Phosphate Transporter PvPht1;2 Enhances PhosphorusAccumulation and Plant Growth without Impacting Arsenic Uptakein PlantsYue Cao,† Dan Sun,† Jun-Xiu Chen,† Hanyi Mei,† Hao Ai,‡ Guohua Xu,‡ Yanshan Chen,*,†

and Lena Q. Ma*,†,§

†State Key Lab of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023,China‡State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Plant Nutrition and Fertilization inLow-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China§Soil and Water Science Department, University of Florida, Gainesville, Florida 32611, United States

ABSTRACT: Phosphorus is an important macronutrient for plant growth and is acquired by plants mainly as phosphate (P).Phosphate transporters (Phts) are responsible for P and arsenate (AsV) uptake in plants including arsenic-hyperaccumulatorPteris vittata. P. vittata is efficient in AsV uptake and P utilization, but the molecular mechanism of its P uptake is largelyunknown. In this study, a P. vittata Pht, PvPht1;2, was cloned and transformed into tobacco (Nicotiana tabacum). In hydroponicexperiments, all transgenic lines displayed markedly higher P content and better growth than wild type, suggesting that PvPht1;2mediated P uptake in plants. In addition, expressing PvPht1;2 also increased the shoot/root 32P ratio by 69−92% and enhancedxylem sap P by 46−62%, indicating that PvPht1;2 also mediated P translocation in plants. Unlike many Phts permeable to AsV,PvPht1;2 showed little ability to transport AsV. In soil experiments, PvPht1;2 also significantly increased shoot biomass withoutelevating As accumulation in PvPht1;2 transgenic tobacco. Taken together, our results demonstrated that PvPht1;2 is a specific Ptransporter responsible for P acquisition and translocation in plants. We envisioned that PvPht1;2 can enhance crop P acquisitionwithout impacting AsV uptake, thereby increasing crop production without compromising food safety.

■ INTRODUCTION

Phosphorus is a major essential macronutrient for plant growth,which is involved in many metabolic pathways. Plants take upphosphorus exclusively in the form of inorganic phosphate (P).Because of its high fixation in soils and slow diffusion to theroot surface, plants have evolved strategies to increase theavailability of soil P.1 In plants, the high-affinity P transporters(Phts) play key roles in P acquisition from soil.2 These Ptransporters are categorized into four subfamilies: Pht1, Pht2,Pht3, and Pht4.3 Over the past decades, many genes thatencode Phts have been identified and cloned from A. thalianaand cereal, legume, and solanaceous species.4−11

In Arabidopsis, Pht1 subfamily is comprised of nine members(AtPht1;1 to 1;9). Among them, AtPht1;1 and AtPht1;4 areresponsible for P acquisition under both high- and low-P

conditions.12,13 The P uptake by atpht1;1/atpht1;4 doublemutant was 75% lower than wild type (WT) plants.13 Inaddition, as high-affinity P transporters, AtPht1;8 and AtPht1;9play key roles in P uptake under P-deficient conditions.10

In rice (Oryza sativa), 13 Pht1 genes are known in thegenome.4 Among them, OsPht1;1, 1;2, 1;4, 1;6, 1;8, 1;9 and1;10 mediate P uptake and translocation in rice.14,15 OsPht1;1 isconstitutively expressed in plants, functioning in P uptake andtranslocation under P-sufficient conditions.9 Similarly, OsPht1;8is expressed in various tissues under both P-sufficient and

Received: December 27, 2017Revised: February 13, 2018Accepted: March 14, 2018Published: March 14, 2018

Article

pubs.acs.org/estCite This: Environ. Sci. Technol. 2018, 52, 3975−3981

© 2018 American Chemical Society 3975 DOI: 10.1021/acs.est.7b06674Environ. Sci. Technol. 2018, 52, 3975−3981

-deficient conditions and is up-regulated in the roots under P-deficient conditions.7 OsPht1;6 is mainly expressed in the roots,involving in P uptake under P-deficient conditions.6 Recently,the function of OsPht1;4 has been characterized, whichfacilitates P acquisition and mobilization in rice.11

Arsenic (As) and P are chemical analogs. However, As is atoxic element and ubiquitous in soils, which can be taken up bycrops, thereby threatening human health through the foodchain.16,17 Due to their similarity, AsV can be taken up andtranslocated via Phts.13,18,19 In Arabidopsis, AsV is taken up viaAtPht1;1 and AtPht1;4.13,20 In rice, OsPht1;1, OsPht1;4, andOsPht1;8 are involved in AsV uptake and translocation, andtheir modulation affects As accumulation in rice.19,21,22 Thoughoverexpression of Phts promotes P acquisition,14 it may alsoincrease As uptake by plants.21,23

Chinese brake fern (Pteris vittata) is the first-known As-hyperaccumulator, it is efficient in As uptake, translocation, anddetoxification.24,25 Besides, the fern is also efficient in acquiringP from insoluble P sources in soils26,27 and efficient in depletingP from hydroponic solution.28 Recently, P. vittata P trans-porters PvPht1;1 to PvPht1;3 have been characterized.29 Yeastexperiments showed that PvPht1;3 is a high-affinity AsVtransporter.29 However, the functions of PvPht1;1 andPvPht1;2 in plants have not been elucidated, so their role inimproving P utilization is unclear.PvPht1;1 and PvPht1;2 encode predicted proteins of 536

amino acids, which share 98.5% identity.29 With only fewnucleotides being different, they can be considered as the samegene. In this work, to study the function of PvPht1;2 and itsrole in P uptake in plants, we transformed PvPht1;2 into modelplant tobacco and investigated its function in P and AsV uptakeand translocation by transgenic tobacco. We believe that thisstudy may provide important insights into the behavior ofPvPht1;2 as well as provide a potential strategy to enhance cropP acquisition.

■ MATERIALS AND METHODSGrowth of P. vittata. Spores of P. vittata were collected

from Florida, USA,24 and preserved in our lab at NanjingUniversity. Their spores were sown on potting soils, watered,and covered with transparent plastic films to keep the soilmoist. After 2 months of cultivation, sporophyte seedlings with2−3 fronds appeared, which were then transplanted intoseparate pots following the work of Fu et al.28 All sporophyteswere cultivated in a greenhouse to 4-frond stage and thenacclimated in 500 mL aerated 0.2 strength (0.2X) Hoaglandnutrient solution (HNS) for 7 d.28 For the transcripts analysis,sporophyte seedlings were transferred to 0.2X HNS containing100 μM KH2PO4 (+P), 0 μM KH2PO4 (−P), or 100 μMKH2PO4/50 μM Na2HAsO4·7H2O (+As) for 3 days. All fernswere grown under a 14 h photoperiod, 26/20 °C day/nighttemperature, 60% relative humidity, and 3000 lx light intensity.Total RNA Preparation and qRT-PCR analysis in P.

vittata. Total RNAs from P. vittata roots and fronds wereisolated using Plant Total RNA Kit (Sigma-Aldrich), reversetranscription and first-strand cDNA was synthesized usingHiScript II One Step RT-PCR Kit (Vazyme Biotech, Nanjing,China). qRT-PCR analysis was performed using SYBR GreenPCR Master Mix (Vazyme Biotech, Nanjing, China), and theCFX Connect Real-Time PCR Detection System (BIO-RAD).Relative expression levels of PvPht1;2 (Accession No.KM192136) were computed by 2−ΔΔC method of relativequantification. P. vittata Actin gene (PvActin) and Histone gene

(PvHistone)30 were used as an internal control. All gene-specificprimers used for qRT-PCR are as follows. PvPht1;2 5′-GCCCTG GTA TTG GCC ACA AG-3′ and 5′-CCT CGA GGGAGC GAC CAT TT-3′; PvActin 5′-GGG CAG TAT TTCCAA GCA TAG TGG G-3′ and 5′-TGC CTC GCT TTGATT GAG CCT CAT C-3′; PvHistone 5′-GGG TTT ACATTC AGC GAA GC-3′ and 5′-GCT TTC CCT CCA GTGGAC TT-3′.

Yeast Vector Construction, Yeast Transformation, andGrowth Assays. PvPht1;2 coding sequence was cloned fromcDNA of P. vittata collected from Florida, USA, using thefollowing primers: 5′-ATG GCA AAA CTA GAG GTC CTCACC G-3′ and 5′-CTA TGA TGT GTG TGT AGC ACCCCC A-3′. Adapters were added to PvPht1;2 CDS using thefollowing primers: 5′-gaa aaa acc ccg gat tct aga ATG GCAAAA CTA GAG GTC CTC ACC G-3′ and 5′-taa cta att acatga ctc gag CTA TGA TGT GTG TGT AGC ACC CCC A-3′(underlining indicates recombination sequences). The PCRproduct was then cloned into the GAL1 promoter cassette ofpAG413GAL-ccdB (Addgene, http://www.addgene.org/) be-tween XbaI and XhoI restriction sites by recombination, usingthe Trelief SoSoo Cloning Kit (TSING KE, Nanjing, China).The yeast (Saccharomyces cerevisiae) strain for heterologousexpression of PvPht1;2 was the Δpho84 mutant (ThermoScientific, https://www.openbiosystems.com) with the BY4741(MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) background.31,32

The methods related to yeast transformations mainly referredto the high efficiency transformation of yeast described by Gietzet al.33

Yeast growth assay was performed according to the work ofChen et al.5 Briefly, yeast cells were grown at 30 °C in syntheticdefined (SD) medium (0.67% yeast nitrogen base) withoutamino acids, containing 2% (w/v) glucose or 2% (w/v)galactose (induction medium), supplemented with yeastsynthetic dropout without histidine at pH 5.8. For AsVtolerance assays, yeast was grown in liquid SD medium (with2% [w/v] glucose) to an OD600 of ∼1.0 and then subjected tocentrifugation and dilution with sterile water. The drop assayswere performed on SD plates (with 2% [w/v] galactose)containing 1.0 mM AsV for Δpho84 expressing PvPht1;2.

Plant Expression Vector Construction and TransgenicPlant Generation and Selection. Adapters were added toPvPht1;2 CDS using the following primers:5′-acg ggg gac tctaga gga tcc ATG GCA AAA CTA GAG GTC CTC ACC G-3′and 5′-ggg aaa ttc gag ctc ggt acc CTA TGA TGT GTG TGTAGC ACC CCC A-3′ (underlining indicates recombinationsequences). The PCR product was then cloned into the 35Spromoter cassette of pSN1301 (pCAMBIA1301, CAMBIA)between BamHI and KpnI restriction sites by recombination,using the CloneEZ PCR Cloning Kit (Genscript, Nanjing,China), with the constructed binary vector being namedpSN1301-PvPht1;2. Agrobacterium strain C58 was transformedwith the binary vector pSN1301-PvPT1;2 by electroporation.Transformation of tobacco leaf explants was carried outfollowing the works of Curtis et al. and Gallois andMarinho.34,35 Transgenic plants were then identified viahygromycin resistance and GUS staining.

Semiquantitative RT-PCR Analysis of TransgenicTobaccos. Total RNA was extracted from tobacco seedlings.The first-strand cDNA was synthesized from 2 μL total RNAusing HiScript II One Step RT-PCR Kit (Vazyme Biotech,Nanjing, China), which was used as RT-PCR templates. ThecDNAs of PvPht1;2 were amplified by PCR for 30 cycles using

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the gene-specific primers 5′-GCC CTG GTA TTG GCC ACAAG-3′ and 5′-CCT CGA GGG AGC GAC CAT TT-3′.Tobacco actin was amplified for 30 cycles as an expressioncontrol using the LeActin primer 5′-TTC CGT TGC CCAGAG GTC CT-3′ and 5′-GGG AGC CAA GGC AGT GATTTC-3′.Growth of Transgenic Tobacco in Different P and As

Conditions. In hydroponic experiments, transgenic tobaccoseeds and wild type (WT) seeds were germinated in 1/5 MSmedia. Uniform 2-week old tobacco seedlings were transferredto 0.2X HNS containing 100 μM KH2PO4 (+P) or 10 μMKH2PO4 (−P) for 14 d. For As accumulation determination,seedlings were exposed to 20 μM AsV (Na2HAsO4·7H2O,Sigmae-Aldrich, USA) for 3 days. For inorganic P determi-nation, PvPht1;2-Ox lines and WT plants were cultured in 0.2XHNS for 14 d under P-deficient condition, and then transferredto P-sufficient (100 μM) solution for 7 d. In soil tests,transgenic and WT tobacco seeds were germinated andcultivated in a garden soil. Then uniform 7-d old tobaccoseedlings were transferred into soils containing 0, 10, 20, or 40mg kg−1 AsV and cultivated for 30 d.

32P Uptake Assay and Xylem Sap Collection inTobacco. After growing in 0.2X HNS for 7 d, tobaccos weretransferred into 0.2X HNS (200 mL) labeled with 8 μCi of 32P(KH2PO4, PerkinElmer, Waltham, MA, USA) and cultivated for12 h. Then, the plant roots were incubated in ice-colddesorption solution (0.5 mM CaCl2, 100 μM NaH2PO4, 2 mMMES, pH 5.5) for 10 min to remove 32P. The plants were thenblotted-dry, the roots and shoots were harvested, and theirfresh weights were measured. Tissues were digested in a HClO4and 30% (v/v) H2O2 mixture at 70 °C for 2−3 h. Scintillationcocktail (3 mL) was added to the digested tissue, and a liquidscintillation counter (Tri−Carb 2100, Packard) was employedto determine 32P activity.Transgenic and WT tobacco seedlings were cultured under

0.2X HNS. Briefly, the stems of tobacco were cut at 2 cm abovethe roots. The cut surfaces were rinsed with deionized waterand blotted dry. The xylem sap was collected by pipet from thecut surface for 2 h. The inorganic P concentration of xylem sapwas determined as described below.P and As Determination in Plants. Total P concen-

trations of plant samples were measured according to the workof Chen et al.5 Briefly, ∼0.05 g of crushed dry samples weredigested with H2SO4−H2O2 at 280 °C. After cooling, thedigested samples were diluted to 100 mL in distilled water. Pconcentration was analyzed by the molybdenum blue methodbased on dry weight.36

For inorganic P in plant, ∼0.5 g fresh samples were used.36

Briefly, the samples were homogenized in 1 mL of 10% (w/v)perchloric acid using an ice-cold mortar and pestle. Thehomogenate was then diluted 10 times with 5% (w/v)perchloric acid and placed on ice for 30 min. Aftercentrifugation at 10 000g for 10 min at 4 °C, the supernatantwas used for P measurement via the molybdenum blue method.The absorption values for the solution at 820 nm weredetermined using a spectrophotometer (SHIMADZUUV-2550).For As analysis, fresh plants were separated into the shoots

and roots, lyophilized (FreezZone 12, LABCONCO) andstored at −80 °C. For total As, freeze-dried plant sample (0.05g) was digested with 50% HNO3 at 105 °C following USEPAMethod 3050B and determined by inductively coupled plasma

mass spectrometry (ICP-MS; PerkinElmer NexION 300X,USA; detection limit at 0.1 μg L−1).

QA/QC and Statistical Analysis. For quality assurance andquality control (QA/QC), indium was used as an internalstandard and was added into the samples, calibration standards,and blanks. During measurement, standard solution at 5 μg L−1

As was measured every 20 samples to monitor the stability ofICP-MS. The check recovery was within 90−110%. In addition,blanks and certified reference material for plant samples (GSB21, Chinese geological reference materials) were included forquality assurance, which were within expected values.37

Data are presented as the mean of 3−5 replicates withstandard error. Analysis of variance (ANOVA) was carried outby SPSS software (SPSS 13.0; SPSS Inc., Chicago, USA).Significant differences were determined with treatment meanscompared by Tukey’s mean grouping tests at p < 0.05.

■ RESULTS AND DISCUSSION

Identification and Expression Pattern of P. vittata PTransporter PvPht1;2. To understand the molecularmechanism of P metabolism in P. vittata, six putative Phtsequences were identified, including PvPht1;2. Then, transcrip-tional expression of PvPht1;2 in P. vittata was investigated byqRT-PCR using actin and histone as reference genes. As shownin Figure 1A, PvPht1;2 was expressed strongly in the roots andfronds, with frond transcripts level of PvPht1;2 being 42%higher than root.It is known that P deficiency induces Pht expression in

plants. Besides, as an analog, AsV is also taken up by Pht inplants, so it may affect Pht transcription in plants. Thus, weinvestigated the expression of PvPht1;2 responding to Pdeficiency (no P) or AsV exposure (50 μM AsV). In theroots, the expression of PvPht1;2 was 8.5-fold higher under P-deficient condition than that under P-sufficient condition(Figure 1B). When P. vittata was exposed to AsV, transcriptslevel of PvPht1;2 in the roots was comparable to no As control(Figure 1B). The results were similar to P. vittata’s expressionpattern in the work of DiTusa et al.29 In the fronds, theexpression of PvPht1;2 transcripts was similar in differenttreatments (Figure 1C). These results showed that PvPht1;2transcripts were induced by P deficiency in P. vittata roots, butnot by AsV, indicating that PvPht1;2 may play a critical role inP acquisition but not As uptake in P. vittata.

Overexpression of PvPht1;2 Increased P Uptake andTranslocation in Tobacco Plants. To characterize itsfunction in P uptake and translocation in plants, we generatedPvPht1;2 transgenic tobacco lines (PvPht1;2-Ox), wherePvPht1;2 was expressed under constitutive CaMV35S pro-moter. Three independent transgenic T2 lines (Ox1, Ox10, andOx21) were selected to assess their effects on P acquisition(Figure 2). RT-PCR analysis showed that PvPht1;2 transcriptswere strongly expressed in PvPht1;2-Ox lines, while it was notdetected in WT plants (Figure 2C).In hydroponic experiments, PvPht1;2-Ox lines and WT

plants were cultured under P-sufficient and -deficientconditions for 14 d (Figure 2). All three transgenic plantsgrew similarly as WT under P-sufficient treatment (Figure2AD). However, Ox1, Ox10, and Ox21 displayed better growththan WT under P-deficient condition, with 26, 50, and 67%higher root biomass and 34, 64, and 66% higher shoot biomass,respectively (Figure 2BE). The results indicated that PvPht1;2may play a crucial role in enhancing P acquisition in transgenic

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tobaccos, thereby promoting plant growth at P-deficientcondition.To test this hypothesis, we measured P concentrations in

PvPht1;2-Ox tobaccos. Under P-sufficient condition, Pconcentrations of Ox1, Ox10, and Ox21 shoots were 21, 29,and 28% higher in the roots, and 17, 14, and 17% higher in theshoots than that of WT, respectively (Figure 2F), indicatingthat expressing PvPht1;2 promoted P acquisition by plants.Under P-deficient conditions, expressing PvPht1;2 enhancedOx1, Ox10, and Ox21 root P concentrations by 13, 22, and21%, respectively (Figure 2G). In contrast, total P concen-tration in the shoots of transgenic lines showed no significantdifference with that in WT (Figure 2G). However, consideringthe increased biomass of Ox1, Ox10, and Ox21 (Figure 2E), weconcluded that heterologous expression of PvPht1;2 increasedP acquisition by tobaccos, thereby promoting plant growthunder P-deficient condition.To further understand the underlying mechanism, 32P

radioisotope assay was employed. After cultivating in 0.2XHNS for 14 d, seedlings of PvPht1;2 transgenic tobaccos andWT were incubated in 0.2X HNS containing 8 μCi of 32P for12 h. The results showed that 32P uptake rates of transgeniclines reached 0.25−0.30 nmol mg−1 root FW, 31−57% higher

than that of WT, further proving that expressing PvPht1;2increased P uptake by transgenic plants (Figure 3A).After plant uptake, P is loaded from root cortical cells into

the xylem and translocated to the shoots, which is alsomediated by P transporters.38 To further investigate whetherPvPht1;2 also mediated P translocation, 32P translocationfactors (shoot/root 32P) were analyzed. The results showedthat 32P translocation factors of PvPht1;2-Ox lines were 0.99−1.1, being 69−92% higher than that of WT (Figure 3B),indicating that PvPht1;2 also facilitated P translocation intransgenic plants. P concentration in the xylem sap is animportant factor to characterize P translocation from the rootsto shoots. The P concentration in the xylem sap of PvPht1;2transgenic lines were 46−62% higher than that of WT (Figure3C), which was consistent with the increased translocationfactors, further proving that PvPht1;2 mediated P translocationin transgenic plants.Besides total P in plant tissues, we also determined the

inorganic P concentration in PvPht1;2-Ox lines. As a mainspecies in plants, inorganic P concentration can be used toindicate their P nutrition. After being cultured in 0.2X HNS for14 d under P-deficient condition, PvPht1;2-Ox lines and WTplants grown in P-sufficient (100 μM) solution for 7 d.Compared with WT plants, the root inorganic P concentrationof PvPht1;2-Ox lines showed no significant difference, butshoot concentrations in Ox1, Ox10, and Ox21 lines were 42,39, and 50% higher (Figure 3D), further confirming the criticalrole of PvPht1;2 in plant P translocation.In plants, P uptake and translocation are mediated by Phts.2

So, increasing number of Phts have been identified andfunctionally characterized, with the Pht1 subfamily being widelystudied.39 In this study, overexpression PvPht1;2 resulted inhigher P uptake, and root to shoot translocation factor (Figure3AB), and increased P accumulation under P-deficient and-sufficient conditions (Figure 2). The results suggested thatPvPht1;2 may play an important role in P uptake, and root tofrond transport in P. vittata. Considering its high expressionlevel in the fronds (Figure 1A), PvPht1;2 might also be involvedin frond P mobilization.

PvPht1;2 Showed Low Arsenate Transport Capacity inHydroponic Solution. Due to their chemical similarity, Ptransporters not only transport P but also AsV. To test whetherPvPht1;2 mediated AsV transport, we examined the growth ofΔpho84 yeast cells expressing PvPht1;2 in the presence of AsV.Compared with empty vector control, Δpho84 expressingPvPht1;2 showed little differences when grown on the SDmedium containing AsV (Figure 4A). Due to the deletion ofyeast P/AsV transporter Pho84, Δpho84 transformed withempty vector accumulated less As than its wild type BY4741(Figure 4B). Moreover, As accumulation in Δpho84 expressingPvPht1;2 was comparable to that with empty vector (Figure4B), suggesting that PvPht1;2 was incapable of complementingpho84 deletion. This was different from P transporter PvPht1;3,which showed high affinity for AsV when expressed in yeast andmay play a critical role in efficient AsV uptake in P. vittata.29

These results indicated that PvPht1;2 was not permeable toAsV, thus conferring little impact on AsV accumulation in yeast.Because PvPht1;2 increased plant P uptake and promoted

plant growth, the PvPht1;2 gene can be used to enhance Pacquisition by food crops to decrease consumption of Pfertilizer and increase crop production. However, consideringAs is ubiquitous in soils and many P transporters also facilitateAsV uptake in plants, it is important to consider As uptake by

Figure 1. Transcriptional patterns of PvPht1;2 in P. vittata sporophytesgrowing in 0.2X Hoagland nutrient solution (HNS) (A) andtranscriptional levels of PvPht1;2 in the roots (B) and fronds (C)responding to P-deficiency or As exposure. P. vittata were grown in0.2X HNS containing 100 μM P (+P), 0 μM P (−P), or 100 μM P/50μM As (+As) for 21 d. Error bars indicate SE of three biologicalreplicates.

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PvPht1;2. Thus, PvPht1;2-Ox lines were exposed to 20 μM AsVhydroponically for 3 d and As accumulation in tobacco weredetermined. The As concentration in PvPht1;2-Ox lines andWT plants were comparable (Figure 4CD). OverexpressionPvPht1;2 did not cause As accumulation in transgenic plants,suggesting that PvPht1;2 may contribute little to As uptake ortranslocation in P. vittata.Taken together, our results showed that PvPht1;2 was an

efficient P transporter but did not mediate AsV uptake by

plants, which is different from known P transporters. Forexample, OsPht1;1 and OsPht1;8 play key roles in P absorption,so they have been used to improve P acquisition by plants viatransgenic approach.9 However, while both OsPht1;1 andOsPht1;8 increase P uptake in transgenic plants, they alsoenhance As accumulation in plants.21,23 For example, inhydroponic solution, overexpression of OsPht1;1 enhanced Asaccumulation in rice by 41−47%.21 Moreover, OsPht1;8overexpression lines accumulated 4.6−5.6 folds higher As.23

Figure 2. Growth performances of PvPht1;2 overexpressing lines (Ox1; 10; and 21) and WT plants under different P levels. 14-d old transgenic andWT plants were grown in 0.2X HNS containing 100 μM P (+P) or 10 μM P (−P) for 14 d. Phenotype of PvPht1;2 overexpressing lines comparedwith WT under + P (A) or − P (B) solution. Relative expression of PvPht1;2 in transgenic lines and WT plants by semi-RT PCR (C). Biomass (DE)and total P concentration (FG) of PvPht1;2 overexpressing lines and WT under + P (DF) or − P (EG) conditions. Error bars represent SE (n = 5).Means marked with different letters indicate significant differences (p < 0.05). FW, fresh weight; DW, dry weight.

Figure 3. Uptake rate and root to shoot translocation of 32P and P concentration in xylem sap, roots, and shoots of PvPht1;2-Ox lines and WTtobaccos. (A) 32P uptake rate of PvPht1;2-Ox lines and WT; (B) shoot-to-root ratios of the 32P taken up by PvPht1;2-Ox lines and WT; (C) Pconcentration in xylem sap of PvPht1;2-Ox lines and WT; and (D) inorganic P concentration in PvPht1;2-Ox lines under P resupply condition. Aftergrown in 0.2X HNS lacking of P for 14 d, plants were transferred to P-sufficient (100 μM) solution 7 d. Error bars represent SE and n = 3 for AB andn = 5 for CD. Means marked with different letters indicate significant differences (p < 0.05). FW, fresh weight.

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Different from OsPht1;1 and OsPht1;8, however, PvPht1;2overexpression lines showed strong transport ability for Pwithout impacting As accumulation under different P regimes.Thus, it could be used as a candidate gene to improve Pabsorption and utilization efficiency in crops.Expression of PvPht1;2 Promoted Plant Growth

without Impacting As Uptake by Tobacco in SoilExperiments. Under hydroponic condition, P was suppliedas KH2PO4, which is soluble and available for plant uptake. Incontrast, in soil, P is often sorbed by Fe/Al oxides, resulting inlow availability.14 Since PvPht1;2 overexpression increased Puptake and translocation without impacting As accumulation inplants under hydroponic cultivation, it is important to validateits effects on plant growth and As accumulation in soils.Therefore, we grew tobaccos for 30 d in a soil, which

contained 8.11 mg kg−1 soluble P and was spiked with 0, 10, 20,or 40 mg kg−1 AsV. Compared with WT, shoot biomass ofPvPht1;2-Ox lines was 54−92, 51−108, 122−285, and 9.1−27% higher in four treatments (Figure 4E). With comparable Pconcentrations and higher biomass (data not shown), total Pcontent of PvPht1;2-Ox lines were higher than that of WT,consistent with hydroponic experiments.Based on their function characterization, expressing P

transporters is a promising approach to engineer low-Ptolerance in transgenic plants.38 Expressing HvPht1;1/6,40

OsPht1;1,9 and AtPht1;58 improved P acquisition andutilization efficiency in barley, rice, and Arabidopsis. However,overexpression of a P transporter does not guarantee bettergrowth. For example, overexpression of OsPht1;8 and OsPht1;2causes P toxicity.6,7 On the other hand, many P transportershave affinity for AsV, with only limited P transporters beingcharacterized for AsV transport. Though they can increase Pconcentration in plants, they may also increase plant As uptake,causing food safety issue. However, in our study, even in As-contaminated soils, expression of PvPht1;2 did not increase Asconcentrations in tobacco shoots (Figure 4F), which is ofsignificance for food safety.In summary, this study showed that the P. vittata P

transporter, PvPht1;2, is efficient in P uptake and translocation

in transgenic tobaccos. Hence, expressing PvPht1;2 increased Pcontent and promoted plant growth in tobacco plants inhydroponic and soil experiments. While many Phts arepermeable to AsV, PvPht1;2 showed little capacity to transportAsV, therefore expressing PvPht1;2 did not increase As uptakein plants. Based on the results, we envisioned that PvPht1;2transgenic approach can be used to enhance crop P acquisitionwithout increasing As uptake, thereby improving cropproduction and food safety.

■ AUTHOR INFORMATIONCorresponding Authors*Phone: + 86 025 8968 0631. E-mail: chenyanshan@nju.edu.cn.*E-mail: lqma@ufl.edu.

ORCIDLena Q. Ma: 0000-0002-8463-9957NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the National Natural ScienceFoundation of China (Grant No. 21637002 and 21707068),Jiangsu Provincial Natural Science Foundation of China (No.BK20160649), and the National Key Research and develop-ment program of China (Grant No. 2016YFD0800801).

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Figure 4. Arsenic concentrations in yeast and tobaccos expressing PvPht1;2. Phenotype (A) and As concentration (B) of yeast mutant Δpho84transformed with vector or vector containing PvPht1;2. Arsenic concentrations in the roots (C) and shoots (D) of PvPht1;2 overexpression lines andWT plants under P-sufficient (+P) and -deficient (−P) conditions, and plant growth (E) and As concentration (F) of PvPht1;2-Ox lines and WTplants after growing for 21 d in soil containing 0, 5, 10, or 20 mg kg−1 of As. Error bars represent SE (n = 5) and means marked with different lettersindicate significant differences (p < 0.05). DW, dry weight.

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