Phosphate transporter PvPht1;2 enhances phosphorus accumulation

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Characterization of Natural and Affected Environments

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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Environmental Science & Technology

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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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ACS Paragon Plus Environment


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TOC

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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

303

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

304

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

305

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

306

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

307

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

308

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

309

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

310

and utilization efficiency in crops.

311

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

312

in soil experiment

313

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

314

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

315

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

316

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

317

its effects on plant growth and As accumulation in soils.

318

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

319

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

320

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

321

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

322

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

323

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

325

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

326

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

327

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

328

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

329

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

330

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

331

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

332

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

333

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

334

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

335

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

336

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

337

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

338

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

339

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

340 341

ACKNOWLEDGEMENTS

342

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

343

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

344

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

345

2016YFD0800801).

346

14


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347 348

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

349

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

350

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

351

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

352

bars indicate SE of three biological replicates.

353

15



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354 355

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

356

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

357

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

358

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

359

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

360

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

361

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

362

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

363 364

16


Page 17 of 21


365 366

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

368

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

369

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

370

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

371

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

373

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

374 375

17



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376 377

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

378

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

379

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

380

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

381

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

382

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

383

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

384 385 386 387

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Phosphate transporter PvPht1;2 enhances phosphorus accumulation

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