/. The location of hydrophobic, isoelectric point, protein molecular weight, and putative transmembrane domains were enabled through the software package mounted at http://expasy.org/tools/protscale.html. Multiple peptide alignments were carried out using DNAman (DNAman v6.0, Lynnon Biosoft, USA). Phylogenetic analyses used MEGA v4.0 software.

Complementation of a yeast mutant strain defective for Pi uptake
Saccharomyces cerevisiae MB192 (MATa pho3-1 pho84::HIS3 ade2 leu2-3, 112 his3-532, trp1-289 ura3-1, 2 can1) defective in the high-affinity Pi transporter gene PHO84 by insertion of an HIS3 DNA fragment was chosen as a heterologous expression yeast for uptakefunctional verification of Pi and As [11,57]. The ORFs of PnPht1;1 and PnPht1;2 were amplified using Trans-Start FastPfu DNA Polymerase (Transgen Biotech, China) with the primer pairs containing restriction enzyme cutting sites ( Table 1). The resulting amplicons were digested with the corresponding enzymes BamH I/ Kpn I and Xba I/Xma I and then introduced into the expression vector YEplac112 with their respective recognition sites using T4 DNA Ligase (NEB, USA) following the manufacturer's protocol. The structure of the resulting recombinant plasmids were defined by restriction enzyme digestion and DNA sequencing with E. coli (DH5α). Two recombinant plasmids and empty vector YEplac112 were transformed into MB192 cells by electrotransformation using the Bio-Rad electroporation equipment (Bio-Rad Laboratories, Richmond, USA) [58]. In total, 3 transformants, including MB192-PnPht1;1, MB192-PnPht1;2 and MB-YEplac112 were yielded. Wild-type (WT) S. cerevisiae was used as a positive control. Positive transformants were picked out through SD-Trp − selective medium. Monoclonal cells were transferred into yeast nitrogen base (YNB) liquid medium supplemented with 4.5 μM Pi, and the recombinant plasmids were verified through plasmid extraction and sequencing.
For the effect of Pi concentration, identified yeasts were re-cultured to the logarithmic phase (OD 600 = 0.6) in the YNB liquid medium. Then, 100 μL of suspension liquid was diluted to 5 mL and cultured at 200 rpm and 30°C for an additional 16 h, in which the medium was adjusted with a range of Pi concentrations (0.002, 0.02, 0.06, and 0.1 mM) and an initial pH of 6.8 [38]. Bromocresol purple was used to indicate the change of pH, which gave a color shift from yellow to purple. During the acidification of the liquid medium, the change correlated well with the growth of the yeast cells and acid phosphatase activity (ACP) [59]. For pH-dependent Pi uptake experiments, the pH value in the medium was in the range of 4.0 to 8.0. In the tests, monoclonal cells were transferred into YNB liquid medium containing 80 μM Pi and cultured for 24 h at 200 rpm and 30°C. For the growth assays, the OD 600 of yeast cells was determined every 3 or 5 h in 5 mL SD-Trp − medium containing 20 μM Pi and 2% glucose at 200 rpm 30°C, adjusting the pH to 6 and the initial concentration of OD 600 to 0.03 with a cell suspension of the logarithmic phase [13]. Thus, growth rate coefficients of the logarithmic growth were calculated via exponential regression.

AsV uptake affected by Pi concentration
For the assays of growth rates and As tolerance, cells expressing PnPht1;1, PnPht1;2 or YEplac112 were washed twice into 10 mL SD-Trp − medium containing 50 μM Pi and 2% glucose, which made an initial concentration of 0.03 (OD 600 ). Then, AsV was added to the medium at a final concentration of 80 μM before culturing at 200 rpm and 30°C for 30 h. The OD 600 of yeast cells was determined every 3 or 5 h to uncover growth rate coefficients and AsV tolerance at the logarithmic phase [13]. The uptake affected by Pi concentration was investigated by determining the OD 600 and As accumulation concentration in cells. First, 1 mL of OD 600 = 0.6 suspensions of transformants and WT were transferred into 50 mL SD-Trp − medium containing 2% glucose, different Pi concentrations (20 or 100 μM) and 80 μM AsV, adjusting the pH to 6.0. The OD 600 of the yeast suspension was measured after cultivation with shaking at 200 rpm for 30 h and at 30°C. Then, yeast cells were collected at 5000 rpm for 5 min, and the pellets were washed twice with 25 mL 10 mM EDTA [62]. After digestion as described above, total As was determined using ICP-MS (Agilent 7500c, USA). Data collected were performed with four biological replicates, and three technical replications of each biological replicate were conducted independently.  [63]. A. tumefaciens EHA105 harboring recombinant plasmids were infiltrated into the leaves of four-week-old N. benthamiana through lower epidermis injection of 1 mL bacterium suspension. The cells were finally induced at 4-6 days after infiltration with 10 μM β-estradiol (Sigma) for 6-12 h and the transient expression analyses were performed as described by Dong et al. [64]. Images were obtained using an UltraVIEW VoX laser doublespinning disk confocal real-time imaging analysis microscope (PerkinElmer, USA). Autoluminescence, GFP, and RFP were excited by a 640, 488 and 561 nm laser, respectively.

Statistical analysis
All data were processed and analyzed statistically with Microsoft Excel 2010, SPSS 17.0, and Sigmaplot 12.0 for Windows. Assumptions of normality and homogeneity of variances were tested prior to all statistical tests. The significant differences were all tested with one-way analysis of variance (ANOVA) followed by Tukey HSD tests at the 0.05 level, including relative expression level (Fig.  4), ACP activity (Fig. 5c), OD 600 (Figs. 5d and 8a), As concentration (Fig. 8b, Figure S1) and growth rate coefficient (Fig. 6d). In addition, an independent-samples ttest at the 0.05 or 0.01 level was also used to analyze the difference, e.g., OD 600 between each treatment (CCCP or 2,4-DNP) and CK (Fig. 5e), OD 600 or As concentration between 20 μM and 100 μM Pi (Fig. 8). All data in figures and tables are expressed as the means ± standard deviation (SD, n ≥ 3).