Phosphate Transporter PvPht1;2 Enhances Phosphorus Accumulation

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Phosphate transporter PvPht1;2 enhances phosphorus accumulation and plant growth without impacting arsenic uptake in plants Yue Cao, Dan Sun, Jun-Xiu Chen, Hanyi Mei, Hao Ai, Guohua Xu, Yanshan Chen, and Lena Q. Ma Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06674 • Publication Date (Web): 14 Mar 2018 Downloaded from http://pubs.acs.org on March 14, 2018

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Phosphate transporter PvPht1;2 enhances phosphorus accumulation and plant growth

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without impacting arsenic uptake in plants

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Yue Cao,† Dan Sun,† Jun-Xiu Chen,† Hanyi Mei,† Hao Ai,‡ Guohua Xu,‡ Yanshan Chen*,†

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Lena Q. Ma†,§

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†State Key Lab of Pollution Control and Resource Reuse, School of the Environment,

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Nanjing University, Jiangsu 210023, China

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‡State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of

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Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of

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Agriculture, Nanjing Agricultural University, 210095, China

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§Soil and Water Science Department, University of Florida, Gainesville, FL 32611, United

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States

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*Corresponding author at State Key Laboratory of Pollution Control and Resource Reuse,

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School of the Environment, Nanjing University, Jiangsu 210023, China; +86 025 8968 0631

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E-mail addresses: [email protected], [email protected]

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ABSTRACT

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Phosphorus is an important macronutrient for plant growth and is acquired by plants

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mainly as phosphate (P). Phosphate transporters (Phts) are responsible for P and arsenate

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(AsV) uptake in plants including arsenic-hyperaccumulator Pteris vittata. P. vittata is

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efficient in AsV uptake and P utilization, but the molecular mechanism of its P uptake is

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largely unknown. In this study, a P. vittata Pht, PvPht1;2, was cloned and transformed into

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tobacco (Nicotiana tabacum). In hydroponic experiments, all transgenic lines displayed

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markedly higher P content and better growth than wild type, suggesting that PvPht1;2

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mediated P uptake in plants. In addition, expressing PvPht1;2 also increased the shoot/root

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mediated P translocation in plants. Unlike many Phts permeable to AsV, PvPht1;2 showed

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little ability to transport AsV. In soil experiments, PvPht1;2 also significantly increased shoot

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biomass without elevating As accumulation in PvPht1;2 transgenic tobacco. Taken together,

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our results demonstrated that PvPht1;2 is a specific P transporter responsible for P acquisition

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and translocation in plants. We envisioned that PvPht1;2 can enhance crop P acquisition

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without impacting AsV uptake, thereby increasing crop production without compromising

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food safety.

P ratio by 69–92% and enhanced xylem sap P by 46–62%, indicating that PvPht1;2 also

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INTRODUCTION

Phosphorus is a major essential macronutrient for plant growth, which is involved in

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many metabolic pathways. Plants take up phosphorus exclusively in the form of inorganic

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phosphate (P). Because of its high fixation in soils and slow diffusion to the root surface,

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plants have evolved strategies to increase the availability of soil P.1 In plants, the high-affinity

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P transporters (Phts/PTs) play key roles in P acquisition from soil.2 These P transporters are

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categorized into four subfamilies: Pht1, Pht2, Pht3, and Pht4.3 Over the past decades, many

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genes that encode PTs have been identified and cloned from A. thaliana and cereal, legume,

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and solanaceous species.4-11

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In Arabidopsis, Pht1 subfamily is comprised of 9 members (AtPht1;1 to 1;9). Among

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them, AtPht1;1 and AtPht1;4 are responsible for P acquisition under both high- and low-P

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conditions.12, 13 The P uptake by atpht1;1/atpht1;4 double mutant was 75% lower than wild

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type (WT) plants.13 In addition, as high-affinity P transporters, AtPht1;8 and AtPht1;9 play

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key roles in P uptake under P-deficient conditions.10

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In rice (Oryza sativa), 13 Pht1 genes are known in the genome.4 Among them, OsPht1;1,

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1;2, 1;4, 1;6, 1;8, 1;9 and 1;10 mediate P uptake and translocation in rice.14, 15 OsPT1 is

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constitutively expressed in plants, functioning in P uptake and translocation under

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P-sufficient conditions.9 Similarly, OsPht1;8 is expressed in various tissues under both

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P-sufficient and -deficient conditions, and is up-regulated in the roots under P-deficient

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conditions.7 OsPht1;6 is mainly expressed in the roots, involving in P uptake under

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P-deficient conditions.6 Recently, the function of OsPht1;4 has been characterized, which

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facilitates P acquisition and mobilization in rice.11

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Arsenic (As) and P are chemical analogs. However, As is a toxic element and ubiquitous

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in soils, which can be taken up by crops, thereby threatening human health through food

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chain.16, 17 Due to their similarity, AsV can be taken up and translocated via PTs.13, 18, 19 In

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Arabidopsis, AsV is taken up via AtPht1;1 and AtPht1;4.13, 20 In rice, OsPht1;1, Os Pht1;4 and

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OsPht1;8 are involved in AsV uptake and translocation, and their modulation affects As

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accumulation in rice.19, 21, 22 Though overexpression of PTs promotes P acquisition,14 it may

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also increase As uptake by plants.21, 23

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Chinese brake fern (Pteris vittata) is the first-known As-hyperaccumulator, it is efficient

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in As uptake, translocation and detoxification.24, 25 Besides, the fern is also efficient in

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acquiring P from insoluble P sources in soils,26, 27 and efficient in depleting P from

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hydroponic solution.28 Recently, P. vittata P transporters PvPht1;1 to PvPht1;3 have been

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characterized.29 Yeast experiments showed that PvPht1;3 is a high-affinity AsV transporter.29

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However, the functions of PvPht1;1 and PvPht1;2 in plants have not been elucidated, so their

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role in improving P utilization is unclear.

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PvPht1;1 and PvPht1;2 encode predicted proteins of 536 amino acids, which share 98.5%

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identity.29 With only few nucleotides being different, they can be considered as the same gene.

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In this work, to study the function of PvPht1;2 and its role in P uptake in plants, we

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transformed PvPht1;2 into model plant tobacco and investigated its function in P and AsV

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uptake and translocation by transgenic tobacco. We believe that this study may provide

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important insights into the behavior of PvPht1;2 as well as provide a potential strategy to

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enhance crop P acquisition.

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MATERIALS AND METHODS

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Growth of P. vittata. Spores of P. vittata were collected from Florida, USA24 and

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preserved in our lab at Nanjing University. Their spores were sown on potting soils, watered

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and covered with transparent plastic films to keep the soil moist. After 2 months of

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cultivation, sporophyte seedlings with 2–3 fronds appeared, which were then transplanted

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into separate pots following Fu et al.28 All sporophytes were cultivated in a greenhouse to

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4-frond stage and then acclimated in 500 mL aerated 0.2 strength (0.2X) Hoagland nutrient

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solution (HNS) for 7 d.28 For the transcripts analysis, sporophyte seedlings were transferred

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0.2X HNS containing 100 µM KH2PO4 (+P) , 0 µM KH2PO4 (–P) or 100 µM KH2PO4/50 µM

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Na2HAsO4·7H2O (+As) for 3 days. All ferns were grown under a 14 h photoperiod, 26/20◦C

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day/night temperature, 60% relative humidity, and 3000 lux light intensity.

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Total RNA Preparation and qRT-PCR analysis in P. vittata. Total RNAs from P.

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vittata roots and fronds were isolated using Plant Total RNA Kit (Sigma-Aldrich), reverse

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transcription and first-strand cDNA was synthesized using HiScript II One Step RT-PCR Kit

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(Vazyme Biotech, Nanjing, China). qRT-PCR analysis was performed using SYBR Green

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PCR Master Mix (Vazyme Biotech, Nanjing, China), and the CFX Connect Real-Time PCR

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Detection System (BIO-RAD). Relative expression levels of PvPht1;2 (Accession No.

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KM192136) were computed by 2-∆∆CT method of relative quantification. P. vittata Actin gene

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(PvActin) and Histone gene (PvHistone)30 were used as an internal control. All gene-specific

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primers used for qRT-PCR are as follows. PvPHT1;2: 5'-GCC CTG GTA TTG GCC ACA

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AG-3' and 5'-CCT CGA GGG AGC GAC CAT TT-3'; PvActin: 5'-GGG CAG TAT TTC

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CAA GCA TAG TGG G-3' and 5'-TGC CTC GCT TTG ATT GAG CCT CAT C-3';

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PvHistone: 5'-GGG TTT ACA TTC AGC GAA GC-3' and 5'-GCT TTC CCT CCA GTG

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GAC TT-3'.

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Yeast Vector Construction, Yeast Transformation and Growth Assays. PvPht1;2

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coding sequence was cloned from cDNA of P. vittata collected from Florida, USA using the

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following primers: 5’-ATG GCA AAA CTA GAG GTC CTC ACC G-3’ and 5’-CTA TGA

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TGT GTG TGT AGC ACC CCC A-3’. Adapters were added to PvPht1;2 CDS using the

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following primers: 5’-gaa aaa acc ccg gat tct aga ATG GCA AAA CTA GAG GTC CTC

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ACC G-3’ and 5’-taa cta att aca tga ctc gag CTA TGA TGT GTG TGT AGC ACC CCC A-3’

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(underlining indicates recombination sequences). The PCR product was then cloned into the

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GAL1 promoter cassette of pAG413GAL-ccdB (Addgene, http://www.addgene.org/) between

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XbaI and XhoI restriction sites by recombination, using the Trelief™ SoSoo Cloning Kit

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(TSING KE, Nanjing, China). The yeast (Saccharomyces cerevisiae) strain for heterologous

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expression of PvPht1;2 was the ∆pho84 mutant (Thermo Scientific,

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https://www.openbiosystems.com) with the BY4741 (MATa his3∆1 leu2∆0 met15∆0 ura3∆0)

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background.31, 32 The methods related to yeast transformations mainly referred to the high

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efficiency transformation of yeast described by Gietz et al.33

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Yeast growth assay was performed according to Chen et al5. Briefly, yeast cells were 6

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grown at 30°C in synthetic defined (SD) medium (0.67% yeast nitrogen base) without amino

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acids, containing 2% (w/v) glucose or 2% (w/v) galactose (induction medium), supplemented

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with yeast synthetic dropout without histidine at pH 5.8. For AsV tolerance assays, yeast was

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grown in liquid SD medium (with 2% [w/v] glucose) to an OD600 of ~1.0 and then subjected

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to centrifugation and dilution with sterile water. The drop assays were performed on SD

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plates (with 2% [w/v] galactose) containing 1.0 mM AsV for ∆pho84 expressing PvPht1;2.

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Plant expression vector construction and transgenic plant generation and selection.

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Adapters were added to PvPht1;2 CDS using the following primers:5’-acg ggg gac tct aga

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gga tcc ATG GCA AAA CTA GAG GTC CTC ACC G-3’ and 5’-ggg aaa ttc gag ctc ggt acc

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CTA TGA TGT GTG TGT AGC ACC CCC A-3’ (underlining indicates recombination

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sequences). The PCR product was then cloned into the 35S promoter cassette of pSN1301

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(pCAMBIA1301, CAMBIA) between BamHI and KpnI restriction sites by recombination,

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using the CloneEZ PCR Cloning Kit (Genscript, Nanjing, China), with the constructed binary

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vector being named pSN1301-PvPT1;2. Agrobacterium strain C58 was transformed with the

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binary vector pSN1301-PvPT1;2 by electroporation. Transformation of tobacco leaf explants

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was carried out following Curtis et al. and Gallois & Marinho et al.34, 35 Transgenic plants

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were then identified via hygromycin resistance and GUS staining.

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Semi-quantitative RT-PCR analysis of transgenic tobaccos. Total RNA was

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extracted from tobacco seedlings. The first-strand cDNA was synthesized from 2 µL total

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RNA using HiScript II One Step RT-PCR Kit (Vazyme Biotech, Nanjing, China), which was

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used as RT-PCR templates. The cDNAs of PvPht1;2 were amplified by PCR for 30 cycles

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using the gene-specific primers 5'-GCC CTG GTA TTG GCC ACA AG-3' and 5'-CCT CGA

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GGG AGC GAC CAT TT-3'. Tobacco actin was amplified for 30 cycles as an expression

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control using the LeActin primer 5'-TTC CGT TGC CCA GAG GTC CT-3' and 5'-GGG

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AGC CAA GGC AGT GAT TTC-3'.

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Growth of transgenic tobacco in different P and As conditions. In hydroponic

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experiments, transgenic tobacco seeds and wild type (WT) seeds were germinated in 1/5 MS

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media. Uniform 2-week old tobacco seedlings were transferred to 0.2X HNS containing 100 7

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µM KH2PO4 (+P) or 10 µM KH2PO4 (–P) for 14 d. For As accumulation determination,

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seedlings were exposed to 20 µM AsV (Na2HAsO4·7H2O, Sigmae-Aldrich, USA) for 3 days.

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For inorganic P determination, PvPht1;2–Ox lines and WT plants were cultured in 0.2X HNS

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for 14 d under P-deficient condition, and then transferred to P-sufficient (100 µM) solution

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for 7 d. In soil test, transgenic and WT tobacco seeds were germinated and cultivated in a

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garden soil. In addition, uniform 7-d old tobacco seedlings were transferred into soils

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containing 0, 10, 20, and 40 mg kg–1 AsV for 30 d. 32

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P uptake assay and xylem sap collection in tobacco. After growing in 0.2X HNS for

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7 d, tobaccos were transferred into 0.2X HNS (200 mL) labeled with 8 µCi of 32P (KH2PO4,

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Perkin-Elmer, Waltham, MA, USA) and cultivated for 12 h. Then the plant roots were

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incubated in ice-cold desorption solution (0.5 mM CaCl2, 100 µM NaH2PO4, 2 mM MES, pH

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5.5) for 10 min to remove 32P. The plants were then blotted-dry, the roots and shoots were

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harvested, and their fresh weights were measured. Tissues were digested in HClO4 and 30%

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(v/v) H2O2 mixture at 70°C for 2–3 h. Scintillation cocktail (3 mL) was added to the digested

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tissue and liquid scintillation counter (Tri–Carb 2100, Packard) was employed to determine

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32

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P activity. Transgenic and WT tobacco seedlings were cultured under 0.2X HNS. Briefly, the stems

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of tobacco were cut at 2 cm above the roots. The cut surfaces were rinsed with deionized

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water and blotted dry. The xylem sap was collected by pipette from the cut surface for 2 h.

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The inorganic P concentration of xylem sap was determined as described below.

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P and As determination in plants. Total P concentrations of plant samples were

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measured according to Chen et al.5 Briefly, ~0.05 g of crushed dry samples were digested

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with H2SO4–H2O2 at 280℃. After cooling, the digested samples were diluted to 100 mL in

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distilled water. P concentration was analyzed by the molybdenum blue method based on dry

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weight.36

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For inorganic P in plant, ~ 0.5 g fresh samples were used.36 Briefly, the samples were

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homogenized in 1 mL of 10% (w/v) perchloric acid using an ice-cold mortar and pestle. The

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homogenate was then diluted 10 times with 5% (w/v) perchloric acid and placed on ice for 30 8

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min. After centrifugation at 10,000 g for 10 min at 4℃, the supernatant was used for P

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measurement via the molybdenum blue method. The absorption values for the solution at 820

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nm were determined using a spectrophotometer (SHIMADZUUV-2550).

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For As analysis, fresh plants were separated into the shoots and roots, lyophilized

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(FreezZone 12, LABCONCO) and stored at -80℃. For total As, freeze-dried plant sample

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(0.05 g ) was digested with 50% HNO3 at 105℃ following USEPA Method 3050B and

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determined by inductively coupled plasma mass spectrometry (ICP-MS; PerkinElmer

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NexION 300X, USA; detection limit at 0.1 µg L-1).

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QA/QC and statistical analysis. For quality assurance and quality control (QA/QC),

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indium was used as internal standards and was added into the samples, calibration standards,

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and blanks. During measurement, standard solution at 5 µg L-1 As was measured every 20

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samples to monitor the stability of ICP-MS. The check recovery was within 90–110%. In

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addition, blanks and certified reference material for plant samples (GSB 21, Chinese

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geological reference materials) were included for quality assurance, which were within

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expected values.37

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Data are presented as the mean of 3–5 replicates with standard error. Analysis of

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variance (ANOVA) was carried out by SPSS software (SPSS 13.0; SPSS Inc, Chicago, USA).

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Significant differences were determined with treatment means compared by Tukey’s mean

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grouping tests at p < 0.05.

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RESULTS AND DISCUSSION

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Identification and Expression pattern of P. vittata P Transporter PvPht1;2

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To understand the molecular mechanism of P metabolism in P. vittata, 6 putative Pht

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sequences were identified, including PvPht1;2. Then transcriptional expression of PvPht1;2

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in P. vittata was investigated by qRT-PCR using actin and histone as reference genes. As

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shown in Figure 1A, PvPht1;2 was expressed strongly in the roots and fronds, with frond

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transcripts level of PvPht1;2 being 42% higher than root.

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It is known that P deficiency induces Pht expression in plants. Besides, as an analog, AsV is also taken up by Pht transporters in plants, so it may affect Pht transcription in plants. 9

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Thus, we investigated the expression of PvPht1;2 responding to P deficiency (no P) or AsV

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exposure (50 µM AsV). In the roots, the expression of PvPht1;2 was 8.5-fold higher under

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P-deficient condition than that under P-sufficient condition (Figure 1B). When P. vittata was

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exposed to AsV, transcripts level of PvPht1;2 in the roots was comparable to no As control

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(Figure 1B).The results were similar to P. vittata’s expression pattern in DiTusa et al.29 In the

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fronds, the expression of PvPht1;2 transcripts was similar in different treatments (Figure 1C).

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These results showed that PvPht1;2 transcripts were induced by P deficiency in P. vittata

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roots, but not by AsV, indicating that PvPht1;2 may play a critical role in P acquisition but

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not As uptake in P. vittata.

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Overexpression of PvPht1;2 increased P uptake and translocation in tobacco plants

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To characterize its function in P uptake and translocation in plants, we generated

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PvPht1;2 transgenic tobacco lines (PvPht1;2-Ox), where PvPht1;2 was expressed under

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constitutive CaMV35S promoter. Three independent transgenic T2 lines (Ox1, Ox10, and

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Ox21) were selected to assess its effects on P acquisition (Figure 2). RT-PCR analysis showed

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that PvPht1;2 transcripts were strongly expressed in PvPht1;2–Ox lines, while it was not

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detected in WT plants (Figure 2C).

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In hydroponic experiments, PvPht1;2-Ox lines and WT plants were cultured under

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P-sufficient and -deficient conditions for 14 d (Figure 2). All three transgenic plants grew

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similarly as WT under P-sufficient treatment (Figure 2AD). However, Ox1, Ox10, and Ox21

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displayed better growth than WT under P-deficient condition, with 26, 50, and 67% higher

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root biomass and 34, 64, and 66% higher shoot biomass, respectively (Figure 2BE). The

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results indicated that PvPht1;2 may play a crucial role in enhancing P acquisition in

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transgenic tobaccos, thereby promoting plant growth at P-deficient condition.

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To test this hypothesis, we measured P concentrations in PvPht1;2-Ox lines. Under

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P-sufficient condition, P concentrations of Ox1, Ox10, and Ox21 shoots were 21, 29, and 28%

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higher in the roots, and 17, 14, and 17% higher in the shoots than that of WT, respectively

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(Figure 2F), indicating that expressing PvPht1;2 promoted P acquisition by plants. Under

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P-deficient conditions, expressing PvPht1;2 enhanced Ox1, Ox10, and Ox21 root P 10

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concentrations by 13, 22, and 21%, respectively (Figure 2G). In contrast, total P

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concentration in the shoots of transgenic lines showed no significant difference with that in

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WT (Figure 2G). However, considering the increased biomass of Ox1, Ox10 and Ox21

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(Figure 2E), we concluded that heterologous expression of PvPht1;2 increased P acquisition

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by plants, thereby promoting plant growth under P-deficient condition.

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To further understand the underlying mechanism, 32P radioisotope assay was employed.

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After cultivating in 0.2X HNS for 14 d, seedlings of PvPht1;2 transgenic lines and WT were

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incubated in 0.2X HNS containing 8 µCi of 32P for 12 h. The results showed that 32P uptake

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rates of transgenic lines reached 0.25–0.30 nmol mg-1 root FW, 31–57% higher than that of

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WT, further proving that expressing PvPht1;2 increased P uptake by transgenic plants (Figure

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3A).

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After plant uptake, P is loaded from root cortical cells into the xylem and translocated to

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the shoots, which is also mediated by P transporters.38 To further investigate whether

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PvPht1;2 also mediated P translocation, 32P translocation factors (shoot/ root 32P) were

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analyzed. The results showed that 32P translocation factors of PvPht1;2-Ox lines were 0.99–

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1.1, being 69–92% higher than that of WT (Figure 3B), indicating that PvPht1;2 also

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facilitated P translocation in transgenic plants. P concentration in the xylem sap is an

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important factor to characterize P translocation from the roots to shoots. The P concentration

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in the xylem sap of PvPht1;2 transgenic lines were 46–62% higher than that of WT (Figure

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3C), which was consistent with the increased translocation factors, further proving that

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PvPht1;2 mediated P translocation in transgenic plants.

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Besides total P in plant tissues, we also determined the inorganic P concentration in

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PvPht1;2-Ox lines. As a main species in plants, inorganic P concentration can be used to

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indicate their P nutrition. After cultured in 0.2X HNS for 14 d under P-deficient condition,

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PvPht1;2–Ox lines and WT plants grown in P-sufficient (100 µM) solution for 7 d.

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Compared with WT plants, the root inorganic P concentration of PvPht1;2-Ox lines showed

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no significant difference, but shoot concentrations in Ox1, Ox10, and Ox21 lines were 42, 39,

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and 50% higher (Figure 3D), further confirming the critical role of PvPht1;2 in plant P

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translocation.

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In plants, P uptake and translocation are mediated by Phts.2 So, increasing number of

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Phts have been identified and functionally characterized, with the Pht1 subfamily being

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widely studied.39 In this study, overexpression PvPht1;2 resulted in higher P uptake, and root

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to shoot translocation factor (Figure 3AB), and increased P accumulation under P-deficient

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and -sufficient conditions (Figure 2). The results suggested that PvPht1;2 may play an

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important role in P uptake, and root to frond transport in P. vittata. Considering its high

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expression level in the fronds (Figure 1A), PvPht1;2 might also be involved in frond P

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mobilization.

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PvPht1;2 showed low arsenate transport capacity in hydroponic solution

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Due to their chemical similarity, P transporters not only transport P but also AsV. To test

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whether PvPht1;2 mediated AsV transport, we examined the growth of ∆pho84 yeast cells

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expressing PvPht1;2 in the presence of AsV. Compared with empty vector control, ∆pho84

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expressing PvPht1;2 showed little differences when grown on the SD medium containing

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AsV (Figure 4A). Due to the deletion of yeast P/AsV transporter Pho84, ∆pho84 transformed

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with empty vector accumulated less As than its wild type BY4741 (Figure 4B). Moreover, As

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accumulation in ∆pho84 expressing PvPht1;2 was comparable to that with empty vector

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(Figure 4B), suggesting that PvPht1;2 was incapable of complementing pho84 deletion. This

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was different from P transporter PvPht1;3, which showed high affinity for AsV when

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expressed in yeast and may play a critical role in efficient AsV uptake in P. vittata.29 These

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results indicated that PvPht1;2 was not permeable to AsV, thus conferring little impact on

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AsV accumulation in yeast.

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Because PvPht1;2 increased plant P uptake and promoted plant growth, PvPht1;2 gene

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can be used to enhance P acquisition by food crops to decrease consumption of P fertilizer

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and increase crop production. However, considering As is ubiquitous in soils and many P

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transporters also facilitate AsV uptake in plants, it is important to consider As uptake by

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PvPht1;2. Thus, PvPht1;2-Ox lines were exposed to 20 µM AsV hydroponically for 3 d and 12

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As accumulation in tobacco were determined. The As concentration in PvPht1;2-Ox lines and

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WT plants were comparable (Figure 4CD). Overexpression PvPht1;2 did not cause As

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accumulation in transgenic plants, suggesting that PvPht1;2 may contribute little to As uptake

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or translocation in P. vittata.

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Taken together, our results showed that PvPht1;2 was an efficient P transporter but

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didn’t mediate AsV uptake by plants, which is different from known P transporters. For

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example, OsPht1;1 and OsPht1;8 play key roles in P absorption, so they have been used to

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improve P acquisition by plants via transgenic approach.9 However, while both OsPht1;1 and

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OsPht1;8 increase P uptake in transgenic plants, they also enhance As accumulation in

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plants.21, 23 For example, in hydroponic solution, overexpression of OsPht1;1 enhanced As

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accumulation in rice by 41-47%.21 Moreover, OsPht1;8 overexpression lines accumulated

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4.6-5.6 folds higher As.23 Different from OsPht1;1 and OsPht1;8, however, PvPht1;2

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overexpression lines showed strong transport ability for P without impacting As accumulation

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under different P regimes. Thus, it could be used as a candidate gene to improve P absorption

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and utilization efficiency in crops.

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Expression of PvPht1;2 promoted plant growth without impacting As uptake by tobacco

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in soil experiment

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Under hydroponic condition, P was supplied as KH2PO4, which is soluble and available

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for plant uptake. In contrast, in soil, P is often sorbed by Fe/Al oxides, resulting in low

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availability.14 Since PvPht1;2 overexpression increased P uptake and translocation without

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impacting As accumulation in plants under hydroponic cultivation, it is important to validate

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its effects on plant growth and As accumulation in soils.

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Therefore, we grew tobaccos for 30 d in a soil, which contained 8.11 mg kg–1 soluble P

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and was spiked with 0, 10, 20, and 40 mg kg–1 AsV. Compared with WT, shoot biomass of

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PvPht1;2-Ox lines was 54–92, 51–108, 122–285 and 9.1–27% higher in 4 treatments (Figure

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4E). With comparable P concentrations and higher biomass (data not shown), total P content

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of PvPht1;2–Ox lines were higher than that of WT, consistent with hydroponic experiments.

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Based on their function characterization, expressing P transporters is a promising 13

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approach to engineer low-P tolerance in transgenic plants.38 Expressing HvPht1;1/6,40

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OsPht1;19 and AtPht1;58 improved P acquisition and utilization efficiency in barley, rice and

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Arabidopsis. However, overexpression of a P transporter does not guarantee better growth.

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For example, overexpression of OsPht1;8 and OsPht1;2 causes P toxicity.6, 7 On the other

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hand, many P transporters have affinity for AsV, with only limited P transporters being

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characterized for AsV transport. Though they can increase P concentration in plants, they

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may also increase plant As uptake, causing food safety issue. However, in our study, even in

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As-contaminated soils, expression of PvPht1;2 didn’t increase As concentrations in plant

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shoots (Figure 4F), which is of significance for food safety.

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In summary, this study showed that the P. vittata P transporter, PvPht1;2, is efficient in P

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uptake and translocation in transgenic tobaccos. Hence, expressing PvPht1;2 increased P

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content and promoted plant growth in tobacco plants in hydroponic and soil experiments.

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While many Phts are permeable to AsV, PvPht1;2 showed little capacity to transport AsV,

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therefore expressing PvPht1;2 didn’t increase As uptake in plants. Based on the results, we

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envisioned that PvPht1;2 transgenic approach can be used to enhance crop P acquisition

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without increasing As uptake, thereby improving crop production and food safety.

340 341

ACKNOWLEDGEMENTS

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This work was supported by the National Natural Science Foundation of China (Grant No.

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21637002 and 21707068), Jiangsu Provincial Natural Science Foundation of China (No.

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BK20160649) and the National Key Research and development program of China (Grant No.

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2016YFD0800801).

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Figure 1. Transcriptional patterns of PvPht1;2 in P. vittata sporophytes growing in 0.2X

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Hoagland nutrient solution (HNS) (A), and transcriptional levels of PvPht1;2 in the roots (B)

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and fronds (C) responding to P-deficiency or As exposure. P. vittata were grown in 0.2X

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HNS containing 100 µM P (+P), 0 µM P (–P) or 100 µM P/50 µM As (+As) for 21 d. Error

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bars indicate SE of three biological replicates.

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Figure 2. Growth performances of PvPht1;2 overexpressing lines (Ox1; 10; and 21) and WT

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plants under different P levels. 14-d old transgenic and WT plants were grown in 0.2X HNS

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containing 100 µM P (+P) or 10 µM P (–P) for 14 d. Phenotype of PvPht1;2 overexpressing

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lines compared with WT under +P (A) or –P (B) solution; Relative expression of PvPht1;2 in

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transgenic lines and WT plants by semi-RT PCR (C); Biomass (DE) and total P

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concentration (FG) of PvPht1;2 overexpressing lines and WT under +P (DF) or –P (EG)

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conditions. Error bars represent SE (n=5). Means marked with different letters indicate

362

significant differences (p < 0.05). FW, fresh weight.

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Figure 3. Uptake rate and root to shoot translocation of 32P and P concentration in xylem sap,

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roots and shoots of PvPht1;2-Ox lines and WT plants. A, 32P uptake rate of PvPht1;2-Ox

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lines and WT; B, shoot-to-root ratios of the 32P taken up by PvPht1;2-Ox lines and WT; C, P

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concentration in xylem sap of PvPht1;2-Ox lines and WT; and D, inorganic P concentration

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in PvPht1;2-Ox lines under P re-supply condition. After grown in 0.2X HNS lacking of P for

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14 d, plants were transferred to P–sufficient (100 µM) solution 7 d. Error bars represent SE

372

and n=3 for AB and n=5 for CD. Means marked with different letters indicate significant

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differences (p < 0.05). FW, fresh weight.

374 375

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Figure 4. Arsenic concentrations in yeast and plants expressing PvPht1;2. Phenotype (A) and

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As concentration (B) of yeast mutant ∆pho84 transformed with vector or vector containing

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PvPht1;2. Arsenic concentrations in the roots (C) and shoots (D) of PvPht1;2 overexpression

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lines and WT plants under P -sufficient (+P) and -deficient (–P) conditions, and plant growth

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(E) and As concentration (F) of PvPht1;2-Ox lines and WT plants after growing for 21 d in

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soil containing 0, 5, 10, or 20 mg kg–1 of As, and Error bars represent SE (n=5) and means

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marked with different letters indicate significant differences (p < 0.05). DW, dry weight.

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